WO2020097048A1

WO2020097048A1 - Systems and methods for sorting particles using hydrodynamic sizing

Info

Publication number: WO2020097048A1
Application number: PCT/US2019/059827
Authority: WO
Inventors: Kyle Christopher SMITH; Ravi Kapur
Original assignee: Micromedicine, Inc.
Priority date: 2018-11-05
Filing date: 2019-11-05
Publication date: 2020-05-14

Abstract

The present invention generally relates to systems and methods for sorting particles. Certain aspects are generally directed to systems and methods for sorting a first type of particle from a second type of particle, e.g., using various microfluidic hydrodynamic sorting techniques. In addition, in some embodiments, particles that differ in other characteristics, and/or particles that generally overlap in size, can be separated by exposing the particles to agents able to alter their hydrodynamic size (e.g., by binding to the particles, directly or through a targeting moiety), sorting the particles, then optionally removing the agents to restore their original hydrodynamic size, for example, by dissolving or reacting at least a portion of the agents and/or the targeting moiety. As examples, the agents may include Ca²⁺-alginate beads, which can be removed using a chelator such as EDTA, or polystyrene beads with cleavable linkers.

Description

SYSTEMS AND METHODS FOR SORTING PARTICLES USING

HYDRODYNAMIC SIZING

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application Serial No. 62/755,678, filed November 5, 2018, entitled“Systems and Methods for Sorting Particles Using Hydrodynamic Sizing,” by Smith, et al., and of U.S. Provisional Patent Application Serial No. 62/758,793, filed November 12, 2018, entitled“Systems and Methods for Sorting Particles Using Hydrodynamic Sizing,” by Smith, et al. Each of these is incorporated herein by reference in its entirety.

FIELD

The present invention generally relates to systems and methods for sorting particles.

BACKGROUND

Particle separation and filtration have been used in numerous applications across industries and fields. Examples of such applications include chemical process and fermentation filtration, water purification/wastewater treatment, sorting and filtering components of blood, concentrating colloid solutions, and purifying and concentrating environmental samples. Many separation and filtration techniques separate particles on the basis of size, in particular, hydrodynamic size, or the effective size of the particle as it flows within a fluid. However, as particle sizes are generally fixed, especially in certain

applications such as biological applications, it can be challenging to separate similar-sized particles from each other. Accordingly, improvements in such techniques are needed.

SUMMARY

The present invention generally relates to systems and methods for sorting particles. The subject matter of the present invention involves, in some cases, interrelated products, alternative solutions to a particular problem, and/or a plurality of different uses of one or more systems and/or articles.

In one aspect, the present invention is generally directed to a method of sorting particles. In accordance with one set of embodiments, the method comprises altering an average hydrodynamic size of at least some particles suspended in a fluid sample by binding agents to the particles able to alter the hydrodynamic size, and sorting the suspended particles based on their hydrodynamic size by flowing the suspended particles through a particle sorting region of a microfluidic device. According to another aspect, the present invention is generally directed to a method of producing a sample enriched in particles of a first type. In one set of embodiments, the method comprises obtaining a fluid sample comprising particles of a first type and particles of a second type, the first particles having a first average hydrodynamic size and the second particles having a second average hydrodynamic size; altering the average hydrodynamic size of the particles of the first type in the fluid sample to provide particles of the first type having an increased effective size in the fluid sample; and sorting the suspended particles based on their hydrodynamic size by flowing the fluid sample containing the particles of the first type having the increased effective size into a particle sorting region of a microfluidic device, and at least partially separating the particles of the first type from particles of the second type.

In yet another aspect, the present invention is generally directed to a method of sorting cells. In one set of embodiments, the method comprises providing cells suspended in a fluid sample, including a first type of cell and a second type of cell, the cells of the first type of cell having a first average hydrodynamic size and the cells of the second type of cell having a second average hydrodynamic size; causing an alteration of the average

hydrodynamic size of the cells of the first cell type, wherein the alteration is 80% selective to the cells of the first type of cell relative to the cells of the second type of cell; and sorting the cells suspended in a fluid sample based on their hydrodynamic size by flowing the suspended cells through a cell sorting region of a microfluidic device.

In another aspect, the present invention encompasses methods of making one or more of the embodiments described herein. In still another aspect, the present invention encompasses methods of using one or more of the embodiments described herein.

Other advantages and novel features of the present invention will become apparent from the following detailed description of various non-limiting embodiments of the invention when considered in conjunction with the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention. In the figures: Figs. 1A-1D are schematics illustrating the use of agents to sort particles on the basis of size, in accordance with one embodiment of the present disclosure;

Figs. 2A-2B are schematics illustrating how binding of agents to particles can be used for sorting, in various embodiments of the present disclosure;

Fig. 3 is a top view of the general architecture of a representative microfluidic device according to one embodiment of the present disclosure;

Fig. 4 is a schematic that illustrates a top view of a portion of a fluid exchange module of the device shown in Fig. 3;

Fig. 5 is a schematic that illustrates a top view of a particle concentration module of the device shown in Fig. 3;

Figs. 6A-6B illustrate one embodiment of a system for the separation, ordering and focusing of particles within microfluidic channels;

Figs. 7A-7B illustrate equilibrium positions for focused streams of particles, in certain embodiments of the invention;

Figs. 8A-8B illustrate symmetric channels for the separation, ordering, and focusing of particles, in certain embodiments of the invention;

Figs. 9A-9B illustrate asymmetric channels for the separation, ordering, and focusing of particles, in certain embodiments of the invention;

Fig. 10 illustrates cells labeled with beads, in accordance with one embodiment of the invention;

Figs. 11 A-l 1B are microscopy images of cells labeled with beads, in another embodiment of the invention;

Figs. 12A-12C illustrate cell count histograms, in yet other embodiments of the invention;

Fig. 13 illustrates flow cytometry results for T cell activation, in one embodiment of the invention;

Fig. 14 illustrates flow cytometry results for effector memory, in another embodiment of the invention;

Fig. 15 illustrates an increase in size of a cell population, in accordance with one embodiment of the invention;

Figs. 16A-16B illustrate the efficacy of the activation of cells, in another embodiment of the invention;

Fig. 17 is a schematic showing activated T cell size sorting, in still another embodiment of the invention; Figs. 18A-18C illustrates a distribution in cell sizes, in yet another embodiment of the invention; and

Fig. 19 illustrates the sorting of activated cells, in still another embodiment of the invention.

DETAILED DESCRIPTION

The present invention generally relates to systems and methods for sorting particles. Certain aspects are generally directed to systems and methods for sorting a first type of particle from a second type of particle, e.g., using various microfluidic hydrodynamic sorting techniques. In addition, in some embodiments, particles that differ in other characteristics, and/or particles that generally overlap in size, can be separated by exposing the particles to agents able to alter their hydrodynamic size (e.g., by binding to the particles, directly or through a targeting moiety), sorting the particles, then optionally removing the agents to restore their original hydrodynamic size, for example, by dissolving or reacting at least a portion of the agents and/or the targeting moiety. As examples, the agents may include Ca²⁺- alginate beads, which can be removed using a chelator such as EDTA, or polystyrene beads with cleavable linkers.

For example, some aspects of the present invention are generally directed to systems and methods for sorting particles, such as cells, based on their size, for example, in a microfluidic device. In some embodiments, the microfluidic devices are able to sort particles by flowing the particles through microfluidic channels and taking advantage of certain geometries to use inertial lift forces and other techniques to cause the particles to separate based on their apparent size (i.e., their hydrodynamic size) as the particles flow through the microfluidic channels. Examples of such microfluidic devices are discussed in more detail below.

However, even with such techniques, particles with similar hydrodynamic sizes may be difficult to separate. As an example, cells with similar or even overlapping ranges in hydrodynamic size may be difficult to separate on the basis of their sizes. Accordingly, certain embodiments of the invention are generally directed to systems and methods for altering the hydrodynamic size of particles, causing separation to occur, and optionally reversing the alterations, e.g., to at least partially restore the particles to their original hydrodynamic size. For example, in certain embodiments, the hydrodynamic sizes of the particles may be altered by binding agents to the particles able to alter the hydrodynamic size. Such binding can be reversed at a later point, e.g., after sorting. For example, the agents may be dissociated from the particles, or at least partially dissolved, reacted with a reaction species, etc. Thus, such alterations can be temporarily applied to the particles in order to facilitate sorting.

Referring now to Fig. 1, Fig. 1A shows a sorting technique in which three types of cells are to be separated: red blood cells and platelets 11, lymphocytes 12, and monocytes and granulocytes 13, as an illustrative non-limiting example of particle sorting. In this schematic figure, it can be seen that red blood cells and platelets are generally smaller than lymphocytes, which are generally smaller than monocytes and granulocytes (the x-axis indicating size). However, it will also be seen that the size ranges of these cell types can overlap, especially lymphocytes 12, and monocytes and granulocytes 13. This figure also shows that by using techniques that allow the sorting of particles based on their

hydrodynamic size, the particles can be sorted into a waste group 21 of red blood cells and platelets, and a product group 22 of lymphocytes, and monocytes and granulocytes. (It should be noted that in this example,“waste” and“product” are arbitrary and are provided here for illustrative purposes only; for example, if the goal was to collect red blood cells and platelets instead, then these labels should be reversed.) Such cells can be sorted, for example, using microfluidic arrangements, including those discussed herein.

These cells can be sorted even further, as is shown in Fig. 1B. In this figure, the lymphocytes, and the monocytes and granulocytes have been sorted into a waste group 21 of lymphocytes, and monocytes and granulocytes, and a product group 22 of lymphocytes, after removing the red blood cells and platelets 11 as waste. (Again, these labels are arbitrary; in other applications, it may be desired to collect monocytes and granulocytes, rather than lymphocytes, or it may be desired to collect both cell types separately). Thus, the cells can be sorted, for example, using microfluidic arrangements such as those discussed herein that are able to separate particles based on their hydrodynamic size, which may be the same or different than that discussed above in Fig. 1A.

In Fig. 1C, the red blood cells and platelets 11 have been removed (e.g., as shown in Fig. 1A), then the average hydrodynamic size of the monocytes and granulocytes 13 is increased. A variety of methods may be used to increase the average hydrodynamic size. For example, an agent may be labeled or bound to those cells to alter the hydrodynamic size, or a cell may be activated in some fashion to cause it to change size. In some cases, the binding may be specific or preferential to the monocytes and granulocytes, relative to the

lymphocytes, as is shown in this example. By increasing the average hydrodynamic size of the monocytes and granulocytes, separation of those cells from the lymphocytes is enhanced, as the overlap in hydrodynamic size is much less, making it easier to distinguish the different cell populations.

In this example, the monocytes and granulocytes 13 are sent to waste. Thus, there is no particular need for removing the agent from those cells after sorting. However, in other embodiments, the monocytes and granulocytes may be kept as product, i.e., in addition to and/or instead of the lymphocytes.

Accordingly, in some embodiments, the agent may be removed from the cells after sorting. For instance, as is shown in the example of Fig. 1D, the average hydrodynamic size of the lymphocytes 12 is increased, relative to the average hydrodynamic size of the monocytes and granulocytes 13. As can be seen in this figure, the change in average hydrodynamic size can be relatively large in some cases, e.g., such that the size of lymphocytes is now greater than the size of the monocytes and granulocytes, rather than smaller. Similar to Fig. 1C, increasing the average hydrodynamic size of the lymphocytes can make it easier to distinguish such cells from monocytes and granulocytes, which can facilitate separation, e.g., using techniques such as those discussed herein. After sorting, the agent can be removed, partially or totally, which may cause the average hydrodynamic size of the cells to decrease (e.g., comparable to values prior to adding the agent), as is shown at the bottom of Fig. 1D. For example, the agent can be removed by dissolving the agent, reacting the agent with a reaction species, etc. In some cases, the agent can be an antibody that binds to cells with specific antigens. Non-limiting example antigens that may be targeted include CD41 for platelets, CD235a for red blood cells, CD45 for leukocytes, CD19 for B

lymphocytes, CD3 for T lymphocytes, CD4 and CD8 for subsets of T lymphocytes, CD56 for natural killer cells, CD 14 for monocytes, CD66b for granulocytes, CD34 for hematopoietic stem cells, and CD146 for endothelial cells. These are widely available from different vendors. The binding event, in certain embodiments, may trigger osmotic swelling or shrinking of the cells to cause a change in hydrodynamic size. Such osmotic shifts can also be reversed after sorting in some cases.

A variety of different agents, and techniques for binding the agents to cells (or other particles) are described herein. In one embodiment, the agents are calcium (Ca²⁺)-alginate beads, which may be made to be relatively solid due to cross-linking of alginate via Ca²⁺ ions. It should be understood that calcium- alginate beads are presented here as a non limiting, illustrative example; other agents are discussed in more detail below. Such beads can be caused to bind to cells, for example, to lymphocytes, or to monocytes and

granulocytes, such as those discussed with respect to Figs. 1A-1D. For example, the Ca²⁺- alginate beads may bind directly to the cells (or other particles), or a targeting moiety may be used, for example, using antibodies specific to the cells of interest, to specifically bind the Ca²⁺-alginate. Binding of such beads to cells, specifically or non-specifically, may cause the cells to exhibit a greater hydrodynamic size, which may be used to facilitate sorting as discussed above. In addition, after sorting, the Ca²⁺-alginate beads can be removed, for example, by applying a chelator (for example, EDTA), which can chelate the Ca²⁺ ions, thereby causing the beads to weaken and/or dissolve, i.e., without sufficient Ca²⁺ to cross-link the alginate. Thus, by applying a chelator, the Ca²⁺-alginate beads may be removed from the cells. Accordingly, such alterations in hydrodynamic size of cells (or other particles) may be reversible in some embodiments.

A non-limiting example is now illustrated in Fig. 2A. In this figure, a plurality of particles 50, including first particles 51 and second particles 52, are to be sorted based on their hydrodynamic size. For example, the particles may be cells, such as those discussed above with reference to Figs. 1A-1D. However, as first particles 51 and second particles 52 are of similar size distributions, it can be hard to do so. In Fig. 2A, agents 55 are then added to the particles, which are able to preferentially bind to first particles 51 over second particles 52. For example, the agents may comprise beads (such as Ca²⁺-alginate beads) that directly bind to the particles, or there may be a targeting moiety present on the agents (e.g., an antibody) that allows them to bind to the particles. Accordingly, after binding, first particles 51 are generally larger in hydrodynamic size than second particles 52, which can thus facilitate sorting of first particles 51 into a first group 61 and second particles 52 into a second group 62. In addition, after sorting, the agents can be removed from particles 51, e.g., dissolved, reacted, etc., to restore particles 51 to their original condition.

Another non-limiting example is now illustrated in Fig. 2B. In this figure, a plurality of particles 50, including first particles 51 and second particles 52, are to be sorted based on their hydrodynamic size. For example, the particles may be cells, such as those discussed above with reference to Figs. 1A-1D. However, as first particles 51 and second particles 52 are of similar size distributions, it can be hard to do so. In Fig. 2B, agents 55 are then added to the particles, which are able to preferentially cluster first particles 51 over second particles 52. For example, the agents may comprise bifunctional antibodies or bispecific antibodies that bind two or more particles 51. Accordingly, after binding, first particles 51 are generally larger in hydrodynamic size than second particles 52, which can thus facilitate sorting of first particles 51 into a first group 61 and second particles 52 into a second group 62. In addition, after sorting, the bifunctional antibodies can be removed from particles 51, to restore particles 51 to their original condition. Accordingly, in some cases, clustering or selective aggregating of particles can be used to cause changes in hydrodynamic size. For example, this can be used to cause clustering of neutrophils or monocytes to effect removal, or clustering of sub population of lymphocytes could be used to facilitate separation. In addition, in some cases, the particles may be de-clustering or de-aggregated.

As other examples, red blood cells or platelets are other potential agents that can be used, e.g., to cause aggregation to occur. For example, in some cases, cells can be labeled with bispecific antibodies or other linked antibodies, e.g., a bead labeled with antibodies against antigens expressed on different cell types.

The above discussion provides non-limiting examples of certain embodiments of the present invention that can be used to sort particles, such as cells, using various agents.

However, other embodiments are also possible. Accordingly, more generally, various aspects of the invention are directed to various systems and methods for sorting particles.

For example, certain aspects are generally directed to systems and methods for sorting particles based on their average hydrodynamic size, based on their apparent size as the particles flow through microfluidic channels. While the particles may be spherical or non- spherical, and may be deformable or non-deformable, the hydrodynamic size of a particle may be taken as a perfect, non-deformable sphere that behaves fluidically in the same fashion as the particle within the microfluidic channels. In some cases, sorting may be enhanced by altering the average hydrodynamic size of some of the particles. Thus, for example, a first particle type having a first average hydrodynamic size may be sorted from a second particle type having a second average hydrodynamic size, or from a population of particle types having different hydrodynamic sizes, by altering the hydrodynamic size of the first particle type. As another example, two particle types within a population of particle types may be separated by altering their hydrodynamic size to make them more readily distinguishable and sortable. In addition, in some cases, after sorting, the alterations may be partially or completely reversed or removed, e.g., to at least partially restore the original particles.

It should be understood that the sorting may not necessarily be perfect. For example, a first particle type and a second particle type may be sorted into a first group containing the first particle type and a second group containing the second particle type. However, there may be some contamination within one or both groups. For example, a group could contain at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of particles of one type, while being contaminated by particles of other types. In some cases, the sorting efficiency may be determined as the number of particles sorted into a particular group, divided by the total number of particles, and such sorting efficiencies may be, e.g., at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%. In addition, in accordance with certain embodiments, two, three, four, or more rounds of sorting may be performed in some cases, e.g., to improve sorting efficiencies. Each of the sorting rounds may be the same or different, and may independently comprise techniques such as those discussed herein, or other techniques.

The particles may include, for example, mammalian cells, blood cells in general as well as fetal blood cells in maternal blood, bone marrow cells, stem cells, circulating tumor cells (CTCs), stem cells, sperm, eggs, bacteria, fungi, virus, algae, any prokaryotic or eukaryotic cells, organelles, exosomes, droplets, bubbles, pollutants, precipitates, lipid vesicles, organic particles, and inorganic particles (for example, plastic particles, ceramic particles, magnetic particles, magnetically labeled analytes, etc.). Examples of blood cells include platelets, red blood cells, and leukocytes, such as neutrophils, eosinophils, basophils, lymphocytes (e.g., B cells, T cells, natural killer cells, etc.), monocytes, or the like. More than one type of particle, including any of the ones described here, may be present in some cases. Alternatively, or in addition, the techniques and devices disclosed herein can be used to extract purified fluid samples, from which particles such as cells have been extracted.

Such fluids can include, e.g., blood, aqueous solutions, oils, or gases. Examples of specific applications are discussed herein. For example, in one embodiment, the fluid is blood taken from a subject, such as a human subject.

As a non-limiting example, in some embodiments, the systems and methods may be used to sort blood cells. This may be done, for example, to separate one type of blood cell from another type of blood cell. Blood cells can come in a range of different sizes. For example, platelets are typically 2 micrometers in diameter, red blood cells are typically about 6-8 micrometers in diameter (however, the hydrodynamic size of a red blood cell is smaller, due to their discoidal shape, typically less than about 5 micrometers), lymphocytes are typically 6-8 micrometers in diameter, neutrophils (and other granulocytes) are typically 6-12 micrometers in diameter, while monocytes are typically 9-11 micrometers in diameter.

Accordingly, based on these differences in size, certain embodiments of the invention are directed to separating one or more cell types from a suitable sample, e.g., a blood sample.

For instance, in one embodiment, it may be desirable to separate lymphocytes from other types of blood cells. In other embodiments, however, it may be desirable to separate other types of cells, such as granulocytes, monocytes, red blood cells, platelets, etc. In one set of embodiments, the average hydrodynamic size of one or more particles (e.g., one or more cell types) may be altered to facilitate sorting of cells using a variety of techniques such as those described herein. For example, as noted above, lymphocytes, monocytes, and granulocytes have overlapping ranges in sizes, which can make it difficult to separate such cells solely on the basis of size. Accordingly, by altering their hydrodynamic size of one or more of these cells, sorting may be easier to perform. For example, a desired cell type may be altered in some fashion to make the hydrodynamic size significantly bigger than other cell types, or undesirable cell types may be altered in some fashion to make the hydrodynamic size significantly bigger than desired cell types.

The particles may be sorted based on a target or desired hydrodynamic size, i.e., particles with a hydrodynamic size greater than the target may be sorted into a first group, while particles with a hydrodynamic size less than the target may be sorted into a second group. Either of these may be considered to be waste and product, depending on the application, or in some cases, both may be considered to be product. In addition, it should be understood that in some embodiments, the particles may be sorted into more than two groups. For example, the particles may be sorted into 3, 4, 5, or more groups. As a non-limiting example, lymphocytes with B, T, and NK subsets that are all similar in size may be sorted with different size particles (for example, by labeling the B cells with 2 micrometer beads and the NK cells with 4 micrometer beads, then sorting into small (T cell), medium (B cell), and large (NK cell) outputs). A variety of other types of particles and/or techniques for altering hydrodynamic size are also discussed herein.

The hydrodynamic size can be chosen to be any of a variety of sizes. Non-limiting examples of such techniques, including how to choose particular hydrodynamic sizes, may be found in references such as U.S. Pat. Nos. 8,186,913, 9,895,694, 9,610,582, or U.S. Pat. Apl. Pub. No. 2016/0123858, each incorporated herein by reference in its entirety. For example, in one set of embodiments, particles may be sorted based on whether they are larger or smaller than about 1 micrometer, 2 micrometers, 3 micrometers, 4 micrometers, 5

micrometers, 6 micrometers, 8 micrometers, 10 micrometers, 12 micrometers, 14

micrometers, 15 micrometers, 16 micrometers, 18 micrometers, 20 micrometers, 22 micrometers, 24 micrometers, 25 micrometers, etc. As a non-limiting example, lymphocytes, monocytes, and granulocytes can be sorted using a target of 9 or 10 micrometers. As another non-limiting example, a target of 17, 18, 19, or 20 micrometers can be used to separate lymphocytes from other cells if the lymphocytes are altered to increase their average hydrodynamic size to be greater than these targets. Thus, the amount of alteration of particle size can be significant in some cases, e.g., to facilitate sorting, as this example shows. In some embodiments, an average hydrodynamic size of a particle type may be increased by at least l.5x, 2x, 3x, 4x, 5x, or more, relative to its initial average hydrodynamic size.

Various techniques can be used to alter hydrodynamic size. For example, with respect to cells, lipid vesicles, and the like, the particle size may be changed by altering the osmolarity of the solution containing the particles, by adding agents to the particles, by activating some of the cells in a manner such that their size changes, etc. For instance, by increasing the osmolarity of the solution (hypertonic), the particle size may be decreased; by decreasing the osmolarlity of the solution (hypotonic), the particle size may be increased. Those of ordinary skill in the art will be aware of various suitable solutions that can be used, such as hypertonic or hypotonic saline. As another example, beads may be nonspecifically bound to the particles, including beads such as those discussed in detail herein.

For example, in one set of embodiments, T-cells or other types of lymphocytes may be activated in some fashion to cause their size to change. T-cells may be activated, for example, by activating T-cell receptors with suitable stimulatory or co- stimulatory agents, such as major histocompatibility complexes (e.g., MHCII), CD3, CD4, CD8a, CD45RA, CD45RO, CD62L, CD 197, CD28, ICOS, or the like. As examples, T-cells may be activated using reagents such as soluble antibodies (e.g., CD3 and/or CD28, etc.). Other examples of activating reagents include, but are not limited to, Dynabeads activation beads (available from ThermoFisher) Cloudz, Miltenyi TransAct colloids, or the like. In some cases, APCs (antigen-presenting cells) may be provided to facilitate activation. In some cases, activation of a T-cell may cause the T-cell to grow and expand in size (thus altering its hydrodynamic size), which accordingly can be used for sorting. For example, T cells may be activated to change from an average size of about 7 micrometers to an average size of about 10 micrometers. In some cases, it may take several hours or days (e.g., 24 to 48 hours) for the cells to increase in size. See also a U.S. patent application filed on November 5, 2019, entitled“T-Cell Activation and Sorting Systems and Methods,” which is incorporated herein by reference in its entirety.

In addition to nonselective techniques such as osmolarity changes, or nonspecific binding of beads or other agents to particles, certain embodiments are directed to techniques that can selectively alter the size of one type of particle, relative to other particle types. The selectivity may be total, or only partial. For instance, in some cases, an agent may preferentially bind to a first type of particle, although there may be some binding to other particles. In some cases, at least 50% of the agents bound to the particles may be bound to a first type of particle, and in some cases, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the agents that are bound to particles are bound to the first type of particle. In some cases, no detectable binding of the agent may occur to other particles.

Thus, some embodiments are generally directed to agents that are able to selectively bind to the particles in order to alter their hydrodynamic size. It should be understood that the agents need not bind perfectly evenly around the particle in order to alter the particle’s hydrodynamic size (although they can be); for example, the agents may all bind to a single portion or side of the particle, the agents may be unevenly distribution on the surface of the particle, etc. As noted above, hydrodynamic size generally relates to the behavior of particles flowing in a sample, and thus, the particles themselves need not be perfectly spherical, nor the agents evenly distributed on the surface of the particles. In some cases, the agents may directly alter the hydrodynamic size of the particles (e.g., due to the size of the agents), although in some cases, the agents may indirectly alter the hydrodynamic size of the particles. For example, the agent may be an agent that allows other components to bind to the particles (e.g., which are able to alter hydrodynamic size). As another example, the agent may cause the particles to bind to each other, thereby producing aggregates with increased

hydrodynamic size. See, e.g., Fig. 2B.

As an example, one or both of a first particle type having a first average

hydrodynamic size and a second particle type having a second average hydrodynamic size (for instance, one that is close to or overlaps with the first average hydrodynamic size) may be altered such that the first and second average hydrodynamic sizes are farther apart after the alteration, e.g., to facilitate particle sorting based on size. Either or both of the particle types may be altered. As a non-limiting example, if the first particle type is smaller than the second particle type, then the second particle type may be altered to increase its average

hydrodynamic size (making it larger and thus farther away from the first average

hydrodynamic size), or in some cases, the first particle type may be altered to increase its average hydrodynamic size such that it is significantly larger than the second average hydrodynamic size to facilitate sorting, i.e., such that the size ordering is reversed between the first and second particle types after the alteration.

A wide variety of agents can be used can be used to alter hydrodynamic size, selectively or non-selectively. In some cases, the agent comprises a bead. The bead may comprise any of a wide variety of materials, including peptides or proteins, polymer (e.g., polystyrene, polypropylene, polyethylene, etc.), glass, ceramic, metal (for example, gold, silver, copper, nickel, etc.), silicon, quantum dots, or the like. In some cases, the bead may be magnetic, e.g., ferromagnetic, ferrimagnetic, or paramagnetic. For example, the bead may comprise iron, nickel, cobalt, iron oxide, etc. Magnetic beads may be dislodged, for example, by application of a suitable magnetic field. In addition, in certain cases, the beads are gels or hydrogels. For example, the beads may comprise polyacrylamide, polyacrylate, agarose, alginate, etc. Gels or hydrogels may comprise certain advantages, e.g., for cells or other types of sensitive particles, for example due to the“soft” or deformable nature of such beads, due to their biocompatibility or biodegradability, etc.

In some cases, the beads may be microbeads, e.g., having average an hydrodynamic size of less than about 1 mm, less than about 500 micrometers, less than about 300

micrometers, less than about 200 micrometers, less than about 100 micrometers, less than about 75 micrometers, less than about 50 micrometers, less than about 30 micrometers, less than about 25 micrometers, less than about 20 micrometers, less than about 15 micrometers, less than about 10 micrometers, less than about 5 micrometers, less than about 3 micrometers, less than about 2 micrometers, less than about 1 micrometer, less than about 500 nm, less than about 300 nm, less than about 100 nm, or less than about 50 nm, etc. The average hydrodynamic sizes may also be at least about 30 nm, at least about 50 nm, at least about 100 nm, at least about 300 nm, at least about 500 nm, at least about 1 micrometer, at least about 2 micrometers, at least about 3 micrometers, at least about 5 micrometers, at least about 10 micrometers, at least about 15 micrometers, or at least about 20 micrometers in certain cases. Combinations of any of these dimensions are also possible in some embodiments, e.g., the beads may range in size between 1 micrometer and 5 micrometers. The beads may also be spherical or non-spherical.

In addition, other types of agents may be used in other embodiments, in addition or instead of beads. Non-limiting examples of agents include cells, proteins, nucleic acids such as DNA or RNA, or the like. A variety of agents thus may be used that are able to alter the hydrodynamic size of the particles in some fashion.

In some embodiments, the agents may be selectively applied to certain particle types (e.g., via specific or preferential binding). For example, the agents may directly bind to certain types of particles, or a targeting moiety may be present on the agent and/or the particles which facilitates binding of the agents to particles. As an example, the targeting moiety may be an antibody which recognizes certain antigens that may be present on the surface of a first cell type but is not present (or is present, but at a significantly lower concentration) on the second cell type. For instance, the targeting moiety may be an antibody selective to a type of blood cell, for example, a lymphocyte.

The targeting moiety may be any entity able to recognize an appropriate target, e.g., the surface of a particle, such as a cell. Thus, binding of the targeting moiety to its target may be used to bind an agent attached to the targeting moiety to a particle. For example, the targeting moiety may comprise a peptide (e.g., one that can recognize a suitable target), an antibody, an antibody fragment, an antigen, a binding partner for a cell-surface receptor, a nucleic acid, or the like. In some cases, the recognition may be specific (e.g., the targeting moiety may recognize one or a small number of targets, such as an antibody that can recognize a target antigen), or the recognition may be relatively non-specific. In some cases, the targeting moiety may allow the agents to be bound to particles at the selectivities discussed herein.

The targeting moiety can be one that binds to the particle essentially permanently, or in some cases, the targeting moiety is not permanent and is instead removable from the particle, e.g., intrinsically (for example, due to diffusion, equilibration, etc.), and/or due to subsequent events (for example, due to the addition of a chemical able to react or dissolve the targeting moiety). For instance, in some cases, a competitive inhibitor may be added to inhibit binding of an antibody on a cell surface, thus allowing the targeting moiety to be at least partially removed.

In some embodiments, the agents may be bifunctional, e.g., able to bind to at least two different particles, such as is shown in Fig. 2B. Such agents may be able to cause the particles to aggregate, thereby increasing their effective hydrodynamic size via aggregation.

In some cases, the agent comprises two or more targeting moieties. For example, the agents may be bispecific antibodies, or be formed from two or more antibodies that are linked together. The antibodies may recognize the same or different particles, depending on the embodiment.

As mentioned, certain aspects of the invention are directed to alterations in hydrodynamic size that can be reversed after sorting, for example, by using agents that can be partially or completely removed from the particles, or otherwise prevented from binding particles. By at least partially removing the agents, the particles (e.g., once sorted) can be restored to their original hydrodynamic size, or at least substantially close to their original hydrodynamic size.

In some cases, the removal may be partial; for example, some agent may be left behind on the particles, although sufficient agent may have been removed to at least partially reverse the alterations in hydrodynamic size. For example, removal of a bead or targeting moiety may be partial, e.g., such that some portions of the bead or targeting moiety remain attached to the particles, although sufficient portions of the bead or targeting moiety may have been removed to at least partially reverse the alterations in hydrodynamic size.

Such techniques may be useful in limiting the amount of stress felt by cells or other types of particles, e.g., during the sorting process, which may prove to be advantageous in certain situations, e.g., in minimizing stress while facilitating the sorting process. In contrast, some techniques, such as osmolarity changes, may be more stressful to cells. For example, in one set of embodiments, the agents may comprise beads chosen due to their ability to be removed from the particles. The beads may be removed mechanically (e.g., using centrifugal force), mechanically (e.g., using an applied magnetic field), or in some cases, the beads can be chemically removed, removed by a change in pH, removed by a change in temperature, or the like. In addition, in some cases, one or more reagents may be added which causes removal of the beads.

There are a variety of different techniques for chemical removal of beads or other agents, and/or targeting moieties (when present) binding beads or other agents to the particles. For example, beads and/or targeting moieties may be dissolved from the particles. As a specific non-limiting example, at least some of the beads may be gel beads comprising alginate that have been cross-linked with a species such as Ca²⁺, forming a gel structure.

Such beads can be reacted or dissolved upon exposure to a suitable chelator, such as EDTA (ethylenediaminetetraacetatic acid), which can bind to Ca²⁺, thereby disrupting the cross- linked structure of the gel and causing the beads to dissolve or disintegrate. Other examples of chelators include, but are not limited to, EGTA (ethylene glycol-bis(beta-aminoethyl ether) -/V,/V,/V’,.V’-tetraacetic acid), iminodisuccinic acid, polyaspartic acid, ethylenediamine- /V,/V’-disuccinic acid, methylglycinediacetic acid, L-glutamic acid-/V,/V-diacetic acid, and the like. In some cases, such removable can be effected without significantly stressing the cells (or other particles). For example, the chelator can be compatible or biocompatible, e.g., with cells or other particles.

As another non-limiting example, an agent and/or a targeting moiety may be reacted with a reaction species, which may ultimately cause removal of the agent from the particles. For example, the reaction species may be an inhibitor able to inhibit binding of the targeting moiety to an agent and/or to the particle. In some cases, the inhibitor may be a competitive inhibitor or a non-competitive inhibitor to a specific targeting moiety, such as an antibody, which may cause the agent to become detached from the particle. As another example, in one embodiment, an agent may be attached to a particle via an interaction between Strep-tag® I (AWRHPQFGG) or Strept-tag® II (WSHPQFEK), and streptavidin or Strept-Tactin® (or other modified streptavidins). Many of these are readily available commercially. Exposure of this system to biotin may weaken the interaction between these components (e.g., due to the biotin- streptavidin binding), which may thus facilitate removal of the agent from the particles.

As yet another example, one or more agents may be attached to a particle via a linker, which can then be cleaved, e.g., enzymatically, upon exposure to a change in pH, exposure to light, or the like. For example, the agent may be attached via acrylic phosphoramidite, which may cleaved using ultraviolet light. As other examples, the cleavable linker may have a cleavable unit such as a disulfide (chemically cleavable), nitrobenzo (a photocleavable unit), or amine, amide or ester (enzyme-sensitive cleavable units).

As mentioned, certain aspects of the present invention are generally directed to systems and methods for sorting particles based on their hydrodynamic size. For instance, particles suspended in a fluid may pass through a particle sorting region of a microfluidic device, and thereby can be sorted based on their size. Examples of such techniques may be found for example, in U.S. Pat. Nos. 8,186,913, 9,895,694, 9,610,582, or U.S. Pat. Apl. Pub. No. 2016/0123858, each incorporated herein by reference in its entirety.

One non-limiting example of a sorting system is illustrated with respect to Fig. 3.

This is a schematic that illustrates a top view of the general architecture of a representative microfluidic device 100. The schematic illustrates, among other things, the outlines of various microfluidic channels, ports, reservoirs, and output receptacles for receiving, transporting, shifting, adjusting and/or storing fluid samples. The device 100 is designed to receive a fluid sample, e.g., blood, containing a suspension of one or more different types of particles, in a fluid exchange module to isolate a subpopulation of particles from the bulk fluid (e.g., by extracting and transferring one or more types of particles from the fluid sample to a second different solution), and then enrich the concentration of the extracted

subpopulation of particles for subsequent analysis and processing in a particle concentration module. Alternatively, the fluid, e.g., if dilute, can first be passed through the particle concentration module and then through the fluid exchange module. The various channels, ports and reservoirs, among other structures for manipulating fluids and particles, can be fabricated within a single device layer. A surface of the device layer is sealed with a lid layer (not shown) that serves as a cover to the channels and reservoirs of the device layer. An optional manifold layer (not shown) can be arranged on a surface of the lid layer to provide simultaneous fluidic coupling of the various through-holes to a macroscopic output/input connection (e.g., tubing). For example, all modules can be arranged and fixed on and/or fabricated on the same substrate, or each module can be arranged and fixed on and/or fabricated on individual substrates and then connected via fluid conduits and/or mechanical connections of the substrates.

The microfluidic device 100 can be sub-divided into separate sections referenced as follows: a fluid sample receiving section 102, a fluid sample filter section 104, a buffer sample receiving section 106, a fluid exchanger module (also referred to herein as a fluid force fractionation (FFF) module or an inertial exchanger) 108, a particle concentration module (also referred to herein as an inertial concentrator) 110, a fluid exchanger module product receptacle section 112, a fluid exchanger module waste receptacle section 114, a fluid exchanger module waste reservoir 116, a particle concentration module input section 118, a particle concentration module waste section 120, and a particle concentration module product output section 122.

In a first step, a fluid sample containing one or more different types of particles enters the chip through the fluid sample receiving section 102. The fluid sample receiving section 102 can include a series of holes into which the fluid sample can be introduced. For instance, each hole can be coupled to corresponding tubing through which the fluid sample is delivered. Alternatively, or in addition, the fluid sample receiving section 102 can include valves that can be opened and closed manually or through an automated process to control over the delivery of the fluid sample to the device 100. Other mechanisms for introducing fluid samples to a microfluidic device as known by those of ordinary skill in the art can also be utilized. The fluid sample can be driven into the device 100 using, e.g., a pump system that applies pressure to the fluid sample and enables continuous flow of the sample through the device 100.

Upon receiving the fluid sample in the device 100, the fluid sample passes to the fluid sample filter section 104 that is configured to filter particles contained in an incoming fluid according to the particle size (e.g., average diameter), such that only particles of a pre-defined size or less are able to pass to the next stage of the system. At the end of the filter section 104, the device 100 includes a buffer sample receiving section 106 configured to receive a second fluid sample (referred to as a buffer sample or buffer stream for the purpose of the example device 100). The buffer sample receiving section 106 includes multiple holes for receiving the buffer sample, in which the holes are arranged just upstream of the fluid exchanger module 108. Similar to the fluid sample receiving section, each hole can be coupled to corresponding tubing through which the fluid sample is delivered. Alternatively, or in addition, the buffer sample receiving section 106 can include valves that can be opened and closed manually or through an automated process to control over the delivery of the buffer fluid sample to the device 100.

In some embodiments, both the filtered fluid sample and the buffer fluid sample then enter the fluid exchanger module 108. In other embodiments, the filtered fluid sample and the buffer fluid sample first enter the particle concentration module. The buffer and fluid sample propagate within the fluid exchanger module 108 under conditions that enable laminar flow. That is, the fluids flow under conditions such that there is no turbulent mixing between the buffer and fluid sample. Rather, both the buffer and fluid sample propagate substantially side by side as parallel streams over the length of the fluid exchanger module 108. While in the module 108, at least a first type of particles are transferred from the fluid sample to the buffer sample so that by the end of the module 108 most, if not all, of the at least first type of particles have been extracted from the fluid sample. As will be explained, the process of transferring particles from the sample fluid to the buffer can rely, in part, on a combination of extracting the fluid sample at openings between island structures within the module 108, as well as inertial lift forces, which force particles away from the extracted fluid and into the buffer sample. Because the inertial lift force is size-dependent, it can be employed to fractionate (e.g., sort) particles based on size. Fractionation is accomplished by repeatedly (1) using the inertial lift force to move large particles away from a channel wall and then (2) shifting the fluid that is free of the large particles into an adjacent channel. After many iterations, the large particles can be moved from the source fluid (e.g., the fluid sample) across streamlines into an adjacent destination fluid (e.g., the buffer fluid sample).

At the end of the fluid exchanger module 108, the fluid sample enters the fluid exchanger module waste station 114. Though referred to as a“waste station,” the fluid sample can be disposed of, re-used for other purposes or processed for further analysis.

On the other hand, the buffer fluid sample, which now contains the transferred particles, enters the fluid exchanger product receptacle section 112. In the present example, the fluid exchanger product receptacle section 112 includes through -holes into which the particle-containing buffer sample passes out of the device 100 and into a manifold layer (not shown) that directs the buffer sample back into the device 100 at the particle concentration module input section 118. In alternative implementations, the buffer sample containing the transferred particles can be fluidly coupled directly to the particle concentration module 110 without having to exit and re-enter the device 100. The particle concentration module 110 contains three regions: a filter region, a focusing region, and a concentrator region. Upon entering the module 110, the buffer sample containing the particles propagates through the filter region, where the filter region is configured to filter particles contained in an incoming fluid according to the particle size (e.g., average diameter), such that only particles of a pre-defined size or less are able to pass to the next stage of the system. The buffer sample then passes to the focusing region. The focusing region employs structures that are configured to induce inertial focusing of the particles within the buffer sample along one or more streamlines. By focusing the particles along defined streamlines, the particles can be positioned at precise locations prior to entering the concentrator region, which enables, in certain implementations, the concentrator to more effectively enrich the particle concentration within the buffer sample. The concentrator region contains an array of structures configured and arranged to increase the concentration of the particles within the buffer. In particular, the particles within the buffer are subject to inertial lift forces that cause them to migrate across fluid streamlines toward equilibrium positions within the channel cross-section. Concentration of the particles is accomplished by repeatedly (1) using the inertial forces to move the particles away from channel walls and then (2) shifting or siphoning particle-free buffer sample into an adjacent channel. This results in two fluid outputs from the particle concentration module 110: an enriched buffer solution containing a high concentration of the extracted particles and a particle-free buffer sample.

At the end of the particle concentration module 110, the enriched buffer fluid passes to the particle concentration module product output 122, where it can be collected for further analysis and/or processing. The particle-free buffer sample passes to the waste section 120. Each of the fluid exchanger module 108 and the particle concentration module 110 employ multiplexing to establish an ultra-high throughput device capable of processing large amounts of sample fluid to obtain highly concentrated subpopulations of particles over relatively short time periods.

Fig. 4 is a schematic that illustrates a top view of a portion of the fluid exchanger module 108. The purpose of fluid exchanger module 108 is to deplete the filtered fluid sample of large particles. That is, the fluid exchanger module 108 is configured to sort a desired sub-population of particles (e.g., relatively large particles) from the filtered fluid sample and transfer those particles to the buffer solution. Thus, the fluid exchanger module 108“exchanges” the fluid in which the desired particles are suspended. This process also can be referred to as“fractionation.” To fractionate the filtered fluid sample, the fluid exchanger 108 includes multiple island structures 300 arranged in one or more arrays, in which each island 300 is separated from an adjacent island in the array by a gap through which fluid can flow. In the example shown here, the fluid exchanger module 108 actually includes two separate arrays, each having three rows of islands 300, in which the arrays are separated from one another by the wall/divider 215. The islands 300 are illustrated as substantially rectangular structures with their elongated sides extending generally in the same direction as fluid flow, though other shapes and orientations can be used instead. Furthermore, the number of rows of islands and the number of island arrays can also be varied from one or more depending on the desired configuration.

Fractionation using the fluid exchanger module 108 is accomplished by repeatedly (1) shifting or extracting portions of the filtered fluid sample that are free from particles through the gaps between the islands, while simultaneously relying on (2) inertial lift forces to move particles within the fluid sample away from the locations where the fluid is extracted. After multiple iterations, the particles in the filtered fluid sample can be moved across fluid streamlines and into a second different fluid propagating alongside the fluid sample (e.g., into the buffer). The inertial forces within the fluid arise from particles flowing at relatively high speeds near microfluidic channel walls. Thus, for example, when the fluid sample propagates near the walls of the islands 300, the particles within the fluid sample will experience inertial forces pushing the particles away from the islands. Fluid extraction or shifting, on the other hand, is controlled by the relative fluidic resistance encountered by the fluid as it propagates through the arrays. For a microfluidic channel in which the fluidic resistance varies over the length of the channel, fluid will tend to follow in a direction towards reduced fluidic resistance, thus leading to portions of the fluid being shifted away from the primary direction of propagation.

In this figure, the fluidic resistance of the channels is controlled by the geometry of the outer boundaries of each channel. For instance, with respect to each array, the distance between the outer channel wall 305 and the islands progressively increases along the direction of fluid flow, leading to lower fluidic resistance. In contrast, the distance between the wall 307 of the divider 215 and the islands 300 progressively decreases along the length of the array in the direction of fluid flow, leading to increased fluidic resistance. As a result, fluid is shifted through the gaps between islands 300 in the directions indicated by arrows 304. For relatively large particles within the fluids, the particles are also subject to inertial lift forces that push the particles away from the gaps, the portions of fluid extracted through the gaps are substantially particle-free. During operation of fluid exchanger 108, the filtered fluid sample enters both island arrays closer to the walls 305 of the channels, whereas the buffer fluid stream enters the island arrays closer to the walls 307 of divider 215. On average, both the filtered fluid sample and buffer fluid follow a horizontal trajectory through the fluid exchanger 108.

Though the fluid sample has been filtered prior to this stage, it can still contain one or more different sub-populations of particles having different sizes. Depending on the size of the gaps between islands 300 and the flow speed of the fluid sample, larger particles can experience a strong repulsive inertial lift force while flowing alongside the islands 300, which causes those particles to follow a trajectory with a component that leads from the filtered fluid sample stream (closer to walls 305) across fluid streamlines and into to the buffer fluid stream (closer to walls 307). Smaller particles can experience a relatively weaker inertial lift force while flowing alongside the islands 300. As a result, the smaller particles can follow the same average trajectory as the filtered fluid sample and cannot be transferred into the buffer fluid stream. At the output of the fluid exchanger 108, the filtered fluid stream will leave the arrays without one or more of the sub-populations of particles (e.g., without the relatively large particles), whereas the buffer fluid stream will have picked up the one or more sub-populations of particles. In some embodiments, one or more of the fluid samples that enter fluid exchange module 108 can come from the particle concentration module 110, described in further detail below.

As indicated above, the inertial lift force is highly size dependent, such that large particles can experience a larger force than small particles. Additionally, the fraction of fluid that is extracted through gaps between islands 300 can be adjusted based on the island design and configuration. Further discussion on the parameters and design principles for such fluid exchangers can be found in U.S. Provisional Application Nos. 62/074,213 and 62/074,315, each of which is incorporated herein by reference in its entirety.

In general,“focusing” particles refers to re-positioning the particles across a lateral extent of the channel and within a width that is less than the channel width. For example, the techniques disclosed herein can localize particles suspended in a fluid within a length of the channel having a width of 1.05, 2, 4, 6, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times the average diameter of the particles. In some implementations, the particles are focused to a streamline of a fluid. In some implementations, a streamline defines a width that is substantially equal to or slightly greater than an average hydraulic diameter of the particle, which can be, but is not limited to, between about 1 micrometer and about 100 micrometers. Further discussion on the parameters and design principles for fabricating inertial focusing structures can be found, for example, in U.S. Pat. No. 8,186,913, and U.S.

Provisional Application Nos. 62/074,213 and 62/074,315, filed Nov. 3, 2014, each of which is incorporated herein by reference in its entirety.

For instance, various channel geometries can require a predetermined particle to volume ratio of the particle to be focused in order to achieve a required interparticle spacing and thereby maintain ordering and focusing of that particle. In particular, the particle to volume ratio of a particle suspended within a fluid can be calculated and adjusted as needed to achieve focusing within certain channel geometries. In general, a maximum particle to volume ratio for a specified particle size and channel geometry can be determined using the formula:

Max Vo lumeFraction =2A¾ta /3 hw

where N is the number of focusing positions in a channel, a is the focused particle diameter, h is the channel height, and w is the channel width. Thus, samples can be diluted or concentrated to attain a predetermined ratio before and/or during introduction of the sample into the system. Particle to volume ratios of a sample within the channels described herein can have any value sufficient to enable ordering and focusing of particles. In general, the particle to volume ratio can be less than about 50%. In other embodiments, particle to volume ratios can be less than about 40%, 30%, 20%, 10%, 8%, or 6%. More particularly, in some embodiments, particle to volume ratios can be in a range of about 0.001% to about 5%, and can preferably be in a range of about 0.01% to about 4%.

In general, there are certain parameters within straight, symmetric, and asymmetric microfluidic channels that lead to optimal ordering and focusing conditions for particles suspended within a sample. These parameters can include, for example, channel geometries, particle size with respect to channel geometries, properties of fluid flow through micro fluidic channels, and forces associated with particles flowing within micro fluidic channels under laminar flow conditions. It is presently believed that the forces acting on the particles can be referred to as inertial forces, however, it is possible that other forces contribute to the focusing and ordering behaviors. Exemplary inertial forces can include, but are not limited to, inertial lift down shear gradients and away from channel walls, Dean drag (viscous drag), pressure drag from Dean flow, and centrifugal forces acting on individual particles. The theory discussed below is meant to be solely descriptive and exemplary and, while the behavior of systems designed using these principles can be predicted using this theory, the theory presented should not be considered as limiting the invention to any of the parameters associated with any of the system embodiments disclosed herein or any particular theory of operation.

In general, inertial lift forces in laminar microfluidic systems, such as those described in the embodiments herein, can act to focus randomly distributed particles continuously and at high rates into a single streamline. Particle geometry dependence can be used to develop systems for high-throughput separations. Channel geometry can be changed to reduce focusing particles from an annulus to four points, to two points, and then to a single point within the channel. Two additional levels of particle ordering can be observed, in particular, longitudinally along the channel length and rotationally (for asymmetric particles). In general, separation, ordering, and focusing is primarily controlled by a ratio of particle size to channel size and the flow characteristics of the system. Advantageously, the focusing is independent of particle density.

Lateral migration of particles in shear flow arises from the presence of inertial lift, attributed mainly to the shear-gradient-induced inertia (lift in an unbounded parabolic flow) that is directed down the shear gradient toward the wall, and the wall induced inertia which pushes particles away from the wall. Particles suspended in fluids are subjected to drag and lift forces that scale independently with the fluid dynamic parameters of the system. Two dimensionless Reynolds numbers can be defined to describe the flow of particles in closed channel systems: the channel Reynolds number (Rc), which describes the unperturbed channel flow, and the particle Reynolds number (Rp), which includes parameters describing both the particle and the channel through which it is translating:

Rc=(U_mD_h)/v and Rp=Rc(a²/D_h ²)=(U_m a^?) (\'D)

Both dimensionless groups depend on the maximum channel velocity, U_m, the kinematic viscosity of the fluid, and ^V T^P (m and p being the dynamic viscosity and density of the fluid, respectively), and D_h, the hydraulic diameter, defined as 2wh/(w+h) (w and h being the width and height of the channel). The particle Reynolds number has an additional dependence on the particle diameter, a. The definition of Reynolds number based on the mean channel velocity can be related to Rc by Rc=2/3 Rc.

Inertial lift forces dominate particle behavior when the particle Reynolds number is of order 1. Typically, particle flow in microscale channels is dominated by viscous interactions with Rp«l. In these systems, particles are accelerated to the local fluid velocity because of viscous drag of the fluid over the particle surface. Dilute suspensions of neutrally buoyant particles are not observed to migrate across streamlines, resulting in the same distribution seen at the inlet, along the length, and at the outlet of a channel. As Rp increases, migration across streamlines occurs in macroscale systems. In a cylindrical tube, particles were observed to migrate away from the tube center and walls to form a focused annulus. The theoretical basis for this“tubular pinch” effect is a combination of inertial lift forces acting on particles at high particle Reynolds numbers. The dominant forces on rigid particles are the “wall effect,” where an asymmetric wake of a particle near the wall leads to a lift force away from the wall, and the shear-gradient-induced lift force that is directed down the shear gradient and toward the wall.

Channels with curvature create additional drag forces on particles. When introducing curvature into rectangular channels, secondary flows develop perpendicular to the streamwise direction due to the nonuniform inertia of the fluid. In a parabolic velocity profile, faster moving fluid elements within the center of a curving channel can develop a larger inertia than elements near the channel edges. These elements can move toward the channel outer edge, and in order to conserve mass at all points where the fluid is recirculated along the top and bottom of the channel. Two dimensionless numbers can be written to characterize this flow, the Dean number (De) based on the maximum velocity in the channel, and the curvature ratio

(d). The Dean number,

and the curvature ratio, ^ J¾/2r^, _wh_Crc r is the average radius of curvature of the channel. For moderate De<75 observed in the microfluidic systems described herein, the secondary rotational flow, or Dean flow, consists of only two vortices. The velocity magnitude of the Dean flow scales as ¾~pD_e /(pD_A) _anc[ therefore, Stokes drag on suspended particles due to this secondary flow becomes significant for large De. In general, the drag due to Dean flow, or Dean drag (FD) scales as:

F_O~(pU_m ²aD_h ²)fr.

In short, three flow regimes can be considered: (1) At low fluid velocities, the ratio of lift to drag forces, R_f, may be larger than 1 over the majority of the channel cross section; however, the magnitudes of F_z and F_D are too low to create focused streams within the length of channel. (2) At intermediate fluid velocities, R_f may be greater or equal to 1 over a limited region of the channel cross section, and the magnitude of forces is large enough to create focusing to one or more streams. (3) For high fluid velocities, R_f is less than 1 over the entire channel cross section, and Dean drag is dominant, leading to particle mixing.

Referring again to device 100, the buffer fluid stream passes from the inertial focusing section of the particle concentration module 110 to microfluidic channels configured to increase the concentration of one or more sub-populations of particles within the buffer stream. Fig. 5 is a schematic that illustrates a top view of the portion of the particle concentration module 110 for enriching particle concentration. The direction of fluid flow through the module 110 is shown at the bottom of the page. In particular, the buffer stream exits the microfluidic channels 504 of the pre-focusing section and passes into microfluidic channels 512 or 514. A first pair of channels 512, 514 remains separated from a second pair of channels 512, 514 by wall 502. Each of channels 512 and 514 are themselves separated from one another by an array of post structures 506. The post structures are spaced apart from one another by gaps through which fluid can be extracted (along, e.g., a direction indicated by arrows 510).

Generally, the pre-focusing positioned the particles within the buffer stream along one or more streamlines that are closer to the surfaces of wall 502 so that, as the buffer stream exits the pre-focuser, the particles are aligned to the channels 512. The design and configuration of the channels 512 are similar to channels 504 in that the pathway is undulating and alternates between areas of relatively high curvature and areas of relatively low curvature. This channel shape again gives rise to inertial forces that focus one or more sub-populations of particles to streamlines within channels 512. At the same time, however, channels 514 are configured to have a decreasing fluidic resistance such that portions of the buffer fluid are repeatedly extracted into channels 514 as indicated by arrows 510, similar to the fluid extraction that takes place in the fluid exchanger module 108. That is, the distance between the outer walls 516 and the island structures 506 progressively increases along the length of the channels 514.

Design parameters that are relevant for establishing the amount of particle-free fluid extracted at each gap between islands 506 and for positioning the focused particles along one or more streamlines include, among others, the lengths of the channels 512, 514, the widths of the channels 512, 514, the spacing between islands 506, and the flow speed of fluid through the particle concentration module 110. For example, the maximum flow rate in which a particle-free layer can form and be extracted through the gaps between islands 506 follows a generally linear relationship with the width of the particle concentration channels (as measured in the x-direction of Fig. 5 between a channel wall 512 and the opposing wall of the divider 502). In another example, the yield of the device 100 can be adversely affected by flow rates through the particle concentration module 110 that are too low (i.e., low

Reynolds numbers) such that inertial forces are not large enough to focus particles to streamlines. In some cases, the fluid exchange module can be used to deplete relatively large particles from a fluid sample and transfer those particles to a second fluid such as a buffer fluid. As a result, the fluid sample that is depleted becomes the waste, whereas the buffer solution becomes the product. It is also possible, in some implementations, to keep the fluid sample that is depleted of the relatively large particles as the product and remove the second fluid to which the relatively large particles have been transferred as the waste. In such cases, the device can be configured to alter (e.g., enrich) the concentration of the fluid sample that is kept as product in the particle concentration module instead of the second fluid sample that contains the transferred particles. For example, the fluid exchange module fractionator can be designed to have a cutoff size for removing white blood cells and red blood cells from a blood sample and then enrich the concentration of the platelets remaining in the blood sample stream.

An example process for fabricating a microfluidic device according to the present disclosure is set forth as follows. A substrate layer is first provided. The substrate layer can include, e.g., glass, plastic or silicon wafer. An optional thin film layer (e.g., Si0₂) can be formed on a surface of the substrate layer using, for example, thermal or electron beam deposition. The substrate and optional thin film layer provide a base in which microfluidic channels 8 can be formed. The thickness of the substrate can fall within the range of approximately 500 micrometers to approximately 10 mm. For example, the thickness of the substrate 210 can be 600 micrometers, 750 micrometers, 900 micrometers, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, or 9 mm. Other thicknesses are possible as well.

The microfluidic channels formed within the substrate include the different fluid flow pathways for the fluid sample and the buffer, such as the straight channels, the filter arrays, the fluidic resistors, the channels within the fluid exchanger module, and the channels within the particle concentration module. The microfluidic channels can be formed, in some implementations, by depositing a polymer (e.g., polydimethylsiloxane (PDMS),

polymethylmethacrylate (PMMA), polycarbonate (PC), or cyclo olefin polymer (COP)) in a mold that defines the fluidic channel regions. The polymer, once cured, then can be transferred and bonded to a surface of a support layer. For example, PDMS can be first poured into a mold (e.g., an SU-8 mold fabricated with two step photolithography) that defines the micro fluidic network of channels. The PDMS then is cured (e.g., heating at 65 °C for about 3 hours). Prior to transferring the solid PDMS structure to the support layer, the surface of the substrate layer is treated with 0₂ plasma to enhance bonding. Alternatively, if the microfluidic channels are fabricated in other substrate materials, such as a glass or silicon wafer, the channels can be formed using standard semiconductor photolithography processing to define the channel regions in combination with wet and/or dry etching techniques to fabricate the channels.

After forming the microfluidic channels within the substrate, the substrate, also referred to as the“fluidic layer,” can be covered with a lid layer. The lid layer seals the fluidic layer microchannels (i.e., forms the“ceiling” of each channel) and is aligned and bonded to the fluidic layer. Bonding can be achieved using, e.g., an adhesive. The lid layer can include through-holes that are aligned with the through-holes formed in the fluidic layer, so as to allow fluids to be introduced and withdrawn from the device. Alternatively, or in addition, the through-holes can be formed in the fluid layer such that, in some

implementations, the lid layer is not necessary. In some implementations, a third interface layer couples to the surface of the lid layer. The interface layer can include a manifold that enables macro-scale connections to the device.

Additional information about microfluidic channel networks and their fabrication can be found, for example, in U.S. Patent App. Publication No. 2011/0091987, U.S. Pat. No. 8,021,614, and U.S. Pat. No. 8,186,913, each of which is disclosed herein by reference in its entirety.

For generally spherical particles being transported through a microfluidic device, the depth and width of the microfluidic channels can be, for example, in the range of about 2 times to about 50 times the diameter of the type of particle for which the device is designed to enrich. With respect to the island structures that form the gaps through which fluid is extracted in the fluid exchanger module or the island structures that form the gaps through which fluid is extracted in the particle concentration module, the width of the structures can be, e.g., up to about 10 times the width of an adjacent microfluidic channel, whereas the length of those structures can be between about 0.25 times the adjacent channel width up to about 50 times the adjacent channel width.

In some implementations, the length of the island structures 300 (as measured generally along the z-direction in Fig. 3) can be between, e.g., about 10 micrometers to about 5 mm long, including about 100 micrometers long, about 250 micrometers long, about 500 micrometers long, about 750 micrometers long, or about 1 mm long. In some

implementations, the width of the structures 300 (as measured generally along the x-direction in Fig. 3) can be between, e.g., about 1 micrometers wide to about 1 mm wide, including about 10 micrometers wide, about 50 micrometers wide, about 100 micrometers wide, about 250 micrometers wide, about 500 micrometers wide, or about 750 micrometers wide. In some implementations, the distance between adjacent islands 300 (as measured generally along the z-direction in Fig. 3) can be between, e.g., about 1 micrometers to about 1 mm, including about 10 micrometers, about 50 micrometers, about 100 micrometers, about 250 micrometers, about 500 micrometers, or about 750 micrometers. In some implementations, the distance between the outermost islands 300 of the arrays and the microfluidic channel walls (e.g., walls 305 or 307) can vary between about 1 micrometers to about 500

micrometers, including, for example, about 10 micrometers, about 20 micrometers, about 30 micrometers, about 40 micrometers, about 50 micrometers, about 60 micrometers, about 70 micrometers, about 80 micrometers, about 90 micrometers, about 100 micrometers, about 150 micrometers, about 200 micrometers, about 250 micrometers, about 300 micrometers, about 350 micrometers, about 400 micrometers, or about 450 micrometers. In some implementations, the length of the fluidic exchanger module 108 (as measured generally along the z-direction from one end of the array of islands 300 to the other end of the array of islands 300) can be between, e.g., about 10 mm to about 100 mm, including about 20 mm, about 30 mm, about 40 mm, about 50 mm, about 60 mm, about 70 mm, about 80 mm, or about 90 mm.

In contrast to the island structures 300, the island structures of the particle

concentration module 110 have a generally triangular prism shape with a maximum width generally corresponding to one base of the triangular face. In some implementations, the maximum length of the island structures 506 can be between, e.g., about 10 micrometers to about 5 mm long, including about 100 micrometers long, about 250 micrometers long, about 500 micrometers long, about 750 micrometers long, or about 1 mm long. In some

implementations, the distance between adjacent islands 506 (as measured generally along the z-direction in Fig. 4) can be between, e.g., about 1 micrometers to about 1 mm, including about 10 micrometers, about 50 micrometers, about 100 micrometers, about 250

micrometers, about 500 micrometers, or about 750 micrometers. In some implementations, the distance between the outermost islands 506 of the arrays and the microfluidic channel walls (e.g., walls 516 or walls of divider 502) can vary between about 1 micrometers to about 500 micrometers, including, for example, about 10 micrometers, about 20 micrometers, about 30 micrometers, about 40 micrometers, about 50 micrometers, about 60 micrometers, about 70 micrometers, about 80 micrometers, about 90 micrometers, about 100 micrometers, about 150 micrometers, about 200 micrometers, about 250 micrometers, about 300 micrometers, about 350 micrometers, about 400 micrometers, or about 450 micrometers. In some implementations, the length of the particle concentration module 110 (as measured generally along the z-direction from one end of the array of islands 506 to the other end of the array of islands 506) can be between, e.g., about 10 mm to about 100 mm, including about 20 mm, about 30 mm, about 40 mm, about 50 mm, about 60 mm, about 70 mm, about 80 mm, or about 90 mm.

As an example, for a generally spherical particle having a diameter of about 8 micrometers, a microfluidic device having two microfluidic channels separated by an array of rigid structures similar to the configuration shown in Figs. 2A-2B can have the following parameters: each microfluidic channel can have a depth about 52 micrometers, each microfluidic channel can have a range of widths between about 10 micrometers to about 5000 micrometers, each island structure can have a width of about 50 micrometers, each island structure can have a length of about 200 micrometers.

In addition, some embodiments feature methods and devices that separate and focus streams of particles to equilibrium positions within a channel flow field based, at least in part, on inertial lift forces. In rectangular channels, this can lead, for example, to four streams of focused particles spaced a distance apart from a center of each of the four rectangular faces. For certain rectangular geometries, this four-fold symmetry can be reduced to a two-fold symmetry, with streams of particles spaced apart from each of two opposed faces of the channel.

Some embodiments of the invention can also include methods and structures that decrease the symmetry of the system using a variety of forces, including, for example, electromagnetic, magnetic, centrifugal, hydrodynamic, thermal, sonic, optical, and/or dielectrophoretic forces or combinations thereof. Although any force may be used to bias a particular potential minimum within the channel flow field, utilizing centrifugal forces with a curved channel structure has certain advantages. In this case, the force will increase with the square of the flow rate based only on a minor geometric change with no additional mechanical or electrical parts required. For example, the symmetry may be reduced by using inertial forces inherent in the flow through an S- shaped rectangular channel to result in a two fold symmetry (down from four-fold) with a majority of the particles aligned with the flow in a periodic manner not corresponding to the period of the underlying channel. The geometry of the channel may also be used to change symmetry either by changing the radius of curvature or the width of the channel in a periodic manner (the channels thus curving asymmetrically) to create a single focused particle stream.

While there are many configurations possible in a system for the self-ordering of particles within microfluidic channels, one non-limiting embodiment of such a system 610 is illustrated in Figs. 6A and 6B. As shown, the system 610 generally includes an inlet 612 that can be configured for introducing a sample 624 having suspended particles 622 into the system. A microfabricated chip 614 can be provided and can have at least one microfluidic channel 616 formed therein for receiving the sample 624 and for ordering and focusing the particles 622 suspended in the sample 624, as shown in Fig. 6B. A plurality of such channels 616 situated in parallel are formed in the exemplary chip 614 illustrated in Fig. 6A.

The plurality of channels 616 formed in the chip 614 can have numerous

configurations which will be described in detail below. In general, however, the plurality of channels 616 can have a specified geometry configured to separate, order, and focus particles of a predetermined size suspended within the sample 624 such that one or more focused streams of particles 622 per channel 616 are provided at an output 626 of the chip 614. An analysis region 618 can be provided in proximity to the output 626 of the channels 616 to monitor, sort, count, image, or otherwise analyze the localized and focused streams of particles 622.

In one embodiment, chip 614 can be, or be part of, a particle enumerating system. In particular, analysis region 618, in which the particles have been focused and ordered, could be subject to interrogation by a detector for the purpose of counting the particles.

One or more microfluidic channels 616 can be formed in the microfabricated chip 614 and can be configured for receiving the sample 624 via one or more inlets 612 in

communication with the channels 616. The channels 616 can be further configured for ordering and focusing particles of a predetermined size suspended within the sample into one or more localized streams or fluxes of particles 622 that is directed into one or more outlets 626. In this way, particles in a dilute solution can be concentrated as illustrated in the figure. As illustrated in Fig. 6B, the localized flux 622 can include three or more particles 620 disposed longitudinally adjacent to one another and can be separated by a substantially constant longitudinal distance. Particles 620 within the flux 622 can also align rotationally relative to the channel 616.

In general,“localization” refers to a reduction in the area of a cross-section of a channel through which a flux of particles passes. In some embodiments, particles can be localized within an area having a width of, at most, 1.05, 2, 3, 4, or 5 times the width of the particles. Localization can occur at any location within the channel, but may occur within an unobstructed portion of the channel. For example, localization can occur in a portion of the channel having less than 50%, 40%, 30%, 20%, 10%, 5%, 2%, 1%, or 0.1% reduction in cross-sectional area. In certain embodiments, localization can occur in a channel having a substantially constant cross-sectional area.

Any number of microfluidic channels can be formed in the chip in any number of ways, described in detail herein. In one exemplary embodiment, a single channel is formed on the chip for focusing particles therein. In other exemplary embodiments, a plurality of channels can be formed in the chip in various configurations of networks for focusing particles. For example, 2, 4, 6, 8, 10, 12, and more channels can be formed in the chip. As shown in Fig. 6A, a tree configuration is particularly convenient for a multiple channel system. Any number of layers can also be included within a microfabricated chip of the system, each layer having multiple channels formed therein.

Various channel geometries can be included on a single chip. As shown in Fig. 6A, straight sections of channels are formed in the chip near the inlet for transporting and dividing flow lines as the sample is introduced into the system. The straight sections of channel can transition to any number of symmetric and/or asymmetric curved channels for focusing particles of a predetermined size as needed. As further shown in Fig. 6A, the chip can also include straight sections of channels at an output region for analysis of focused particles, collection of focused particles, and/or for recombining stream lines. As will be appreciated by those skilled in the art, any number of curves or straight sections can be included as needed within the chip. Additional curved sections of channels can serve as“off ramps” for focused particle streams to facilitate additional separation based on labels or tags associated with the particles. Channel forks or splits can be included at any positions within the channels to further facilitate manipulation of focused particles as needed for various applications.

Various channel dimensions can also be included within a single chip. Channel dimensions can decrease over the length of the chip to facilitate filtering of the sample, or for other reasons specific to an application. Channel dimensions can be larger at the input area or at the output area to enable forks or valve systems to be positioned within the channels, or to enable multiple stream lines to be separated and directed to different locations for analysis or collection. In a similar way, cross-sections of various channels can also be changed as needed within a single chip to manipulate stream lines of focused particles for particular applications. In general, any combination of channel geometries, channel cross-sections, and channel dimensions can be included on a single chip as needed to sort, separate, order, and focus particles of a predetermined size or particles of multiple predetermined sizes. The channels used in the systems described herein can have various geometries and cross-sections for focusing particles of a predetermined size suspended within a fluid. For example, in one embodiment illustrated in Figs. 7A and 7B, a straight channel 630 is provided having a rectangular cross-section with an aspect ratio of substantially 1 to 1. As will be described in more detail below, particles of a predetermined size flowing within such a channel geometry will be separated, ordered, and focused into four streamlines 632a, 632b, 632c, 632d corresponding to four equilibrium points or potential minimums at a distance from each face of the four channel walls. In another embodiment, a straight channel 636 is provided having a rectangular cross-section with an aspect ratio of substantially 2 to 1.

Particles of a predetermined size flowing within such a channel geometry can be separated, ordered, and focused into two focused streamlines 638a, 638b corresponding to two equilibrium points or potential minimums along top and bottom walls across the width of the channel. In one embodiment, an aspect ratio of 1 to 2 can also be used.

The channels may also be curved as shown in Figs. 8A and 8B. For example, symmetrically curved channels can be provided such as S-shaped, sinusoidal, or sigmoidal shaped channel 640 having a rectangular cross-section. Particles of a predetermined size flowing within such a channel geometry will be generally focused into two streamlines 642a, 642b corresponding to two equilibrium points or potential minimums at a distance from left and right side faces of the channel. An aspect ratio of a sigmoidal channel 640 can be substantially 1 to 1 and/or can vary along a length thereof. For example, the aspect ratio of a sigmoidal channel can vary over the length of the channel between 1 to 1 and 2 to 1 depending on the configuration chosen.

In another embodiment, asymmetrically curved channels are provided as shown in Figs. 9A and 9B. While asymmetrically curved channels can have various shapes and configurations as needed for a particular application, in one embodiment an asymmetric channel 646 can generally have the shape of a wave having large and small turns, where a radius of curvature can change after each inflection point of the wave. Each large and small turn can have a specified width of the channel associated with the turn. In particular as shown in Fig. 9A, one-half of a wavelength of the wave can have a large curve with a radius Ri_a, Ri_b defining a width Wi. A second half of the wavelength can have a curve with a radius R_2a, Ri_b defining a width W₂, where Ri_a and Ri_b can be greater than R_2a and R_¾ and vice versa (and where Ri_a=R_2a and Ri_b=R_2b would be a sinusoidal, symmetric shaped channel as indicated above). In addition, Wi can be greater than W₂, and vice versa. The wavelength having a first half with the radius Ri_a, Ri_b and the second half with the radius R_2a, R_2b can then be repeated as many times as needed, varying after each inflection point, to provide a specified length of channel with an asymmetric curve. The asymmetrically curved channel 46 can also have a rectangular cross-section with an aspect ratio that can vary as needed over the channel length depending on the nature of the asymmetry in the curves. In one embodiment, the aspect ratio can vary between 1 to 1 and 2 to 1. In this case, a single focused stream 648 of particles is created corresponding to a single equilibrium point or potential minimum within the channel 646.

Aspect ratios of all channels described above and herein, including straight, symmetric, and asymmetric, can vary as needed from one application to another and/or as many times as needed over the course of a channel. In embodiments illustrated in Fig. 9, aspect ratios are shown as 1 to 1 and 2 to 1, however, a person of ordinary skill will recognize that a variety of aspect ratios could be employed. In addition, the choice of width to height as the standard for determining the aspect ratio is somewhat arbitrary in that the aspect ratio can be taken to be the ratio of a first cross-sectional channel dimension to a second cross- sectional channel dimension, and for rectangular channels this would be either width to height or height to width.

Other channel cross-sections can also be included in each of the geometries noted above. Channel cross-sections can include, but are not limited to, circular, triangular, diamond, and hemispherical. Particles of a predetermined size can be focused in each of these exemplary cross-sections, and the equilibrium positions will be dependent on the geometry of the channel. For example, in a straight channel having a circular or

hemispherical cross-section, an annulus or arc of focused particles can be formed within the channel. In a straight channel having a triangular or diamond cross-section, particles can be focused into streamlines corresponding to equilibrium positions at a distance from the flat faces of each wall in the geometry. As symmetric and asymmetric curving channels are included having each of the exemplary cross-sections noted above, focusing streams and equilibrium positions can generally correspond to that described above with respect to the channels having a rectangular cross-section.

In general, there are certain parameters within straight, symmetric, and asymmetric microfluidic channels which lead to ordering and focusing conditions for particles suspended within a sample. These parameters can include, for example, channel geometries, particle size with respect to channel geometries, properties of fluid flow through microfluidic channels, and forces associated with particles flowing within microfluidic channels under laminar flow conditions. It is presently believed that the forces acting on the particles can be referred to as inertial forces, however, it is possible that other forces contribute to the focusing and ordering behaviors. Exemplary inertial forces can include, but are not limited to, inertial lift down shear gradients and away from channel walls, Dean drag (viscous drag), pressure drag from Dean flow, and centrifugal forces acting on individual particles.

Any number of samples can be introduced into the system for particle focusing and should not be limited to those samples described herein. A sample can generally include any suspensions, liquids, and/or fluids having at least one type of particle, cellular, droplet, or otherwise, disposed therein. Further, focusing can produce a flux of particles enriched in a first particle based on size. In some embodiments, a sample can be derived from an animal such as a mammal. In a preferred embodiment, the mammal can be a human. Exemplary fluid samples derived from an animal can include, but are not limited to, whole blood, sweat, tears, ear flow, sputum, bone marrow suspension, lymph, urine, brain fluid, cerebrospinal fluid, saliva, mucous, vaginal fluid, ascites, milk, secretions of the respiratory, intestinal and genitourinary tracts, and amniotic fluid. In other embodiments, exemplary samples can include fluids that are introduced into a human body and then removed again for analysis, including all forms of lavage such as antiseptic, bronchoalveolar, gastric, peritoneal, cervical, arthroscopic, ductal, nasal, and ear lavages. Exemplary particles can include any particles contained within the fluids noted herein and can be both rigid and deformable. In particular, particles can include, but are not limited to, cells, alive or fixed, such as adult red blood cells, fetal red blood cells, trophoblasts, fetal fibroblasts, white blood cells, epithelial cells, tumor cells, cancer cells, hematopoietic stem cells, bacterial cells, mammalian cells, protists, plant cells, neutrophils, T lymphocytes, CD4+, B lymphocytes, monocytes, eosinophils, natural killers, basophils, dendritic cells, circulating endothelial, antigen specific T-cells, and fungal cells; beads; viruses; organelles; droplets; liposomes; nanoparticles; and/or molecular complexes. In some embodiments, one or more particles such as cells, may stick, group, or clump together within a sample. In such a configuration, a grouping or clumping of particles can be considered to be“a particle.” More particularly, a grouping or clumping of particles may act and be treated as a single particle and can thus be sorted, ordered, separated, and focused in the same way as a single particle.

Non-biological samples can include, for example, any number of various industrial and commercial samples suitable for particle separating, ordering, and focusing. Exemplary industrial samples that can be introduced into the system can include, but are not limited to, emulsions, two-phase chemical solutions (for example, solid-liquid, liquid-liquid, and gas- liquid chemical process samples), waste water, bioprocess particulates, and food industry samples such as juices, pulps, seeds, etc. Similarly, exemplary commercial samples can include, but are not limited to, bacteria/parasite contaminated water, water with particulates such as coffee grounds and tea particles, cosmetics, lubricants, and pigments.

The following documents are incorporated herein by reference in their entireties: U.S. Pat. Nos. 8,186,913, 8,784,012, 8,807,879, 9,347,595, 9,610,582, 9,808,803, and 9,895,694; U.S. Pat. Apl. Pub Nos. 2018/0161775, 2017/0144159, and 2016/0123858; and Int. Pat. Apl. Nos. WO 2008/130977, WO 2016/073448, WO 2016/073481, and WO 2016/073486.

In addition, U.S. Provisional Patent Application Serial No. 62/755,678, filed

November 5, 2018, entitled“Systems and Methods for Sorting Particles Using

Hydrodynamic Sizing,” by Smith, et al., and U.S. Provisional Patent Application Serial No. 62/758,793, filed November 12, 2018, entitled“Systems and Methods for Sorting Particles Using Hydrodynamic Sizing,” by Smith, et al. are each incorporated herein by reference in its entirety. Also incorporated herein by reference in its entirety is a U.S. patent application, filed on November 5, 2019, entitled“T-Cell Activation and Sorting Systems and Methods.”

The following examples are intended to illustrate certain embodiments of the present invention, but do not exemplify the full scope of the invention.

EXAMPLE 1

This example illustrates leukocyte subpopulation selection by inertial microfluidics, in accordance with certain embodiments of the invention.

Since two chimeric antigen receptor T cell therapies were approved by FDA in 2017, immunotherapy, therapies that utilize and strengthen a patient’s own immune system to attack cancer cells, has quickly emerged as the so-called fifth pillar of cancer treatments following surgery, radiotherapy, chemotherapy, and targeted therapies. As demands for safe, robust and effective cell therapies grow, the need for simple, inexpensive, and high- throughput separation of peripheral blood mononuclear cells (PBMCs) into its distinct subpopulations such as T cells, B cells, and NK cells, also has grown. Because the average size of lymphocytes is smaller than those of granulocytes and monocytes, size-based cell sorting technologies such as those discussed herein, e.g., based on microfluidic inertial focusing, could be employed to isolate lymphocytes from PBMC or leukocyte starting material. To further select subpopulations of lymphocytes such as T cells, B cells or NK cells, which have very small size differences, unwanted cells could be labeled with beads to increase their apparent sizes and therefore sorted out. This example thus demonstrates T cell and NK cell enrichment from leukocytes by removing granulocytes, monocytes, and bead- labeled B cells using inertial microfluidics. Various materials and methods are now provided.

Reagents and buffers. PE anti-human CD 19 antibody, PerCP/Cyanine5.5 anti-human IgM antibody, APC anti-human CD66b antibody, biotin anti-human CD 19 antibody, and PE Streptavidin were acquired from Biolegend. SuperAvidin coated microspheres (3 micrometers in diameter) were acquired from Bangs Lab. lx BLB buffer included 0.1% BSA, 0.1% F68, 2 mM EDTA, and lx PBS. IX FB buffer included 1% F68, 2 mM EDTA, and lx PBS.

Cells and subpopulation quantification on flow cytometer. Leukocytes were obtained by fractionation of 10 ml fresh whole blood (StemExpress) on Sorterra (MicroMedicine).

The collected white blood cells (WBCs) were counted to be 24 million cells/ml on the Sysmex XP-30 Automated Hematology Analyzer. About 0.42 million WBCs were stained with 2 microliters of PE CD 19 in 400 microliters of lx BLB buffer for 15 min in the dark at room temperature, and then run on the Attune flow cytometer (Thermo Fisher Scientific). To differentiate subpopulations of WBCs, forward scatter (FSC) and side scatter (SSC) at 488 nm excitation were used.

B cell labeling. SuperAvidin-biotin chemistry was used to label B cells with beads. This is a 2-step process, as illustrated in the top part of Fig. 10. First, B cells were labeled with biotinylated CD 19 antibody and washed; second, biotinylated B cells were labeled with SuperAvidin coated beads. Following bead labeling process, there were two fraction steps, one to remove free beads in the solution, the other to separate smaller cells such as T cells and NK cells from the larger cells like granulocytes, monocytes, and bead decorated B cells, as shown in the bottom part of Fig. 10.

Fig. 10 shows a schematic of the bead labeling steps and the subsequent two step fractionation. In this figure, step 1 shows labeled B cells with biotin-CDl9; step 2 shows labeled biotinylated B cells with SuperAvidin covered beads; step 3 shows the removal of free beads from cells; and step 4 shows the removal of unwanted larger cells.

In more detail, 1.4 ml WBCs at a concentration of 24 million cells/ml was spun down at 350 g for 7 min, then resuspended to the same volume in lx BLB buffer. 17.5 microliters of 0.5 micrograms/microliters biotin-CDl9 antibody was added to 0.7 ml of the cell solution to target a ratio of -0.5 micrograms of antibody per million total cells. The solution was gently mixed by flipping the tube a few times and then loaded onto a Roto-Shaker at 4 °C for 15 min. After incubation, 0.8 ml of lx BLB buffer was added to the cell solution, gently mixed, and spun down at 350 g for 7 min. After most of the supernatant was removed without disturbing the cell pellet, 0.4 ml lx BLB buffer was gently laid on top of the cell pellet, spun down at 350 g for 2 min, and then resuspended in 0.7 ml lx BLB buffer. To target a bead to total cell ratio of 8:1, 0.22 ml of SuperAvidin beads at a concentration of 640 million beads/ml was added into lx BLB buffer to make a 1.5 ml solution, spun down at 3000 g for 5 min, and resuspended in 0.22 ml lx BLB buffer. It was then mixed gently with 0.7 ml resuspended biotinylated cell solution and incubated on the shaker at 4 °C for 25 min. All the reagents used until this step were kept at 4 °C. Once labeling was done, room temperature lx FB buffer was added to the bead-cell solution to make a 3 ml solution and was filtered through a 10 micrometer pluriStrainer (pluriS elect).

Devices. Microfluidic chips with varying shift, varying main channel width, and a channel depth of 52 micrometers were manufactured of cyclic olefin polymer (COP) by Stratec Biomedical AG (Germany). Two different chips were selected for T/NK cell selection. Chip 1 has a shift of 3.6% and a 50 micrometer main channel width. It was used to remove the 3 micrometer free beads after cell-bead linking step. Chip 2 had a shift of 4.4% and a 50 micrometer main channel width. It was used to remove beads labeled B cells, granulocytes, and monocytes. Both chips have two inputs and two outputs. The two inputs are for sample injection and buffer injection, and the two outputs are for small cells and large cells. By balancing the hydraulic resistances of the two output channels in the chip design, the output for small cells (SO) had a volume fraction of -70%, whereas the output for large cells (LO) has a volume fraction of -30%.

Fractionation proceeded as follows in this example.

Step 1: Unbound 3 micrometer bead removal. 30 ml lx FB buffer at room

temperature was loaded into a 30 ml plastic BD syringe and pumped into the buffer input side of Chipl at 1.0 ml/min by a Harvard syringe pump (Harvard Apparatus) with all other ports on the chip completely open. Once the sample side was primed, it was pinched off so the rest of the chip could be primed properly. The 3 ml cell-bead solution prepared in the bead labeling step was then loaded into a 3 ml plastic BD syringe, and pumped into the sample input side of the chip by another Harvard syringe pump. The flow rate of the buffer was set to a constant volume flow rate of 1.063 ml/min, and the sample to 0.233 ml/min for the whole fractionation process. ~3ml output from the LO side was collected, 300 microliters of which was taken out and diluted 2x in lx FB buffer. For flow cytometer quantification, 300 microliters of the 2x diluted LO output was stained for 15 min in the dark at room

temperature with 1 microliter of PE Streptavidin, and 1 microliter of APC CD66b antibody. CD66b was used to count the granulocytes, in addition to the standard FSC-SSC

quantification. This was to help resolve the cell count ambiguity caused by the overlap of the granulocyte cluster and the bead bound lymphocyte cluster on FSC-SSC plot. The rest of the LO output was loaded into a 3 ml plastic BD syringe for step 2.

Step 2: Granulocytes, monocytes, and bead labeled B cell removal. Chip 2 was primed and the LO output from Step 1 was loaded through the sample input side, the same way as for Chip 1. The flow rate of the buffer was again set to 1.063 ml/min, and the sample to 0.233 ml/min. The outputs from both SO and LO were collected. For flow cytometer quantification, 300 microliters of the SO and LO outputs each were stained for 15 min in the dark at room temperature with 1 microliter of PE Strep tavidin and 1 microliter of APC CD66b antibody.

Microscope imaging was performed as follows. 10 microliters of the SO and LO outputs were loaded into a hemocytometer C-Chip (SKC, Inc.), and imaged with bright field transillumination on Nikon Ti2 microscope with a 20X objective lens (Nikon CFI S Plan Fluor ELWD 20X).

Results and discussion. Fig. 11 shows two of the microscope images taken for the SO and LO outputs of Chip 2, where the granulocytes, monocytes and bead-labeled B cells were expected to sort to the LO output, whereas the non-B cell lymphocytes to the SO output. On the SO side, there were occasional cells with 1 bead on it, some with 2 beads, which when combined with flow cytometer data discussed later, were assigned to non-specific binding of beads to the lymphocytes. On the LO side, there were cells with varying amount of bead coverage; some of the more densely decorated ones are shown in Fig. 1 IB.

Fig. 11 illustrates 20x microscope images of fractionation outputs. Fig. 11A shows SO output. Fig. 11B shows LO output where some densely decorated cells are shown (inserts).

Flow cytometry with FSC, SSC, CD19, and CD66b was used to quantify

granulocytes, monocytes, and B cells in both the pre-sorting and post-sorting samples. For the pre-sorting WBCs, granulocytes were the most frequent at 54.0%, lymphocytes come in second at 32.8%, and monocytes the least at 8.9%. Post-sorting the lymphocyte fraction jumped to 89.1%, whereas granulocytes dropped to 6.7% and monocytes to 0.06%. Fig. 12 shows the ID histograms of CD19 marker for B cell counts before and after sorting. The B cell fraction were -3.9% of the total WBCs in the pre-sorting sample, and it dropped to -0.12% for the post-sorting SO output.

Fig. 12 shows ID histograms of CD19 marker for B cell count. Fig. 12A shows pre sorting, Fig. 12B shows post-sorting SO output, and Fig. 12C shows post-sorting LO output. Because of the observation of non-specific binding of bead to lymphocytes in the microscope imaging experiment, granulocytes were also counted by CD66b marker, and the results indicated that only -3.0% of the post-sorting cells was CD66b+. Separate

experiments were performed with a CD3 marker to confirm lymphocytes with bead overlap with granulocytes in the FSC-SSC signals. From this analysis, the granulocyte fraction in the post-sorting sample was determined to be 3%, lymphocyte 93%, monocyte 0.1%, and B cells 0.1%. In short, removal of granulocytes, monocytes, and B cells was very efficient; for example, the granulocyte fraction in the sample was decreased by l7-fold.

In conclusion, this example demonstrated lymphocytes enrichment from WBCs using inertial microfluidics. A target cell size can be changed by labeling it with beads, allowing its removal with inertial microfluidics.

EXAMPLE 2

This example illustrates a procedure for activating and expanding a purified population of T lymphocytes, in accordance with one embodiment of the invention.

For each donor or condition tested, a negative activation control with no beads may be used to determine the background activation level of the isolation or purification process if any appears. In addition, for each run, full single stain (SS) and fluorescence minus one (FMO) controls may be run using a pooled population of cells from each condition. SS controls may be used for compensation along with an unstained sample. FMO controls may be used to determine background signal as well as to verify appropriate compensation.

Media and Buffer Preparation. Aseptically dilute IL-2 to a lOOx working

concentration of 10,000 IU/mL in sterile PBS. Exact reagent amounts depend on COA for specific lot. Aliquot 550 microliters each into sterile 1.5 mL microcentrifuge tubes. Store at -80 °C and discard after thaw. Thaw 100 mL of FBS at 4 °C overnight along with a 5 mL aliquot of lOOx Pen/Strep. FBS is stable at 4 °C for 2-4 weeks. Pen/Strep is stable at 4 °C for 1 week. Working under aseptic conditions in the BSC (biosafety cabinet), open a 1000 mL bottle of RPMI and remove 100 mL of media. Aliquot into 2 x 50 mL centrifuge tubes and store at 4 °C. Add 100 mL of thawed FBS. Pipette up and down several times to mix using a 50 mL serological pipette and then aliquot into several 50 mL centrifuge tubes.

On day 0 and day 1, to one of the 50 mL centrifuge tube, add 500 microliters of lOOx Pen/Strep, and 50 microliters of lOOOx 2-mercaptoethanol. On day 2 onward, in addition to antibiotic and 2-Me, add 500 microliters of a 10,000 IU/mL IL-2 Stock. Working in the BSC, prepare“flow buffer” by taking a 1000 mL bottle of PBS and add 1 gram of BSA powder and 4 mL of 500 mM EDTA to a final concentration of 0.1% BSA and 2 mM EDTA. Sterile filter the flow buffer through a 1000 mL bottle top 0.22 micrometer PES vacuum filter system.

Activation of Cells on Day 0. Prepare media as described above. Warm media to 37 °C immediately before use. Remove activation beads from storage and prepare according to manufacturer’s recommendations, resuspending to a final concentration of 1 x 10⁶ beads per 25 microliters in complete cell culture media. Open a 24 well tissue culture plate and label activation and control wells as appropriate. Add 1 x 10⁶ beads in a 25 microliter volume to each well for activation. Place cells in humidified 5% C0₂ incubator at 37 °C for two days.

Flow Cytometric Investigation of Activation, Day 2. Working with one sample at a time, pipette up and down vigorously 10 times with one pipette set to 1000 microliters.

Using the other pipette set to 500 microliters, remove 500 microliters of the culture volume and transfer to a microcentrifuge tube. Repeat one by one until all samples have been collected. Once all samples have been harvested, place tubes on the Dynabead magnet.

Allow beads to be captured by magnet for 2 minutes, then transfer suspension to a clean 2 mL microcentrifuge tube. Place this in a magnet for 2 min, then transfer cells to a second clean microcentrifuge tube. Remove approximately half of the remaining volume from each sample after taking CBC (complete blood count) and pool together in a larger tube. Mix well and distribute evenly amongst FMO and SS controls. Spin all tubes at 500g for 5 min to pellet cells and remove supernatant by inverting open vial over a waste beaker. Add 1 mL of PBS to each tube, resuspend cells, and spin at 500g for 5 min, and discard supernatant.

Table 1. Activation panel for beads

Formulate zombie dye following product insert instructions. Dilute zombie stain 1:100 in plain PBS and add 100 microliters to each sample, mix, and incubate for 10-15 minutes at room temperature in the dark. Formulate blocking reagent by diluting the FcX reagent 1:10 into Flow Buffer (PBS +BSA +EDTA) and add 100 microliters to each sample and mix. Incubate for 10-15 minutes at room temperature in the dark. Formulate stain master mix by adding 1 microliter of each antibody to 100 microliters of cold Flow Buffer. Add 100 microliters to each sample and mix. Incubate for 30 minutes at 4 °C in the dark.

Add 1 mL of cold Flow Buffer to each tube and spin at 500g for 5 min and discard supernatant. Resuspend cells in appropriate volume of Flow Buffer to be used for analysis and run samples on flow cytometer.

Proliferation and Maintenance of Activated Cells - Day 2+. Make media as outlined above with BME (beta-mercaptoethanol) and IL-2 and warm to 37 °C in a water bath. Split cells as appropriate into media containing 100 U/mL of IL-2. Maintain cells between 1 x 10⁵ and 1 x 10⁶ cells per mL, splitting every 2-4 days as necessary. Typically, a 1:8 split is appropriate. On day 4, it is advised to split up into a 6 well plate or larger vessel. Count cells by pipetting up and down and debeading as described above except only remove a volume of 200 microliters as necessary for CBC. Count cells on CBC.

Effector Memory Phenotyping - Day 7+. Harvest cells as described in above, except remove and debead 1 mL from each well. Take the 1 mL debeaded sample and take a CBC of each sample for proliferation analysis. Follow staining instructions as discussed above for the Activation panel. Use antibodies outlined in table below.

Table 2. Effector memory panel.

Activation Panel Gating Strategy and Analysis. Gate lymphocytes on FSC-A vs SSC-

A. From the lymphocyte gate, gate singlets on FSC-A vs FSC-H. From the singlets gate, gate live CD3+ events on Viability-NIR vs CD3-BV421. From this Live CD3+ gate, gate CD4+ and CD8+ events on CD4-FITC vs CD8-BV605. From each of these gates,“Live CD3,”“CD4+” and“CD8+” generate 3 identical plots of CD25-PE vs CD69-APC and apply an identically placed quadrant gate on each plot. Use a sample with a distinct dual negative and positive population to place this gate and verify placement of gate and compensation by checking for appropriate placement on activated and unactivated samples. Once all gates have been set, check background signal in the unstained control as well as in the FMO controls. There should be little to no background (-1% or less) in the positive gate in the corresponding FMO. Significant signal in the positive gate of an FMO indicates

inappropriate compensation values or gate placement.

Effector Memory Panel Gating Strategy and Analysis. There are many different ways to determine effector memory phenotypes of T cells and many different approaches have been published. One simple example approach using a dual parameter CD45RA and CD 197 (also known as CCR7) plot and a quadrant gate to determine phenotype is as follows. Gate lymphocytes on FSC-A vs SSC-A. From the lymphocyte gate, gate singlets on FSC-A vs FSC-H. From the singlets gate, gate Live Lymphocytes on a viability-aqua negative histogram. From this Live gate, gate CD4+ and CD8+ events on CD4-FITC vs CD8-BV605. From each of these gates,“Live Lymphocytes,”“CD4+” and“CD8+” generate 3 identical plots of CD197-APC vs CD45RA-PE and apply an identically placed quadrant gate on each plot. Use a sample with a distinct dual negative and positive population to place this gate and verify placement of gate and compensation by checking for appropriate placement on multiple samples. Gate placement can be challenging because of the diffuse expression of CD45RA and CD 197. Once all gates have been set, check background signal in the unstained control as well as in the FMO controls. There should be little to no background (-1% or less) in the positive gate in the corresponding FMO. Significant signal in the positive gate of an FMO indicates inappropriate compensation values or gate placement.

Fig. 13 shows the gating strategy process for a T Cell Activation Panel. Top graphic shows the gating strategy as applied to activated cells and bottom pane shows the strategy as applied to unactivated cells. Note the FSC and SSC increase of activated cells, as well as the increased variability in the CD3 and CD8a markers. Fig. 14 shows the gating strategy process for the Effector Memory Panel. In this example, naive cells fall in the upper right, effector memory cells in the lower right, central memory in the lower left, and effector memory RA+ cells in the upper left.

EXAMPLE 3

This example illustrates lymphocyte and activated T cell selection.

The T cells were activated and the CD3/CD28 Dynambeads were prepared as discussed in Example 2. Activated T Cell Sorting Procedure. On day 2 after activation, the 48 hr cultured activated lymphocyte population was then processed on a fractionator study disc using a fixed flow rate system. A COP fractionator array device was used with a shift of 4.4% and channel width of 50 micrometers for the purposes of selecting for larger activated T cells.

The fractionator array device was processed at an 18% sample injection and with a running buffer flow rate of 597 microliters/min and a sample flow rate of 131 microliters/min.

Harvard apparatus syringe pumps were used to drive the input fluid flow, with the sample input containing the cultured activated T cell population. Prior to processing the input, the sample was washed in order to remove the culture media from the sample, and was then resuspended in the FB diluent. The sample was resuspended in FB diluted to 3.6 M/mL WBC, and the running buffer used was FB filtered using a 0.22 micrometer filter. Prior to processing on the fractionator study disc, the input sample was filtered using a 20 micrometer pluriselect filter in order to remove large cell aggregates and debris.

The data show a size increase in activated T cells after about 48 hours after activation. To monitor T cell size change as a function of time post activation, a Z2 Coulter Counter Analyzer (Beckman Coulter Life Sciences) was used. Fig. 15 displays T cell size distribution 48 hours post activation alongside the non-activated controls. The unactivated control sample is shown with a peak cell diameter of roughly 7 micrometers. The paired day 2 post activation sample is shown with a peak cell diameter of roughly 10 micrometers. The larger activated cells are the cells of interest in this context.

To demonstrate that the cells with larger diameter were in fact the activated cell population of interest CD25 and CD69 activation markers were used on the Attune NxT flow cytometer in order to assess the populations. The forward scatter (FSC) flow cytometry metric was also used as an association to the cell size, which is common practice in flow cytometry applications. Fig. 16 demonstrates the efficacy of the activation process, demonstrating that a large percentage of the cells are shown to be activated (dual CD25 and CD69 positive) post-activation procedure on day 2 compared to the control non-activated condition. Additionally, this figure shows that the activated cells (CD25+/CD69+) are larger in FSC compared to the control non-activated condition.

Fig. 16A shows an unactivated control sample. The left plot displays the bivariate activation marker plot for the unactivated control sample in which negligible activation marker expression of CD25 and CD69 is seen. The right plot displays forward scatter (FSC- A) histogram of the unactivated control sample. Fig. 16B shows an activated day 2 sample. The left plot displays the bivariate activation marker plot for the activated sample in which a higher percentage of events are expressing CD25 and CD69 activation markers. The middle plot displays forward scatter (FSC-A) histogram of the activated sample. The right plot displays only the subset of dual CD25 and CD69 activated cells, which are shown to be higher on the FSC axis, indicating that they are larger in size.

Fig. 17 shows activated T cell size sorting. This figure summarizes the workflow of the activated T cell sorting process, starting from a lymphocyte population to the sorted activated cell population. On the first day of the process, the isolated lymphocyte population is attained. Following isolation, the population is activated using activation beads. On day 2 after activation, the activated cell population has increased in size dramatically. Finally, the activated population is sorted through the fractionator array sorting process in order to isolate the larger activated cells of interest. On day 2 after activation of isolated lymphocyte population, the activated cell sample was processed on the fractionator array following methods detailed above.

Prior to collection the sample was processed to allow the processing to reach steady state flow. Processing then was conducted for 11:50 in which the outputs were collected. Table 3 details the fluid processing results. The sample injection was measured as the percent of fluid in the output that resulted from the input sample, measured by the amount of processed input sample divided by the total output volume. The LO product fraction is the percent of fluid volume that goes to the LO output compared to all volume in the outputs.

The flow rate per fractionator row is the flow rate through each functional row of the fractionator array. The measured values reported in this table are within the expected error of the expected values given measurement noise which indicates there were no significant issues with the run processing, including no pump issues or clogging. This was confirmed through post run chip inspection of the input filters which confirmed that there was no clogging material buildup.

Table 3: Summary of key fluid flow metrics.

Fig. 18 shows the size distribution breakdown of each relevant solution, the input sample, the LO output and the SO output, as measured by Coulter counter. The size distribution difference between the LO sample (Fig. 18B) and the SO sample (Fig. 18C) can be identified. The LO sample can be seen to have a larger sized cell population. Table 4 identifies some relevant statistics related to the size distribution curves. Fig. 18A shows a size distribution plot of the input activated T cell sample. Fig. 18B shows the LO output sample resulting from the fractionation run. Fig. 18C shows the SO output sample resulting from the fractionation run. Table 4: Statistics related to size distribution of cells

In addition to Coulter counter characterization, flow cytometry was utilized in order to characterize subpopulation phenotyping on the input and output samples. Fig. 19 depicts the input sample, the LO sample (mainly isolated activated cells), and the SO sample (mostly of undesired waste fluid). A table summarizing the results is provided in Table 6.

This figure shows a summary schematic of demonstrated activated T cell sorting.

Dual positive CD69 and CD25 are indicators of activated cells and are indicated by the upper right box. Post fractionation large output is shown to have a high percentage of activated cells while the small output is shown to have a much smaller percentage of the activated cells. The small output sample displayed in this figure shows the relatively low loss of activated cells to the waste stream with majority of larger activated cells going to the large output stream, demonstrating the successful selection of specifically activated cell subtypes.

Table 6: Summary of sample phenotyping and activation expression

Summarizing the findings to the key output performance metrics, the viability of the CD3 positive cells, the relative yield of the activated viable CD3 cells, and the purity of the activated viable CD3 cells is reported in Table 7. The CD3 Viable metric was calculated by the percentage of CD3 positive cells which were not stained positive for the Zombie NIR viability marker. The activated Viable CD3 Relative Yield metric was calculated as the number of activated viable CD3 cells which resided in the LO compared to the total number of activated viable CD3 cells in both the LO and SO. The Activated Viable CD3 Purity metric was calculated as the percentage of the of viable CD3 cells which were shown to be activated through flow cytometry panel in the LO.

Table 7: Key performance metrics from the processing run

While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the scope of the present invention.

In cases where the present specification and a document incorporated by reference include conflicting and/or inconsistent disclosure, the present specification shall control. If two or more documents incorporated by reference include conflicting and/or inconsistent disclosure with respect to each other, then the document having the later effective date shall control.

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

The indefinite articles“a” and“an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean“at least one.”

The phrase“and/or,” as used herein in the specification and in the claims, should be understood to mean“either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e.,“one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the“and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to“A and/or B”, when used in conjunction with open-ended language such as“comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims,“or” should be understood to have the same meaning as“and/or” as defined above. For example, when separating items in a list,“or” or“and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as“only one of’ or“exactly one of,” or, when used in the claims,“consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term“or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e.“one or the other but not both”) when preceded by terms of exclusivity, such as“either,”“one of,”“only one of,” or “exactly one of.”

As used herein in the specification and in the claims, the phrase“at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase“at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example,“at least one of A and B” (or, equivalently,“at least one of A or B,” or, equivalently“at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another

embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one,

B (and optionally including other elements); etc.

When the word“about” is used herein in reference to a number, it should be understood that still another embodiment of the invention includes that number not modified by the presence of the word“about.”

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

In the claims, as well as in the specification above, all transitional phrases such as “comprising,”“including,”“carrying,”“having,”“containing,”“involving,”“holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases“consisting of’ and“consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

Claims

What is claimed is: CLAIMS

1. A method of sorting particles, the method comprising:

altering an average hydrodynamic size of at least some particles suspended in a fluid sample by binding agents to the particles able to alter the hydrodynamic size; and

sorting the suspended particles based on their hydrodynamic size by flowing the suspended particles through a particle sorting region of a microfluidic device.

2. The method of claim 1, further comprising removing the agents from at least some of the suspended particles.

3. The method of any one of claims 1 or 2, wherein the particles comprise cells.

4. The method of claim 3, wherein the particles comprise mammalian cells.

5. The method of any one of claims 3 or 4, wherein the particles comprise blood cells.

6. The method of any one of claims 1-5, wherein flowing the suspended particles

through a particle sorting region of a microfluidic device comprises causing inertial forces to act on the particles.

7. The method of any one of claims 1-6, wherein the fluid sample is a blood sample taken from a subject.

8. The method of any one of claims 1-7, wherein sorting the suspended particles based on their hydrodynamic size comprises sorting particles having a hydrodynamic size less than about 20 micrometers from particles having a hydrodynamic size greater than about 20 micrometers.

9. The method of any one of claims 1-8, wherein sorting the suspended particles based on their hydrodynamic size comprises sorting particles having a hydrodynamic size less than about 10 micrometers from particles having a hydrodynamic size greater than about 10 micrometers.

10. The method of any one of claims 1-9, wherein sorting the suspended particles

comprises sorting the suspended particles into a first type of particles and a second type of particles.

11. The method of claim 10, wherein sorting the suspended particles comprises sorting the first type and second type of particles to first and second regions, respectively, at a separation efficiency of at least 50%.

12. The method of any one of claims 10 or 11, wherein sorting the suspended particles comprises sorting the first type and second type of particles to first and second regions, respectively, at a separation efficiency of at least 75%.

13. The method of any one of claims 10-12, wherein the first type of particles comprises lymphocytes and the second type of particles comprises monocytes and granulocytes.

14. The method of any one of claims 10-13, wherein the agents bind preferentially to the first type of particle relative to the second type of particle.

15. The method of claim 14, wherein at least 80% of the agents bound to the particles are bound to the first type of particle.

16. The method of any one of claims 10-15, wherein the first type of particle has a first average hydrodynamic size, and the second type of particle has a second average hydrodynamic size greater than the first average hydrodynamic size.

17. The method of claim 16, wherein after binding the agents to the particles, the first type of particles have a first average hydrodynamic size greater than the second average hydrodynamic size.

18. The method of any one of claims 1-17, wherein the agents comprise beads.

19. The method of claim 18, wherein the beads have an average hydrodynamic size of less than about 5 micrometers.

20. The method of any one of claims 18 or 19, wherein the beads have an average

hydrodynamic size of less than about 1 micrometer.

21. The method of any one of claims 18-20, wherein removing the agents comprises

dissolving at least a portion of the beads.

22. The method of any one of claims 18-21, wherein removing the agents comprises

reacting at least a portion of the beads with a reaction species.

23. The method of any one of claims 18-22, wherein at least some of the beads are

magnetic.

24. The method of any one of claims 18-23, wherein at least some of the beads comprise alginate.

25. The method of claim 24, wherein at least some of the alginate is cross-linked via Ca²⁺.

26. The method of any one of claims 1-25, wherein at least some of the agents further comprise a targeting moiety attached thereto.

27. The method of claim 26, wherein the agents are able to bind to at least some of the particles via the targeting moiety.

28. The method of any one of claims 26 or 27, wherein the targeting moiety binds

specifically to at least some of the particles.

29. The method of any one of claims 26-28, wherein the targeting moiety binds

nonspecifically to at least some of the particles.

30. The method of any one of claims 26-29, wherein the targeting moiety comprises an antibody.

31. The method of any one of claims 1-30, wherein removing the agents comprises exposing the agents to a magnetic field.

32. The method of any one of claims 1-31, wherein removing the agents comprises applying centrifugal force to the suspended particles.

33. The method of any one of claims 1-32, wherein removing the agents comprises exposing the agents to a chelator.

34. The method of claim 33, wherein the chelator comprises ethylenediaminetetraacetic acid (EDTA).

35. The method of any one of claims 1-34, wherein removing the agents comprises dissolving at least a portion of the agents.

36. The method of any one of claims 1-35, wherein removing the agents comprises reacting at least a portion of the agents with a reaction species.

37. The method of any one of claims 1-36, wherein altering the average hydrodynamic size comprises increasing the average hydrodynamic size.

38. The method of any one of claims 1-37, wherein binding agents to the particles comprises binding agents to the particles sufficiently to increase the average hydrodynamic size by at least about 2x.

39. The method of any one of claims 1-38, wherein binding agents to the particles comprises binding agents to the particles sufficiently to increase the average hydrodynamic size by at least about 3x.

40. The method of any one of claims 1-39, wherein the particle sorting region of the microfluidic device comprises:

a first outer wall of the particle sorting region;

a second outer wall of the particle sorting region; a first microfluidic channel extending longitudinally along the particle sorting region between the first outer wall and the second outer wall;

a second microfluidic channel extending longitudinally between the first microfluidic channel and the first outer wall;

a first array of islands separating the first microfluidic channel from the second microfluidic channel, wherein each island in the first array is separated from an adjacent island in the first array by an opening that fluidly couples the first microfluidic channel to the second microfluidic channel;

a third microfluidic channel extending longitudinally between the first microfluidic channel and the second outer wall;

a second array of islands separating the first microfluidic channel from the third microfluidic channel, wherein each island in the second array is separated from an adjacent island in the second array by an opening that fluidly couples the first microfluidic channel to the third microfluidic channel;

wherein, over a length of the particle sorting region that includes a plurality of islands from the first array and from the second array, a distance from the first outer wall to the first array of islands increases so that a fluidic resistance of the second microfluidic channel decreases along a longitudinal direction of the particle sorting region relative to the fluidic resistance of the first microfluidic channel, such that a portion of fluid from the fluid sample in the first microfluidic channel passes through the first array into the second microfluidic channel, and

wherein, over the length of the particle sorting region that includes the plurality of islands from the first array and from the second array, a distance from the second outer wall to the second array of islands decreases so that a fluidic resistance of the third microfluidic channel increases along the longitudinal direction of the particle sorting region relative to the fluidic resistance of the first microfluidic channel, such that a portion of fluid from the fluid sample in the third microfluidic channel passes through the second array into the first microfluidic channel.

41. The method of claim 40, wherein a cross-sectional area of the first microfluidic

channel between the first array of islands and the second array of islands is substantially constant along a longitudinal direction of the microfluidic device.

42. The method of any one of claims 40 or 41, wherein the first array of islands consists of a single row of islands.

43. The method of any one of claims 40-42, wherein the first array of islands comprises an array of two or more rows of islands.

44. The method of any one of claims 40-43, wherein the second array of islands consists of a single row of islands.

45. The method of any one of claims 40-44, wherein the second array of islands

comprises array of two or more rows of islands.

46. A method of producing a sample enriched in particles of a first type, the method

comprising:

obtaining a fluid sample comprising particles of a first type and particles of a second type, the first particles having a first average hydrodynamic size and the second particles having a second average hydrodynamic size;

altering the average hydrodynamic size of the particles of the first type in the fluid sample to provide particles of the first type having an increased effective size in the fluid sample; and

sorting the suspended particles based on their hydrodynamic size by flowing the fluid sample containing the particles of the first type having the increased effective size into a particle sorting region of a microfluidic device, and at least partially separating the particles of the first type from particles of the second type.

47. The method of claim 46, further comprising altering the particles of the first type having the increased effective size to provide particles of the first type having the first average hydrodynamic size.

48. The method of any one of claims 46 or 47, wherein the particles comprise cells.

49. The method of claim 48, wherein the particles comprise mammalian cells.

50. The method of any one of claims 48 or 49, wherein the particles comprise blood cells.

51. The method of any one of claims 48-50, wherein the particles comprise T-cells.

52. The method of claim 51, wherein altering the average hydrodynamic size of the

particles comprises activating at least some of the T-cells.

53. The method of any one of claims 48-52, wherein at least partially separating the

particles of the first type from particles of the second type comprises causing inertial forces to act on at least one of the particles of the particles of the first type and the particles of the second type.

54. The method of any one of claims 46-53, wherein the fluid sample is a blood sample taken from a subject.

55. The method of any one of claims 46-54, wherein sorting the suspended particles comprises sorting the first type and second type of particles to first and second regions, respectively, at a separation efficiency of at least 50%.

56. The method of any one of claims 46-55, wherein the first type of particles comprises lymphocytes and the second type of particles comprises monocytes and granulocytes.

57. The method of any one of claims 46-55, wherein the first type of particles comprises monocytes and granulocytes and the second type of particles comprises lymphocytes.

58. The method of any one of claims 46-57, wherein the agents bind preferentially to the first type of particle relative to the second type of particle.

59. The method of claim 58, wherein at least 80% of the agents bound to the particles are bound to the first type of particle.

60. The method of any one of claims 46-59, wherein the second average hydrodynamic size is greater than the first average hydrodynamic size.

61. The method of claim 60, wherein after binding the agents to the particles, the first type of particles have a first average hydrodynamic size greater than the second average hydrodynamic size.

62. The method of any one of claims 46-61, wherein the agents comprise beads.

63. The method of claim 62, wherein removing the agents comprises dissolving at least a portion of the beads.

64. The method of any one of claims 62 or 63, wherein removing the agents comprises reacting at least a portion of the beads with a reaction species.

65. The method of any one of claims 62-64, wherein at least some of the beads comprise alginate.

66. The method of claim 65, wherein at least some of the alginate is cross-linked via Ca²⁺.

67. The method of any one of claims 46-66, wherein at least some of the agents further comprise a targeting moiety attached thereto.

68. The method of claim 67, wherein the agents are able to bind to at least some of the particles via the targeting moiety.

69. The method of any one of claims 67 or 68, wherein the targeting moiety binds

specifically to at least some of the particles.

70. The method of any one of claims 67-69, wherein the targeting moiety binds

nonspecifically to at least some of the particles.

71. The method of any one of claims 67-70, wherein the targeting moiety comprises an antibody.

72. The method of any one of claims 46-71, wherein removing the agents comprises

exposing the agents to a chelator.

73. The method of claim 72, wherein the chelator comprises ethylenediaminetetraacetic acid (EDTA).

74. The method of any one of claims 46-73, wherein removing the agents comprises dissolving at least a portion of the agents.

75. The method of any one of claims 46-74, wherein removing the agents comprises reacting at least a portion of the agents with a reaction species.

76. The method of any one of claims 46-75, wherein the particle sorting region of the microfluidic device comprises:

a first outer wall of the particle sorting region;

a second outer wall of the particle sorting region;

a first microfluidic channel extending longitudinally along the particle sorting region between the first outer wall and the second outer wall;

77. The method of claim 76, wherein inertial lift forces cause the particles of the first type to remain within the first microfluidic channel.

78. The method of any one of claims 76 or 77, wherein the inertial lift forces are

insufficient to prevent the particles of the second type from flowing with the portion of the fluid sample passing through one or more of the openings between adjacent islands into the second microfluidic channel.

79. The method of any one of claims 76-78, further comprising focusing the particles of the first type having the increased effective size along one or more streamlines within the first microfluidic channel.

80. The method of any one of claims 76-79, wherein a cross-sectional area of the first microfluidic channel between the first array of islands and the second array of islands is substantially constant along a longitudinal direction of the microfluidic device.

81. The method of any one of claims 76-80, wherein the first array of islands consists of a single row of islands.

82. The method of any one of claims 76-81, wherein the first array of islands comprises an array of two or more rows of islands.

83. The method of any one of claims 76-82, wherein the second array of islands consists of a single row of islands.

84. The method of any one of claims 76-83, wherein the second array of islands comprises array of two or more rows of islands.

85. A method of sorting cells, the method comprising:

providing cells suspended in a fluid sample, including a first type of cell and a second type of cell, the cells of the first type of cell having a first average

hydrodynamic size and the cells of the second type of cell having a second average hydrodynamic size;

causing an alteration of the average hydrodynamic size of the cells of the first cell type, wherein the alteration is 80% selective to the cells of the first type of cell relative to the cells of the second type of cell; and

sorting the cells suspended in a fluid sample based on their hydrodynamic size by flowing the suspended cells through a cell sorting region of a microfluidic device.

86. The method of claim 85, further comprising reversing the alteration from the cells of the first type of cell.

87. The method of any one of claims 85 or 86, wherein the cells comprise mammalian cells.

88. The method of any one of claims 85-87, wherein the cells comprise blood cells.

89. The method of any one of claims 85-88, wherein the cells comprise T-cells.

90. The method of claim 89, wherein causing an alteration of the average hydrodynamic size of the cells of the first cell type comprises activating at least some of the T-cells.

91. The method of any one of claims 85-90, wherein the fluid sample is a blood sample taken from a subject.

92. The method of any one of claims 85-91, wherein flowing the suspended cells through a cell sorting region of a microfluidic device comprises causing inertial forces to act on the suspended cells.

93. The method of any one of claims 85-92, wherein sorting the cells comprises sorting the first type and second type of cells to first and second regions, respectively, at a separation efficiency of at least 50%.

94. The method of any one of claims 85-93, wherein the first type of cells comprises lymphocytes and the second type of cells comprises monocytes and granulocytes.

95. The method of any one of claims 85-93, wherein the first type of cells comprises monocytes and granulocytes and the second type of cells comprises lymphocytes.

96. The method of any one of claims 85-95, wherein the agents bind preferentially to the first type of cell relative to the second type of cell.

97. The method of claim 96, wherein at least 80% of the agents bound to the particles are bound to the first type of cell.

98. The method of any one of claims 85-97, wherein the second average hydrodynamic size is greater than the first average hydrodynamic size.

99. The method of claim 98, wherein after binding the agents to the cells, the first type of cells have a first average hydrodynamic size greater than the second average hydrodynamic size.

100. The method of any one of claims 85-99, wherein the agents comprise beads.

101. The method of claim 100, wherein removing the agents comprises dissolving at least a portion of the beads.

102. The method of any one of claims 100 or 101, wherein removing the agents comprises reacting at least a portion of the beads with a reaction species.

103. The method of any one of claims 100-102, wherein at least some of the beads

comprise alginate.

104. The method of claim 103, wherein at least some of the alginate is cross-linked via Ca²⁺.

105. The method of any one of claims 85-104, wherein at least some of the agents further comprise a targeting moiety attached thereto.

106. The method of claim 105, wherein the agents are able to bind to at least some of the cells via the targeting moiety.

107. The method of any one of claims 105 or 106, wherein the targeting moiety binds specifically to at least some of the cells.

108. The method of any one of claims 105-107, wherein the targeting moiety binds

nonspecifically to at least some of the cells.

109. The method of any one of claims 105-108, wherein the targeting moiety comprises an antibody.

110. The method of any one of claims 85-109, wherein removing the agents comprises exposing the agents to a chelator.

111. The method of claim 110, wherein the chelator comprises ethylenediaminetetraacetic acid (EDTA).

112. The method of any one of claims 85-111, wherein removing the agents comprises dissolving at least a portion of the agents.

113. The method of any one of claims 85-112, wherein removing the agents comprises reacting at least a portion of the agents with a reaction species.

114. The method of any one of claims 85-113, wherein the cell sorting region of the

microfluidic device comprises:

a first outer wall of the cell sorting region;

a second outer wall of the cell sorting region; a first microfluidic channel extending longitudinally along the cell sorting region between the first outer wall and the second outer wall;

wherein, over a length of the cell sorting region that includes a plurality of islands from the first array and from the second array, a distance from the first outer wall to the first array of islands increases so that a fluidic resistance of the second microfluidic channel decreases along a longitudinal direction of the cell sorting region relative to the fluidic resistance of the first microfluidic channel, such that a portion of fluid from the fluid sample in the first microfluidic channel passes through the first array into the second microfluidic channel, and

wherein, over the length of the cell sorting region that includes the plurality of islands from the first array and from the second array, a distance from the second outer wall to the second array of islands decreases so that a fluidic resistance of the third microfluidic channel increases along the longitudinal direction of the cell sorting region relative to the fluidic resistance of the first microfluidic channel, such that a portion of fluid from the fluid sample in the third microfluidic channel passes through the second array into the first microfluidic channel.

115. The method of claim 114, wherein inertial lift forces cause the cells of the first type to remain within the first microfluidic channel.

116. The method of any one of claims 114 or 115, wherein the inertial lift forces are

insufficient to prevent the cells of the second type from flowing with the portion of the fluid sample passing through one or more of the openings between adjacent islands into the second microfluidic channel.

117. The method of any one of claims 114-116, further comprising focusing the cells of the first type having the increased effective size along one or more streamlines within the first microfluidic channel.

118. The method of any one of claims 114-117, wherein a cross-sectional area of the first microfluidic channel between the first array of islands and the second array of islands is substantially constant along a longitudinal direction of the microfluidic device.

119. The method of any one of claims 114-118, wherein the first array of islands consists of a single row of islands.

120. The method of any one of claims 114-119, wherein the first array of islands comprises an array of two or more rows of islands.

121. The method of any one of claims 114-120, wherein the second array of islands

consists of a single row of islands.

122. The method of any one of claims 114-121, wherein the second array of islands

comprises an array of two or more rows of islands.

123. A method of sorting cells, the method comprising:

activating cells within a sample to alter the average hydrodynamic size of the cells; and

sorting the cells based on their hydrodynamic size by flowing the suspended particles through a particle sorting region of a microfluidic device.

124. The method of claim 123, wherein at least some of the cells are T-cells.