WO2023277943A1

WO2023277943A1 - Tapered obstacle arrays for particle processing

Info

Publication number: WO2023277943A1
Application number: PCT/US2021/063468
Authority: WO
Inventors: Alison Skelley; Khushroo Gandhi; Anthony Ward; Yasna BEHMARDI
Original assignee: Gpb Scientific, Inc.
Priority date: 2021-07-01
Filing date: 2021-12-15
Publication date: 2023-01-05
Also published as: CN115555062A

Abstract

Disclosed herein is a particle separation device comprising an array of obstacles spaced apart along a flow channel with each obstacle in the array of obstacles comprising a tapered profile, wherein a difference in gap sizes between adjacent obstacles along opposing tapered profiles is controlled to achieve a critical size range that permits the array of obstacles to separate target particles of a predetermined size from contaminants of a different size in a sample when the sample is transported along the flow channel.

Description

TAPERED OBSTACLE ARRAYS FOR PARTICLE PROCESSING

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63/217,730, filed July 1, 2021, which is incorporated herein by reference in its entirety.

BACKGROUND

[0002] The preparation of cells for personalized therapy often requires the collection of biological material from a patient, the purification of a specific cell type from the material collected and the engineering or growth of the purified cells. Microfluidic deterministic lateral displacement (DLD) arrays offer a size-based separation process that is rapid, gentle and versatile. However, these arrays utilize carefully designed array geometries to achieve size- based separation. Imperfections or poor design in array geometry can lead to undesirable effects, including the deposit of biological debris during the operation of the arrays, lower throughput and poor separations due to mixing within the array. Thus, the development of better device geometries that are more tolerant to manufacturing practices are of considerable interest.

SUMMARY

[0003] Described herein are DLD arrays utilizing tapered obstacles to provide size-based separations of particles of a predetermined size from a mixture containing particles of a plurality of sizes.

BRIEF DESCRIPTION OF THE DRAWINGS [0004] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “figure” and “FIG.” herein), of which: [0005] FIG. 1 illustrates tapered obstacles having various shapes for use in DLD arrays. [0006] FIG. 2A illustrates the effect of tapered obstacles on the gap between adjacent obstacles.

[0007] FIG. 2B illustrates the effect of tapered obstacles on the gap between adjacent obstacles of three different aspect ratios.

[0008] FIG. 3 illustrates symmetric and asymmetric obstacle placements for example obstacle shapes.

[0009] FIG. 4 illustrates the effects of utilizing tapered obstacles in an asymmetric layout DLD array on gap spacing.

[0010] FIG. 5 illustrates a comparison of mixing effects between two tapered-obstacle DLD-array geometries.

[0011] FIG. 6 illustrates the effects of tapered-obstacle geometry on product contamination in a blood separation application.

[0012] FIG. 7 illustrates the effect of critical size range on white blood cell recover in a blood separation application of tapered-obstacle DLD arrays.

[0013] FIG. 8 illustrates a scanning electron micrograph of a DLD array with tapered hexagonal obstacles.

[0014] FIG. 9 illustrates the manufacturing advantages of parts with angled walls during injection molding.

[0015] FIG. 10 illustrates scanning electron micrographs of DLD arrays with tapered obstacles made by etching.

[0016] FIG. 11 illustrates the effect of input pressure on white blood cell recovery and throughput in a blood separation application using tapered-obstacle DLD arrays.

DETAILED DESCRIPTION

[0017] The present disclosure relates to size based microfluidic separations, and especially with the use of DLD in preparing cells that are of therapeutic value. The disclosure herein describes the manufacturing/manufacturability and use of microfluidic devices and the use of DLD for carrying out separations involving biological materials.

[0018] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0019] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0020] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

Definitions

[0021] Apheresis: As used herein this term refers to a procedure in which blood from a patient or donor is separated into its components, e.g., white blood cells, platelets and red blood cells. An "apheresis sample" is the product that is the end result of this procedure. More specific terms are "plateletpheresis" (referring to the separation of platelets) and "leukapheresis" (referring to the separation of leukocytes). In this context, the term "separation" refers to the obtaining of a product that is enriched in a particular component compared to whole blood or other starting material and does not mean that absolute purity has been attained. [0022] CAR T cells: The term "CAR" is an acronym for "chimeric antigen receptor." A "CAR T cell" is therefore a T cell that has been genetically engineered to express a chimeric receptor.

[0023] CAR T cell therapy: This term refers to any procedure in which a disease or condition is treated with CAR T cells. Diseases that may be treated include hematological and solid tumor cancers, autoimmune diseases and infectious diseases.

[0024] The terms “obstacle array” and “DLD array” are used synonymously herein and describe an ordered array of obstacles that are disposed in a flow channel through which a cell or particle-bearing fluid can be passed. An obstacle array comprises a plurality of obstacles arranged in a column (along the path of fluid flow). Gaps are formed between the obstacles (along the path of the fluid flow) that allows the passage of cells or other particles. Such arrays or columns can be arranged into one or more repeating rows (perpendicular to the path of fluid flow).

[0025] As described herein “particles” may include human or biological cells. In some instances, particles include nonbiological materials such as beads, spheres, or other discrete objects.

[0026] As described herein a “channel” or “lane” refers to a fluidic pathway comprising a plurality of obstacles that are arranged into a discrete separation unit. Such channels may be bounded on either side by walls such that discrete lanes are separated. Channels may run in parallel from one or more common inputs to one or more common outputs. Channels may be fluidly connected in series.

[0027] Deterministic Lateral Displacement: As used herein, the term "Deterministic Lateral Displacement" or "DLD" refers to a process in which particles are deflected on a path through a microfluidic obstacle array deterministically, based on their size. This process can be used to separate cells, which is generally the context in which it is discussed herein. However, it is important to recognize that DLD can also be used to concentrate cells and for buffer exchange. Processes are generally described herein in terms of continuous flow (DC conditions; i.e., bulk fluid flow in only a single direction). However, DLD can also work under oscillatory flow (AC conditions; i.e., bulk fluid flow alternating between two directions).

[0028] Critical size: The "critical size," "critical diameter" or “predetermined size” of particles passing through an obstacle array describes the size limit of particles that are able to follow the bulk fluid flow. Particles larger than the critical size can be diverted from the flow path of the bulk fluid while particles having sizes lower than the critical size (or predetermined size) will not be displaced. The "critical size," "critical diameter" or “predetermined size” may be associated with size-based discrimination and behavior of particles passing through an obstacle array. For example, at above a critical size, certain particles may no longer follow the bulk fluid path. At below the critical size, certain particles will follow the bulk fluid path. A critical size range may refer to an optimal or preferred range for which target particles of a certain size can be separated from contaminants of another size in a sample, while maintaining at least a predefined flow throughput rate along a flow channel comprising the obstacle array. [0029] Fluid flow: The terms "fluid flow" and “bulk fluid flow” as used herein in connection with DLD refer to the macroscopic movement of fluid in a general direction across an obstacle array. These terms do not take into account the temporary displacements of fluid streams for fluid to move around an obstacle in order for the fluid to continue to move in the general direction.

[0030] Tilt angle e: In an obstacle array device, the tilt angle is the angle between the direction of bulk fluid flow and the direction defined by alignment of rows of sequential obstacles in the array. Tilt angle can be specified in degrees, radian, or as a fractional unit indicating the frequency of a repeated row position (i.e. a 1/60 tilt angle refers to an arrangement where each sequential row is shifted relative to the first row such that at the 60^th shift iteration results in a row with an identical arrangement to the first row). [0031] Wall angle Q: The wall angle is the angle formed between a given wall of an obstacle and the surface normal of a bottom surface of the obstacle, where the bottom surface is defined as the surface positioned opposite to the surface with the smallest surface area on the volume of the obstacle (e.g. see FIG. 1) .

[0032] Tapered obstacle: A tapered obstacle is an obstacle having at least one wall wherein at least one wall angle (Q) is not equal to zero degrees. Tapered profile as described herein refers to a side profile or cross-section profile of a tapered obstacle.

[0033] Array Direction: In an obstacle array device, the “array direction” is a direction defined by the alignment of rows of sequential obstacles in the array. A particle is "deflected” in an obstacle array if, upon passing through a gap and encountering a downstream obstacle, the particle's overall trajectory follows the array direction of the obstacle array (i.e., travels at the tilt angle e relative to bulk fluid flow). A particle is not deflected if its overall trajectory follows the direction of bulk fluid flow under those circumstances.

[0034] Sample: The term “sample,” as used herein, generally refers to any sample containing or suspected of containing a nucleic acid molecule or cells. For example, a sample can be a biological sample containing one or more nucleic acid molecules or cells. The biological sample can be obtained (e.g., extracted or isolated) from or include blood (e.g., whole blood), plasma, serum, urine, saliva, mucosal excretions, sputum, stool and tears. The sample may contain blood, a blood product (such as a leukapheresis or apheresis product) also containing an anti-coagulant (e.g., EDTA, EGTA, heparin, citrate, ACD-A, or a thrombin inhibitor). The biological sample can be a fluid or tissue sample (e.g., skin sample). In some examples, the sample is obtained from a cell-free bodily fluid, such as whole blood. In some examples, the sample can include circulating tumor cells. In some examples, the sample is an environmental sample (e.g., soil, waste, ambient air and etc.), industrial sample (e.g., samples from any industrial processes), and food samples (e.g., dairy products, vegetable products, and meat products). The sample may be processed prior to loading into the microfluidic device. The sample may suitably be an apheresis product or a leukapheresis product (e.g., leukopak).

[0035] Target cells: As used herein "target cells" are the cells that various procedures described herein require or are designed to purify, collect, engineer etc. What the specific cells are will depend on the context in which the term is used. For example, if the objective of a procedure is to isolate a particular kind of stem cell, that cell would be the target cell of the procedure.

[0036] Isolate or purify: Unless otherwise indicated, these terms, as used herein, are synonymous and refer to the enrichment of a desired product relative to unwanted material. The terms do not necessarily mean that the product is completely isolated or completely pure. For example, if a starting sample had a target cell that constituted 2% of the cells in a sample, and a procedure was performed that resulted in a composition in which the target cell was 60% of the cells present, the procedure would have succeeded in isolating or purifying the target cell.

[0037] About: As used herein the term about refers to an amount near a stated amount that is within 10% of the stated value.

[0038] The present disclosure is directed to microfluidic devices in which size-based purifications are performed by passing a biological sample through an array of obstacles in a microfluidic channel. Size-based separations in a DLD array require that mixing of a raw sample undergoing separation and a buffer used to recover a target particle does not occur as the two coflowing streams traverse the DLD array. Manufacturing tolerances and the use of certain manufacturing practices for construction of a DLD array can result in deviations from the ideal array geometry, particularly with respect to the obstacle shape and arrangement, which are critical to the functioning of DLD array -based separations.

[0039] In general, the microfluidic devices will be used to separate target particles or target cells having a size larger than the critical size of the device from contaminants with sizes smaller than the critical size. When a sample containing the target cells or particles is applied to a device through a sample inlet and fluidically passed through the channel, the target cells or target particles will flow to one or more product outlets where a product enriched in target cells or target particles is obtained. The term "enriched" as used in this context means that the ratio of target cells or particles to contaminants is higher in the product than in the sample.

Contaminants with a size smaller than the critical size will flow predominantly to one more waste outlets where they may be either collected or discarded.

[0040] Although the objective of a separation will generally be to separate target cells or particles from smaller contaminants, there may be times when a user wants to separate target cells or particles from larger contaminants. In these instances, a microfluidic device may be used with a critical size larger than the target cells or particles but smaller than the contaminants. Combinations of two or more obstacle arrays with different critical sizes, either on a single device or on multiple devices, may also be used in separations. For example, a device may have channels with a first array of obstacles that has a critical size larger than T cells but smaller than granulocytes and monocytes and a second array with a critical size smaller than T cells but larger than platelets and red blood cells. Processing of a blood sample on such a device allows for the collection of a product in which T cells have been separated from granulocytes, monocytes, platelets and red blood cells. The order of the obstacle arrays should not be of major importance to the result, i.e., an array with a smaller critical size could come before or after an array with a larger critical size. Also arrays with different critical sizes can be on separate devices that cells pass through.

[0041] The sample may be obtained from an individual or a patient, especially a patient with cancer, an autoimmune disease or an infectious disease. In a certain embodiment, the sample is blood or is derived from blood ( e.g ., an apheresis or leukapheresis sample), and the target cells are dendritic cells, leukocytes (especially T cells), stem cells, B-cells, NK-cells, monocytes or progenitor cells. The contaminants in these instances will typically include red blood cells and/or platelets. The purification should result in a product enriched in target cells and in which at least 80% (preferably 90% and more preferably 95%) of the platelets and/or red blood cells from the sample have been removed.

[0042] Methods for making and using CAR T and natural killer (NK) cells are well known in the art. Procedures have been described in, for example, US 9,629,877; US 9,328,156; US 8,906,682; US 2017/0224789; US 2017/0166866; US 2017/0137515; US 2016/0361360; US 2016/0081314;US 2015/0299317; and US 2015/0024482; each of which is incorporated by reference herein in its entirety.

[0043] The present disclosure provides microfluidic cartridges (i.e. devices, chips, cassettes, plates, microfluidic devices, cartridges, DLD devices, etc.) and methods for purifying particles or cells, which may comprise chimeric antigen receptor (CAR) T and NK cells. The microfluidic cartridges (i.e. devices, chips, cassettes, plates, microfluidic devices, cartridges, DLD devices, etc.) may be any of those described herein. The use of the described cartridges may allow for production of more highly effective CAR T or NK cells by providing a purer T orNK cell product for downstream genetic engineering and CAR T or NK cell production. A more effective CAR T or NK cell may be produced by removing platelets that other methods for producing CAR T or NK cells cannot accomplish.

[0044] A method for producing chimeric antigen receptor (CAR) T or NK cells may comprise obtaining sample comprising T or NK cells and separating the T or NK cells from contaminants. Contaminants may comprise platelets, or other contaminants described herein. Separating contaminants may comprise applying the sample to the one or more sample inlets of any of the cartridges or devices described herein., flowing the sample to the outlets of the cartridge, obtaining a product enriched in T or NK cells from the product outlet, and genetically engineering the T cells in the enriched product to product chimeric antigen receptors on the surface of the T NK cells. The sample of the method may include an apheresis product or a leukapheresis product. The genetically engineering of the method may comprise genetic engineering methods as described herein. The method may further comprise expanding the

CAR T or NK cells by growing the cell in vitro.

[0045] Some commercial examples of CAR T cell therapeutics that can be engineered according to the device and methods herein include axicabtagene ciloleucel, tisagenlecleucel, and brexucabtagene autoleucel.

[0046] The target particles or target cells of the method may comprise stem cells, thrombocytes, synoviocytes, fibroblasts, beta cells, liver cells, megakaryocytes, pancreatic cells, DE3 lysogenized cell, yeast cells, plant cells, algae cells, monocytes, T cells, B cells, regulatory T cells, macrophages, dendritic cells, granulocytes, innate lymphoid cells, natural killer cells, leukocytes, peripheral blood mononuclear cells, CD3+ cells, neurons, platelets, cancer cells, muscle cells, or epithelial cells. The method may comprise enriching target particles or target cells to produce enriched target cells comprising stem cells, thrombocytes, synoviocytes, fibroblasts, beta cells, liver cells, megakaryocytes, pancreatic cells, DE3 lysogenized cell, yeast cells, plant cells, algae cells, monocytes, T cells, B cells, regulatory T cells, macrophages, dendritic cells, granulocytes, innate lymphoid cells, natural killer cells, leukocytes, peripheral blood mononuclear cells, CD3+ cells, neurons, platelets, cancer cells, muscle cells, or epithelial cells. The contaminants of the method may comprise stem cells, thrombocytes, synoviocytes, fibroblasts, beta cells, liver cells, megakaryocytes, pancreatic cells, DE3 lysogenized cell, yeast cells, plant cells, algae cells, monocytes, T cells, B cells, regulatory T cells, macrophages, dendritic cells, granulocytes, innate lymphoid cells, natural killer cells, leukocytes, peripheral blood mononuclear cells, CD3+ cells, neurons, platelets, cancer cells, muscle cells, or epithelial cells. For example, the target cells may be peripheral blood mononuclear cells and the contaminants may be platelets. For example, the target cells may be CD3+ cells and the contaminants may be platelets. The method may result in the removal of more than 90% of the platelets. The method may result in the removal of about 50 % of the platelets to about 99 % of the platelets. The method may result in the removal of about 50 % of the platelets to about 75 % of the platelets, about 50 % of the platelets to about 80 % of the platelets, about 50 % of the platelets to about 90 % of the platelets, about 50 % of the platelets to about 95 % of the platelets, about 50 % of the platelets to about 99 % of the platelets, about 75 % of the platelets to about 80

% of the platelets, about 75 % of the platelets to about 90 % of the platelets, about 75 % of the platelets to about 95 % of the platelets, about 75 % of the platelets to about 99 % of the platelets, about 80 % of the platelets to about 90 % of the platelets, about 80 % of the platelets to about 95

% of the platelets, about 80 % of the platelets to about 99 % of the platelets, about 90 % of the platelets to about 95 % of the platelets, about 90 % of the platelets to about 99 % of the platelets, or about 95 % of the platelets to about 99 % of the platelets. The method may result in the removal of about 50 % of the platelets, about 75 % of the platelets, about 80 % of the platelets, about 90 % of the platelets, about 95 % of the platelets, or about 99 % of the platelets. The method may result in the removal of at least about 50 % of the platelets, about 75 % of the platelets, about 80 % of the platelets, about 90 % of the platelets, or about 95 % of the platelets.

The method may result in the removal of at most about 75 % of the platelets, about 80 % of the platelets, about 90 % of the platelets, about 95 % of the platelets, or about 99 % of the platelets.

[0047] The method may comprise modifying the enriched target cells. The method may comprise genetically engineering the enriched target cells to obtain genetically engineered target cells. Genetically engineering includes transfecting or transducing the target cells with a recombinant nucleic acid. Methods of genetic engineering may include the use of TALENs, Zinc

Finger Nucleases, CRISPR-Cas associated proteins, homologous recombination, viral vectors, or heterologous plasmids. The method may also include expanding the enriched target cells or genetically engineered cells by culturing them in vitro.

[0048] The present invention relates to DLD array geometries, that are compatible with existing manufacturing processes (for example, injection molding), and remain functional regardless of the manufacturing process used to construct them. The devices herein include DLD arrays which incorporate obstacles with tapered profiles. [0049] Tapered obstacles can include the following. For example, FIG. 1 illustrates examples of obstacles of three different shapes (diamond, hexagon, and triangle) in a tapered configuration (left) and a non-tapered configuration (right). The wall taper on the tapered obstacles can be described in terms of wall angle (Q). In some embodiments, the wall angle is between 0.1 and 6 degrees.

[0050] In some embodiments, the wall angle may be about 0.1 degrees to about 6 degrees. In some embodiments, the wall angle may be about 0.1 degrees to about 0.3 degrees, about 0.1 degrees to about 0.6 degrees, about 0.1 degrees to about 0.9 degrees, about 0.1 degrees to about 1 degree, about 0.1 degrees to about 1.5 degrees, about 0.1 degrees to about 2 degrees, about 0.1 degrees to about 2.5 degrees, about 0.1 degrees to about 3 degrees, about 0.1 degrees to about 3.5 degrees, about 0.1 degrees to about 4 degrees, about 0.1 degrees to about 6 degrees, about 0.3 degrees to about 0.6 degrees, about 0.3 degrees to about 0.9 degrees, about 0.3 degrees to about 1 degree, about 0.3 degrees to about 1.5 degrees, about 0.3 degrees to about 2 degrees, about 0.3 degrees to about 2.5 degrees, about 0.3 degrees to about 3 degrees, about 0.3 degrees to about 3.5 degrees, about 0.3 degrees to about 4 degrees, about 0.3 degrees to about 6 degrees, about 0.6 degrees to about 0.9 degrees, about 0.6 degrees to about 1 degree, about 0.6 degrees to about 1.5 degrees, about 0.6 degrees to about 2 degrees, about 0.6 degrees to about 2.5 degrees, about 0.6 degrees to about 3 degrees, about 0.6 degrees to about 3.5 degrees, about 0.6 degrees to about 4 degrees, about 0.6 degrees to about 6 degrees, about 0.9 degrees to about 1 degree, about 0.9 degrees to about 1.5 degrees, about 0.9 degrees to about 2 degrees, about 0.9 degrees to about 2.5 degrees, about 0.9 degrees to about 3 degrees, about 0.9 degrees to about 3.5 degrees, about 0.9 degrees to about 4 degrees, about 0.9 degrees to about 6 degrees, about 1 degree to about 1.5 degrees, about 1 degree to about 2 degrees, about 1 degree to about 2.5 degrees, about 1 degree to about 3 degrees, about 1 degree to about 3.5 degrees, about 1 degree to about 4 degrees, about 1 degree to about 6 degrees, about 1.5 degrees to about 2 degrees, about 1.5 degrees to about 2.5 degrees, about 1.5 degrees to about 3 degrees, about 1.5 degrees to about 3.5 degrees, about 1.5 degrees to about 4 degrees, about 1.5 degrees to about 6 degrees, about 2 degrees to about 2.5 degrees, about 2 degrees to about 3 degrees, about 2 degrees to about 3.5 degrees, about 2 degrees to about 4 degrees, about 2 degrees to about 6 degrees, about

2.5 degrees to about 3 degrees, about 2.5 degrees to about 3.5 degrees, about 2.5 degrees to about 4 degrees, about 2.5 degrees to about 6 degrees, about 3 degrees to about 3.5 degrees, about 3 degrees to about 4 degrees, about 3 degrees to about 6 degrees, about 3.5 degrees to about 4 degrees, about 3.5 degrees to about 6 degrees, or about 4 degrees to about 6 degrees. In some embodiments, the wall angle may be about 0.1 degrees, about 0.3 degrees, about 0.6 degrees, about 0.9 degrees, about 1 degree, about 1.5 degrees, about 2 degrees, about 2.5 degrees, about 3 degrees, about 3.5 degrees, about 4 degrees, or about 6 degrees. In some embodiments, the wall angle may be at least about 0.1 degrees, about 0.3 degrees, about 0.6 degrees, about 0.9 degrees, about 1 degree, about 1.5 degrees, about 2 degrees, about 2.5 degrees, about 3 degrees, about 3.5 degrees, or about 4 degrees. In some embodiments, the wall angle may be at most about 0.3 degrees, about 0.6 degrees, about 0.9 degrees, about 1 degree, about 1.5 degrees, about 2 degrees, about 2.5 degrees, about 3 degrees, about 3.5 degrees, about

4 degrees, or about 6 degrees.

[0051] In some embodiments, the wall angle may be about 0.3 degrees to about 1.4 degrees. In some embodiments, the wall angle may be about 0.3 degrees to about 0.4 degrees, about 0.3 degrees to about 0.5 degrees, about 0.3 degrees to about 0.6 degrees, about 0.3 degrees to about 0.7 degrees, about 0.3 degrees to about 0.8 degrees, about 0.3 degrees to about 0.9 degrees, about 0.3 degrees to about 1 degree, about 0.3 degrees to about 1.1 degrees, about 0.3 degrees to about 1.2 degrees, about 0.3 degrees to about 1.3 degrees, about 0.3 degrees to about 1.4 degrees, about 0.4 degrees to about 0.5 degrees, about 0.4 degrees to about 0.6 degrees, about 0.4 degrees to about 0.7 degrees, about 0.4 degrees to about 0.8 degrees, about 0.4 degrees to about 0.9 degrees, about 0.4 degrees to about 1 degree, about 0.4 degrees to about 1.1 degrees, about 0.4 degrees to about 1.2 degrees, about 0.4 degrees to about 1.3 degrees, about 0.4 degrees to about 1.4 degrees, about 0.5 degrees to about 0.6 degrees, about 0.5 degrees to about 0.7 degrees, about 0.5 degrees to about 0.8 degrees, about 0.5 degrees to about 0.9 degrees, about

0.5 degrees to about 1 degree, about 0.5 degrees to about 1.1 degrees, about 0.5 degrees to about

1.2 degrees, about 0.5 degrees to about 1.3 degrees, about 0.5 degrees to about 1.4 degrees, about 0.6 degrees to about 0.7 degrees, about 0.6 degrees to about 0.8 degrees, about 0.6 degrees to about 0.9 degrees, about 0.6 degrees to about 1 degree, about 0.6 degrees to about 1.1 degrees, about 0.6 degrees to about 1.2 degrees, about 0.6 degrees to about 1.3 degrees, about 0.6 degrees to about 1.4 degrees, about 0.7 degrees to about 0.8 degrees, about 0.7 degrees to about 0.9 degrees, about 0.7 degrees to about 1 degree, about 0.7 degrees to about 1.1 degrees, about 0.7 degrees to about 1.2 degrees, about 0.7 degrees to about 1.3 degrees, about 0.7 degrees to about 1.4 degrees, about 0.8 degrees to about 0.9 degrees, about 0.8 degrees to about 1 degree, about 0.8 degrees to about 1.1 degrees, about 0.8 degrees to about 1.2 degrees, about 0.8 degrees to about 1.3 degrees, about 0.8 degrees to about 1.4 degrees, about 0.9 degrees to about

1 degree, about 0.9 degrees to about 1.1 degrees, about 0.9 degrees to about 1.2 degrees, about 0.9 degrees to about 1.3 degrees, about 0.9 degrees to about 1.4 degrees, about 1 degree to about 1.1 degrees, about 1 degree to about 1.2 degrees, about 1 degree to about 1.3 degrees, about 1 degree to about 1.4 degrees, about 1.1 degrees to about 1.2 degrees, about 1.1 degrees to about

1.3 degrees, about 1.1 degrees to about 1.4 degrees, about 1.2 degrees to about 1.3 degrees, about 1.2 degrees to about 1.4 degrees, or about 1.3 degrees to about 1.4 degrees. In some embodiments, the wall angle may be about 0.3 degrees, about 0.4 degrees, about 0.5 degrees, about 0.6 degrees, about 0.7 degrees, about 0.8 degrees, about 0.9 degrees, about 1 degree, about 1.1 degrees, about 1.2 degrees, about 1.3 degrees, or about 1.4 degrees. In some embodiments, the wall angle may be at least about 0.3 degrees, about 0.4 degrees, about 0.5 degrees, about 0.6 degrees, about 0.7 degrees, about 0.8 degrees, about 0.9 degrees, about 1 degree, about 1.1 degrees, about 1.2 degrees, or about 1.3 degrees. In some embodiments, the wall angle may be at most about 0.4 degrees, about 0.5 degrees, about 0.6 degrees, about 0.7 degrees, about 0.8 degrees, about 0.9 degrees, about 1 degree, about 1.1 degrees, about 1.2 degrees, about 1.3 degrees, or about 1.4 degrees.

[0052] In some embodiments, the wall angle may be about 1.5 degrees to about 2.6 degrees. In some embodiments, the wall angle may be about 1.5 degrees to about 1.6 degrees, about 1.5 degrees to about 1.7 degrees, about 1.5 degrees to about 1.8 degrees, about 1.5 degrees to about 1.9 degrees, about 1.5 degrees to about 2 degrees, about 1.5 degrees to about 2.1 degrees, about

1.5 degrees to about 2.2 degrees, about 1.5 degrees to about 2.3 degrees, about 1.5 degrees to about 2.4 degrees, about 1.5 degrees to about 2.5 degrees, about 1.5 degrees to about 2.6 degrees, about 1.6 degrees to about 1.7 degrees, about 1.6 degrees to about 1.8 degrees, about

1.6 degrees to about 1.9 degrees, about 1.6 degrees to about 2 degrees, about 1.6 degrees to about 2.1 degrees, about 1.6 degrees to about 2.2 degrees, about 1.6 degrees to about 2.3 degrees, about 1.6 degrees to about 2.4 degrees, about 1.6 degrees to about 2.5 degrees, about

1.6 degrees to about 2.6 degrees, about 1.7 degrees to about 1.8 degrees, about 1.7 degrees to about 1.9 degrees, about 1.7 degrees to about 2 degrees, about 1.7 degrees to about 2.1 degrees, about 1.7 degrees to about 2.2 degrees, about 1.7 degrees to about 2.3 degrees, about 1.7 degrees to about 2.4 degrees, about 1.7 degrees to about 2.5 degrees, about 1.7 degrees to about 2.6 degrees, about 1.8 degrees to about 1.9 degrees, about 1.8 degrees to about 2 degrees, about 1.8 degrees to about 2.1 degrees, about 1.8 degrees to about 2.2 degrees, about 1.8 degrees to about 2.3 degrees, about 1.8 degrees to about 2.4 degrees, about 1.8 degrees to about 2.5 degrees, about 1.8 degrees to about 2.6 degrees, about 1.9 degrees to about 2 degrees, about 1.9 degrees to about 2.1 degrees, about 1.9 degrees to about 2.2 degrees, about 1.9 degrees to about 2.3 degrees, about 1.9 degrees to about 2.4 degrees, about 1.9 degrees to about 2.5 degrees, about 1.9 degrees to about 2.6 degrees, about 2 degrees to about 2.1 degrees, about 2 degrees to about 2.2 degrees, about 2 degrees to about 2.3 degrees, about 2 degrees to about 2.4 degrees, about 2 degrees to about 2.5 degrees, about 2 degrees to about 2.6 degrees, about 2.1 degrees to about 2.2 degrees, about 2.1 degrees to about 2.3 degrees, about 2.1 degrees to about 2.4 degrees, about 2.1 degrees to about 2.5 degrees, about 2.1 degrees to about 2.6 degrees, about 2.2 degrees to about 2.3 degrees, about 2.2 degrees to about 2.4 degrees, about 2.2 degrees to about 2.5 degrees, about 2.2 degrees to about 2.6 degrees, about 2.3 degrees to about 2.4 degrees, about

2.3 degrees to about 2.5 degrees, about 2.3 degrees to about 2.6 degrees, about 2.4 degrees to about 2.5 degrees, about 2.4 degrees to about 2.6 degrees, or about 2.5 degrees to about 2.6 degrees. In some embodiments, the wall angle may be about 1.5 degrees, about 1.6 degrees, about 1.7 degrees, about 1.8 degrees, about 1.9 degrees, about 2 degrees, about 2.1 degrees, about 2.2 degrees, about 2.3 degrees, about 2.4 degrees, about 2.5 degrees, or about 2.6 degrees.

In some embodiments, the wall angle may be at least about 1.5 degrees, about 1.6 degrees, about

1.7 degrees, about 1.8 degrees, about 1.9 degrees, about 2 degrees, about 2.1 degrees, about 2.2 degrees, about 2.3 degrees, about 2.4 degrees, or about 2.5 degrees. In some embodiments, the wall angle may be at most about 1.6 degrees, about 1.7 degrees, about 1.8 degrees, about 1.9 degrees, about 2 degrees, about 2.1 degrees, about 2.2 degrees, about 2.3 degrees, about 2.4 degrees, about 2.5 degrees, or about 2.6 degrees.

[0053] In some embodiments, the wall angle may be about 2.7 degrees to about 3.5 degrees. In some embodiments, the wall angle may be about 2.7 degrees to about 2.8 degrees, about 2.7 degrees to about 2.9 degrees, about 2.7 degrees to about 3 degrees, about 2.7 degrees to about 3.1 degrees, about 2.7 degrees to about 3.2 degrees, about 2.7 degrees to about 3.3 degrees, about 2.7 degrees to about 3.4 degrees, about 2.7 degrees to about 3.5 degrees, about 2.8 degrees to about 2.9 degrees, about 2.8 degrees to about 3 degrees, about 2.8 degrees to about 3.1 degrees, about 2.8 degrees to about 3.2 degrees, about 2.8 degrees to about 3.3 degrees, about

2.8 degrees to about 3.4 degrees, about 2.8 degrees to about 3.5 degrees, about 2.9 degrees to about 3 degrees, about 2.9 degrees to about 3.1 degrees, about 2.9 degrees to about 3.2 degrees, about 2.9 degrees to about 3.3 degrees, about 2.9 degrees to about 3.4 degrees, about 2.9 degrees to about 3.5 degrees, about 3 degrees to about 3.1 degrees, about 3 degrees to about 3.2 degrees, about 3 degrees to about 3.3 degrees, about 3 degrees to about 3.4 degrees, about 3 degrees to about 3.5 degrees, about 3.1 degrees to about 3.2 degrees, about 3.1 degrees to about 3.3 degrees, about 3.1 degrees to about 3.4 degrees, about 3.1 degrees to about 3.5 degrees, about

3.2 degrees to about 3.3 degrees, about 3.2 degrees to about 3.4 degrees, about 3.2 degrees to about 3.5 degrees, about 3.3 degrees to about 3.4 degrees, about 3.3 degrees to about 3.5 degrees, or about 3.4 degrees to about 3.5 degrees. In some embodiments, the wall angle may be about 2.7 degrees, about 2.8 degrees, about 2.9 degrees, about 3 degrees, about 3.1 degrees, about 3.2 degrees, about 3.3 degrees, about 3.4 degrees, or about 3.5 degrees. In some embodiments, the wall angle may be at least about 2.7 degrees, about 2.8 degrees, about 2.9 degrees, about 3 degrees, about 3.1 degrees, about 3.2 degrees, about 3.3 degrees, or about 3.4 degrees. In some embodiments, the wall angle may be at most about 2.8 degrees, about 2.9 degrees, about 3 degrees, about 3.1 degrees, about 3.2 degrees, about 3.3 degrees, about 3.4 degrees, or about 3.5 degrees.

[0054] Alternatively, wall taper can be described in terms of the change in the width, AW, of a single obstacle traveling from the bottom surface to the top surface (AW = Wbottom-Wtop). Similarly, the taper can be described by the change in the gap, AG, between two adjacent obstacles in an array going from the top of the array to the bottom (AG = Gmax-Gmin), as depicted in FIG. 2A. Gmin and Gmax are also useful in calculation of the maximum wall angle a DLD device can utilize, while remaining functional for separations. Examples of these calculations for obstacle heights of 50 pm and 100 pm are also described in FIG. 2A.

[0055] In some embodiments, the height of the obstacles may be about 20 pm to about 200 pm. In some embodiments, the height of the obstacles may be about 20 pm to about 25 pm, about 20 pm to about 30 pm, about 20 pm to about 35 pm, about 20 pm to about 40 pm, about 20 pm to about 50 pm, about 20 pm to about 75 pm, about 20 pm to about 100 pm, about 20 pm to about 125 pm, about 20 pm to about 150 pm, about 20 pm to about 175 pm, about 20 pm to about 200 pm, about 25 pm to about 30 pm, about 25 pm to about 35 pm, about 25 pm to about 40 pm, about 25 pm to about 50 pm, about 25 pm to about 75 pm, about 25 pm to about 100 pm, about 25 mih to about 125 mih, about 25 mih to about 150 mih, about 25 mih to about 175 mih, about 25 mih to about 200 mih, about 30 mih to about 35 mih, about 30 mih to about 40 mih, about 30 mih to about 50 mih, about 30 mih to about 75 mih, about 30 mih to about 100 mih, about 30 mih to about 125 mih, about 30 mih to about 150 mih, about 30 mih to about 175 mih, about 30 mih to about 200 mih, about 35 mih to about 40 mih, about 35 mih to about 50 mih, about 35 mih to about 75 mih, about 35 mih to about 100 mih, about 35 mih to about 125 mih, about 35 mih to about 150 mih, about 35 mih to about 175 mih, about 35 mih to about 200 mih, about 40 mih to about 50 mih, about 40 mih to about 75 mih, about 40 mih to about 100 mih, about 40 mih to about 125 mih, about 40 mih to about 150 mih, about 40 mih to about 175 mih, about 40 mih to about 200 mih, about 50 mih to about 75 mih, about 50 mih to about 100 mih, about 50 mih to about 125 mih, about 50 mih to about 150 mih, about 50 mih to about 175 mih, about 50 mih to about 200 mih, about 75 mih to about 100 mih, about 75 mih to about 125 mih, about 75 mih to about 150 mih, about 75 mih to about 175 mih, about 75 mih to about 200 mih, about 100 mih to about 125 mih, about 100 mih to about 150 mih, about 100 mih to about 175 mih, about 100 mih to about 200 mih, about 125 mih to about 150 mih, about 125 mih to about 175 mih, about 125 mih to about 200 mih, about 150 mih to about 175 mih, about 150 mih to about 200 mih, or about 175 mih to about 200 mih. In some embodiments, the height of the obstacles may be about 20 mih, about 25 mih, about 30 mih, about 35 mih, about 40 mih, about 50 mm, about 75 mih, about 100 mih, about 125 mm, about 150 mih, about 175 mm, or about 200 mih. In some embodiments, the height of the obstacles may be at least about 20 mih, about 25 mih, about 30 mih, about 35 mih, about 40 mih, about 50 mih, about 75 mih, about 100 gm, about 125 gm, about 150 gm, or about 175 gm. In some embodiments, the height of the obstacles may be at most about 25 gm, about 30 gm, about 35 gm, about 40 gm, about 50 gm, about 75 gm, about 100 gm, about 125 gm, about 150 gm, about 175 gm, or about 200 gm.

[0056] Many different variations of obstacle height can be utilized without changing the function or nature of the invention. For example, FIG. 2B shows two adjacent obstacles having tapered walls from both top and cross-sectional views in three different aspect ratios example aspect ratios. Any of these aspect ratios could be made and utilized to separate particles, if the wall angle limits calculated according to the equations of FIG. 2A are not exceeded. In some embodiments, the height of obstacles is in the range from 20 pm to 200 pm. In some embodiments, the aspect ratio is between 0.5:1 and 5:1.

[0057] DLD arrays having tapered obstacles may further incorporate either symmetric or asymmetric arrangements of obstacles. In some embodiments, asymmetric arrangements are preferred because they reduce the shear forces experienced by separated particles relative to symmetric array layouts. Lengthening obstacle gaps perpendicular to the direction of fluid flow and decreasing the length of gaps parallel to fluid flow allows cells of a given size to be processed more rapidly. This can be achieved with a range of obstacle shapes, with the most preferred obstacles being diamond or hexagonally shaped. Hexagonally shaped obstacles may be preferred because they provide the same processing advantages as diamonds but can result in a device that is easier to manufacture and more resistant to biofouling.

[0058] Using asymmetric gaps can improve throughput and allow devices to operate more efficiently. Polygonal-shaped, elongated obstacles that have vertices pointing toward one another in parallel gaps but where the vertices are offset from one another (as opposed to being directly opposite one another) are preferred. This design reduces flow through the parallel gaps (also called minor flux) by making the gap longer, not narrower. In contrast, vertices in perpendicular gaps preferably are directly opposite from one another. Thus, a primary characteristic of the devices disclosed herein is the presence of obstacle arrays in which perpendicular gaps and parallel gaps are asymmetric, i.e., they are not the same size. By varying spacing, it is possible to decrease resistance to flow compared to devices that separate particles and cells in the same size range but that have perpendicular and parallel gaps of the same length. [0059] Examples of both symmetric and asymmetric obstacle placements are shown in FIG. 3. Arrangements are described in terms of the gaps between obstacles and the tilt angle e, which describes the offset between adjacent rows of obstacles. In some embodiments the tilt angle is between 0.1 degree and 6 degrees. In the left arrangement, a symmetric configuration is depicted, meaning that the gap is uniform between all obstacles (i.e. the same spacing is used for placement of obstacles in both the vertical and horizontal directions of the diagram). In the center arrangement the placement of the obstacles is asymmetric, meaning that the gaps between array obstacles along perpendicular axes differ, where Gap 1 is larger than Gap 2 in this particular example. FIG. 3 further provides examples of obstacle shape which are symmetric or asymmetric with respect to the axis of flow. The left and center arrangements both demonstrate symmetric obstacles, since the symmetry axis of the diamond-shaped obstacles is aligned with the axis of fluid flow. The right arrangement provides an additional example of an asymmetric obstacle layout (i.e. Gap 1 is not equal to Gap 2), which further demonstrates an asymmetric obstacle geometry. While the shape of the triangles in the particular example of the right arrangement is symmetric in of itself, the axis of symmetry is out of phase with the axis of flow, making the obstacle geometry asymmetric.

[0060] When utilizing tapered obstacles in a DLD array, the placement of obstacles in the array (i.e. either symmetrically or asymmetrically, such as in the examples given in FIG. 3) may be a function of the median gap between adjacent obstacles. FIG. 4 depicts two example arrays of tapered obstacles in asymmetric layouts, tapered diamond-shaped obstacles (top) and hexagonal tapered obstacles (bottom). The tapered walls of the obstacles in both cases result in the gaps between adjacent posts having gaps that increase in size going from the bottom of the array to the top, where for Gap 1 the minimum gap is Gi_b and the maximum gap is Gi_t. Similarly, for Gap 2 the minimum gap is G2_b and the maximum gap is G2_t. Herein, except where specified otherwise, in the case that a single value or a single range of values is provided for Gap 1 or Gap 2, the value or range is understood to refer to the mean gap. In some embodiments the Gap 1 is in the range of 5-150 pm and Gap 2 is in the range of 5-150 pm. [0061] In some embodiments, Gap 1 and Gap 2 may each independently be about 5 pm to about

150 pm. In some embodiments, Gap 1 and Gap 2 may each independently be about 5 pm to about 10 pm, about 5 pm to about 15 pm, about 5 pm to about 20 pm, about 5 pm to about 25 pm, about 5 pm to about 30 pm, about 5 pm to about 35 pm, about 5 pm to about 40 pm, about

5 pm to about 50 pm, about 5 pm to about 100 pm, about 5 pm to about 125 pm, about 5 pm to about 150 pm, about 10 pm to about 15 pm, about 10 pm to about 20 pm, about 10 pm to about

25 pm, about 10 pm to about 30 pm, about 10 pm to about 35 pm, about 10 pm to about 40 pm, about 10 pm to about 50 pm, about 10 pm to about 100 pm, about 10 pm to about 125 pm, about 10 pm to about 150 pm, about 15 pm to about 20 pm, about 15 pm to about 25 pm, about

15 pm to about 30 pm, about 15 pm to about 35 pm, about 15 pm to about 40 pm, about 15 pm to about 50 pm, about 15 pm to about 100 pm, about 15 pm to about 125 pm, about 15 pm to about 150 pm, about 20 pm to about 25 pm, about 20 pm to about 30 pm, about 20 pm to about

35 pm, about 20 pm to about 40 pm, about 20 pm to about 50 pm, about 20 pm to about 100 pm, about 20 pm to about 125 pm, about 20 pm to about 150 pm, about 25 pm to about 30 pm, about 25 pm to about 35 pm, about 25 pm to about 40 pm, about 25 pm to about 50 pm, about

25 pm to about 100 pm, about 25 pm to about 125 pm, about 25 pm to about 150 pm, about 30 pm to about 35 pm, about 30 pm to about 40 pm, about 30 pm to about 50 pm, about 30 pm to about 100 pm, about 30 pm to about 125 pm, about 30 pm to about 150 pm, about 35 pm to about 40 pm, about 35 pm to about 50 pm, about 35 pm to about 100 pm, about 35 pm to about

125 pm, about 35 pm to about 150 pm, about 40 pm to about 50 pm, about 40 pm to about 100 pm, about 40 pm to about 125 pm, about 40 pm to about 150 pm, about 50 pm to about 100 pm, about 50 pm to about 125 pm, about 50 pm to about 150 pm, about 100 pm to about 125 pm, about 100 pm to about 150 pm, or about 125 pm to about 150 pm. In some embodiments,

Gap 1 and Gap 2 may each independently be about 5 pm, about 10 pm, about 15 pm, about 20 pm, about 25 pm, about 30 pm, about 35 pm, about 40 pm, about 50 pm, about 100 pm, about

125 pm, or about 150 pm. In some embodiments, Gap 1 and Gap 2 may each independently be at least about 5 mih, about 10 mih, about 15 mih, about 20 mih, about 25 mih, about 30 mih, about

35 mih, about 40 mih, about 50 mih, about 100 mih, or about 125 mih. In some embodiments, Gap

1 and Gap 2 may each independently be at most about 10 pm, about 15 pm, about 20 pm, about

25 pm, about 30 pm, about 35 pm, about 40 pm, about 50 pm, about 100 pm, about 125 pm, or about 150 pm.

[0062] In some embodiments, Gap 1 and Gap 2 may each independently be about 7 pm to about 18 pm. In some embodiments, Gap 1 and Gap 2 may each independently be about 7 pm to about

8 pm, about 7 pm to about 9 pm, about 7 pm to about 10 pm, about 7 pm to about 11 pm, about 7 pm to about 12 pm, about 7 pm to about 13 pm, about 7 pm to about 14 pm, about 7 pm to about 15 pm, about 7 pm to about 16 pm, about 7 pm to about 17 pm, about 7 pm to about 18 pm, about 8 pm to about 9 pm, about 8 pm to about 10 pm, about 8 pm to about 11 pm, about 8 pm to about 12 pm, about 8 pm to about 13 pm, about 8 pm to about 14 pm, about 8 pm to about 15 pm, about 8 pm to about 16 pm, about 8 pm to about 17 pm, about 8 pm to about 18 pm, about 9 pm to about 10 pm, about 9 pm to about 11 pm, about 9 pm to about 12 pm, about

9 pm to about 13 pm, about 9 pm to about 14 pm, about 9 pm to about 15 pm, about 9 pm to about 16 pm, about 9 pm to about 17 pm, about 9 pm to about 18 pm, about 10 pm to about 11 pm, about 10 pm to about 12 pm, about 10 pm to about 13 pm, about 10 pm to about 14 pm, about 10 pm to about 15 pm, about 10 pm to about 16 pm, about 10 pm to about 17 pm, about

10 pm to about 18 pm, about 11 pm to about 12 pm, about 11 pm to about 13 pm, about 11 pm to about 14 pm, about 11 pm to about 15 pm, about 11 pm to about 16 pm, about 11 pm to about 17 pm, about 11 pm to about 18 pm, about 12 pm to about 13 pm, about 12 pm to about 14 pm, about 12 pm to about 15 pm, about 12 pm to about 16 pm, about 12 pm to about 17 pm, about 12 pm to about 18 pm, about 13 pm to about 14 pm, about 13 pm to about 15 pm, about 13 pm to about 16 pm, about 13 pm to about 17 pm, about 13 pm to about 18 pm, about 14 pm to about 15 pm, about 14 pm to about 16 pm, about 14 pm to about 17 pm, about 14 pm to about 18 pm, about 15 pm to about 16 pm, about 15 pm to about 17 pm, about 15 pm to about 18 mih, about 16 mih to about 17 mih, about 16 mih to about 18 mih, or about 17 mih to about 18 mih. In some embodiments, Gap 1 and Gap 2 may each independently be about 7 pm, about 8 pm, about 9 pm, about 10 pm, about 11 pm, about 12 pm, about 13 pm, about 14 pm, about 15 pm, about 16 pm, about 17 pm, or about 18 pm. In some embodiments, Gap 1 and Gap 2 may each independently be at least about 7 pm, about 8 pm, about 9 pm, about 10 pm, about 11 pm, about 12 pm, about 13 pm, about 14 pm, about 15 pm, about 16 pm, or about 17 pm. In some embodiments, Gap 1 and Gap 2 may each independently be at most about 8 pm, about 9 pm, about 10 pm, about 11 pm, about 12 pm, about 13 pm, about 14 pm, about 15 pm, about 16 pm, about 17 pm, or about 18 pm.

[0063] A variety of tapered obstacle shapes (circles, triangles, ovals, hexagons, diamonds, trapezoids, teardrops, polygons, and many other shapes conceivable to one skilled in the art) can be employed in the DLD arrays described herein. Tapered obstacles with symmetric cross- sectional shapes with respect to the axis of flow exhibit reduced or no mixing, which can lead to proper separation of particles. Two DLD array devices with tapered obstacles are depicted in FIG. 5. The device on the right incorporates diamond-shaped tapered obstacles in an asymmetric arrangement similar to the one shown in FIG. 3, center arrangement. The device on the left incorporates tapered triangle obstacles in an asymmetric layout, where the triangles are rotated such that they are asymmetric with respect to the axis of flow, as shown in FIG. 3, right arrangement. In FIG. 5, food dye is added to the sample stream (502, outer flow), but not to the buffer stream (504, inner flow) in both devices. In the right device, the two streams remain laminar, parallel and coflowing as they travel down the device and exit without mixing. In the left device, the two streams are mixed by three-dimensional currents as they traverse the DLD array, resulting in a visible blurring of the boundary between the two flows (506). In some embodiments, tapered obstacles arrays can be as separation devices which employ flows that are nonlaminar. [0064] Mixing can greatly hinder the ability of a DLD array to perform size-based separations, due to contaminant particles and/or diffusible species smaller than the critical size (D_c) getting mixed with the buffer meant to collect the target particles, resulting in contamination. FIG. 6 demonstrates the contamination effects of mixing, by applying DLD array separations to isolation of white blood cells from blood, removing the smaller red blood cell and platelet contaminants. The x-axis of the graph shows the effective D_c value for a given DLD array, while the y-axis shows level of platelet contamination in the product fraction for DLD arrays with geometries that correspond to each of the three example layouts detailed in FIG. 3. The data are further divided by the degree to which the obstacles are tapered. Markedly, the asymmetric shaped triangles with Q > 0.5 produce the worst contamination, due to the mixing effects demonstrated for this geometry in FIG. 5.

[0065] The critical size of a given DLD array is set by the geometry of the gap spacings between the obstacles. For straight-walled obstacles, a single critical size value governs the separation process. Particles above that size are deflected into the product fraction flow stream, while particles below that size are carried by the sample flow to the waste outlet. In an array utilizing tapered obstacles, the gap size varies from the bottom of the array to the top of the array (see FIG. 2A and FIG. 4). This results in variability in the effective critical size which depends on the height at which the particles encounter the obstacles. If the critical size is encountered by the particles is too large, poor recoveries result since fewer particles will be deflected to the product fraction outlet. If the critical size encountered is too low, contamination will result since smaller particles will also be deflected to the product fraction outlet. Careful consideration of the separation application is needed to design an array using tapered obstacles that has an appropriate effective critical size range.

[0066] In the instance of separating white blood cells from smaller blood cells for therapeutic applications using a tapered-obstacle DLD array, as in most applications, maximizing recovery and minimizing contamination is essential. FIG. 7 shows white blood cell recoveries as a function of critical size, where error bars on the x-axis represent the range of possible effective critical sizes available in a particular tapered-DLD array. All arrays shown on the graph utilize diamond shape posts, which are symmetric with respect to the axis of flow. [0067] A further parameter, which is an important consideration for all separations, is throughput. Improving the throughput of DLD devices was explored using asymmetrically arranged tapered-hexagonal, which are expected to allow the highest throughput while minimizing the shear forces experienced by particles as they pass through the DLD array. A scanning electron micrograph depicting tapered hexagonal obstacles is shown in FIG. 8.

[0068] -The 40 x 20 pm hexagon design resulted from a need to process a more complex sample (apheresis vs. blood) and manufacturing considerations around obstacle/gap aspect ratios (post height to post width, or post height to gap). This design was optimized to be manufacturable in plastic, using high-volume manufacturing, particularly by injection molding a plastic design off a rigid tool. This process requires surfaces to be angled to facilitate release of the part from the tool. The angle also reduces wear by prevents the plastic part dragging on the tool (see FIG. 9). [0069] The initial design was a diamond array (20 x 20 pm diamonds with 16-17 um gaps). To process more sample, and process faster, the asymmetric arrays were designed (larger gaps perpendicular to flow, smaller gaps parallel to flow). This gave the same net D_c, but a bigger gap, which was more resistant to biofouling, and also produced less resistance to flow, allowing operation at higher flow rate. Smaller gaps are more difficult to manufacture, so DLD arrays were designed to have a minimum gap size of 12-14 um, to facilitate manufacturing. In order to maintain the asymmetric geometry (and the processing benefits) a design was needed where instead of pinching the small gap to set the minor flux, the pathway is lengthened at a reasonable gap size. This results in the same net minor flux, which is critical to determine the D_c.

[0070] Through the process of optimizing the design for manufacturability (keeping minimum gap size above 12 pm) and for throughput, a series of designs were made with the same principle, but with varying D_c values (i.e. 3, 3.5, 4, 4.5 um D_c, all with a 20 x 40 pm hexagon). These are set by changing the two gaps (Gap 1 and Gap 2) to adjust the major/minor flux. The same blood sample matrix was run in all of the devices fabricated, and the fall off points for recovery were determined (i.e. Dc » optimal, WBCs go to waste). Contamination cutoff points were further determined where contamination problems began (i.e. Dc « optimal, and platelets (PLT)s and red blood cells (RBC)s deflect to the product outlet). This resulted in a range of D_c values where reliable separations can be performed.

[0071] Theoretical wall angle limitations were validated by construction of tapered obstacle devices with a variety of wall angles in silicon through a DRIE etch process. Scanning electron micrographs of these devices are shown in FIG. 10. Separation performance of the tapered devices was compared to straight-walled devices for blood sample separations in terms of recovery and depletion (i.e. lack of contamination) to determine the effects of tapered devices having a range of D_c values from lid to floor. The tolerable limits for a device with a 50 pm obstacle height were calculated to be 2-3 degrees of wall angle, which results in a maximum of about 0.5 pm to 1 pm Dc shift (AD_C) from the top of the array to the bottom of the array.

[0072] In one embodiment, the posts are hexagonal with dimensions of 20 x 40 pm, sharp comer radii, heights of 50 pm, gap values at the top of the device (16-17 um), and gaps at the bottom of the device (12-14 um) driven by a 2-3 degree wall angle, where gaps are the minor flux gaps. At 50 pm in height a 13 pm gap at the bottom of an obstacle and a 17 pm gap at the top of an obstacle yields a wall angle (Q) of 2.3 degrees. A 13 pm gap yields a D_c of 3.7 pm in a straight-walled device, while a 17 pm gap yields a D_c of 4.7 pm, meaning that a 50 pm device with a 2.3 degree wall angle and a G_min of 13 pm will have an effective D_c in the range of 3.7 pm to 4.7 pm.

[0073] A blood separation was performed, and white blood cell (WBC) recoveries were examined as a function of pressure in using three different array geometries incorporating tapered hexagonal obstacles with dimensions of 40 pm length x 20 pm width x 50 pm height, as depicted in FIG. 11. Pressures of 10, 15, and 20 psi were tested for each array. The three leftmost bars of the left graph show that high recoveries are maintained at all pressures for the array where the tilt angle is 1/60, and Gap 1 and Gap 2 are 21 and 15 pm, respectively, such that the D_evalue of the array is 4.1 pm. This particular array is a preferred configuration due to its versatility. The rightmost plot shows a measurement of throughput of this array at 10 and 20 psi, which resulted in processing rates of 1.8 and 3.4 mL/hr, where operating at higher pressures leads to faster throughput.

[0074] The pressure behavior of an arrangement with a tilt angle of 1/60, Gap 1 and Gap 2 of 18 and 15 pm, respectively, such that the D_evalue of the array is 3.6 pm, is explored in the middle three bars of the leftmost graph in FIG. 11. This array also displays versatility with respect to pressure. The right three bars describe the pressure behavior of an array where the tilt angle is 1/100, Gap 1 and Gap 2 are 30 and 15 pm, respectively, such that the D_evalue of the array is 4.7 pm. This array is less robust to pressure, where lower WBC recoveries were observed at higher pressures.

[0075] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0076] All references cited herein are fully incorporated by reference. Having now fully described the invention, it will be understood by one of skill in the art that the invention may be performed within a wide and equivalent range of conditions, parameters and the like, without affecting the spirit or scope of the invention or any embodiment thereof.

Claims

CLAIMS What is Claimed is:

1. A particle separation device comprising: an array of obstacles spaced apart along a flow channel with each obstacle in the array of obstacles comprising a tapered profile, wherein a difference in gap sizes between adjacent obstacles along opposing tapered profiles is controlled to achieve a critical size range that permits the array of obstacles to separate target particles of a predetermined size from contaminants of a different size in a sample when the sample is transported along the flow channel.

2. The particle separation device of claim 1, wherein the tapered profile extends along at least one sidewall of each obstacle.

3. The particle separation device of claim 1, wherein an angle of the tapered profile is at least 0.1 degree.

4. The particle separation device of claim 1, wherein an angle of the tapered profile is 1 to 3 degrees.

5. The particle separation device of claim 1, wherein an angle of the tapered profile is no more than 5 degrees.

6. The particle separation device of claim 1, wherein the tapered profile has a positive taper such that a width of a top portion of each obstacle is less than a width of a bottom portion of said obstacle.

7. The particle separation device of claim 1, wherein the tapered profile has a negative taper such that a width of a top portion of each obstacle is greater than a width of a bottom portion of said obstacle.

8. The particle separation device of claim 1, wherein a height of each obstacle is at least 20 pm.

9. The particle separation device of claim 1, wherein a height of each obstacle is less than 150 pm.

10. The particle separation device of claim 1, wherein the gap sizes are different between top portions and bottom portions of the adjacent obstacles.

11. The particle separation device of claim 10, wherein the gap sizes are larger at the top portions compared to the bottom portions of the adjacent obstacles.

12. The particle separation device of claim 10, wherein the gap sizes are smaller at the top portions compared to the bottom portions of the adjacent obstacles.

13. The particle separation device of claim 1, wherein the gap sizes are different between a longitudinal axis and a transverse axis of the flow channel.

14. The particle separation device of claim 13, wherein the gap sizes are greater along the transverse axis compared to the longitudinal axis of the flow channel.

15. The particle separation device of claim 1, wherein adjacent rows or columns of the obstacles are offset from each other by an effective tilt.

16. The particle separation device of claim 15, wherein the effective tilt is 0.5 degree to 6 degrees.

17. The particle separation device of claim 15, wherein the effective tilt depends at least on a symmetry or asymmetry of (i) the gaps between the obstacles and (ii) a shape of the obstacles.

18. The particle separation device of claim 15, wherein a maximum allowable angle of the tapered angle depends in part on the effective tilt, in order to achieve the critical size range.

19. The particle separation device of claim 1, wherein the critical size range is further based on a height of the obstacles and an angle of the tapered profile.

20. The particle separation device of claim 1, wherein the critical size range is from 3 to 4.5.

21. The particle separation device of claim 1, wherein the critical size range comprises (i) a first critical size at a bottom portion of the gaps between adjacent obstacles and (ii) a second critical size at a top portion of the gaps between adjacent obstacles.

22. The particle separation device of claim 21, wherein the first critical size is smaller than the second critical size.

23. The particle separation device of claim 21, wherein the first critical size is greater than the second critical size.

24. The particle separation device of claim 21, wherein a difference between the first critical size and the second critical size is no more than 1 pm.

25. The particle separation device of claim 21, wherein a difference between the first critical size and the second critical size is no more than 2 pm.

26. The particle separation device of claim 1, wherein the critical size range is within 5% of an average or median critical size.

27. The particle separation device of claim 1, wherein the critical size range is within 10% of an average or median critical size.

28. The particle separation device of claim 1, wherein the critical size range is within 20% of an average or median critical size.

29. The particle separation device of claim 1, wherein the critical size range has an effect on laminar flow and/or mixing as the sample is transported along the flow channel.

30. The particle separation device of claim 1, wherein the critical size range is controlled to prevent or reduce mixing of the target particles with the contaminants as the sample is transported along the flow channel.

31. The particle separation device of claim 1, wherein the critical size range is controlled to reduce a contamination level of a product enriched with the target particles, which said product is generated using the array of obstacles.

32. The particle separation device of claim 1, wherein each obstacle has a shape that is symmetrical with respect to a longitudinal axis of the flow channel.

33. The particle separation device of claim 32, wherein the shape comprises a diamond shape, a polygonal shape, a circular shape, or an ellipsoidal shape.

34. The particle separation device of claim 33, wherein the polygonal shape comprises a regular hexagon.

35. The particle separation device of claim 33, wherein the polygonal shape comprises an elongated hexagon.

36. The particle separation device of claim 35, wherein the elongated hexagon is elongated along an axis that is substantially perpendicular to a longitudinal direction of the flow channel.

37. The particle separation device of claim 1, wherein none of the obstacles has a shape that is asymmetrical with respect to a longitudinal axis of the flow channel.

38. The particle separation device of claim 1, wherein at least one of the obstacles has a shape that is asymmetrical with respect to a longitudinal axis of the flow channel.

39. The particle separation device of claim 1, wherein the array of obstacles is used for a particle separation process, wherein a contamination level of the particle separation process is more sensitive to changes in an angle of the tapered profile when using asymmetric-shaped obstacles compared to symmetric-shaped obstacles.

40. The particle separation device of claim 39, wherein the contamination level is reduced using symmetric-shaped obstacles compared to asymmetric-shaped obstacles.

41. The particle separation device of 39, wherein the contamination level is lower when using the symmetric-shaped obstacles compared to the asymmetric-shaped obstacles, for a same angle of the tapered profile.

42. The particle separation device of claim 39, wherein the contamination level is lower when using the symmetric-shaped obstacles compared to the asymmetric-shaped obstacles, for a same increase in the angle of the tapered profile.

43. The particle separation device of claim 39, wherein a recovery yield of the target particles is higher when using symmetric-shaped obstacles compared to asymmetric-shaped obstacles.

44. The particle separation device of any one of claims 39 through 43, wherein a symmetry of the symmetric-shaped obstacles or a lack of symmetry of the asymmetric-shaped obstacles are defined with respect to a longitudinal axis of the flow channel.

45. The particle separation device of claim 1, wherein the array of obstacles comprise a plurality of posts.

46. The particle separation device of claim 45, wherein the plurality of posts are elongated in a vertical direction that is perpendicular to a longitudinal axis of the flow channel.

47. The particle separation device of claim 1, wherein the array of obstacles are formed using a molding process.

48. The particle separation device of claim 47, wherein the molding process comprises injection molding.

49. The particle separation device of claim 1, wherein the array of obstacles are made of a polymer.

50. The particle separation device of claim 1, wherein the array of obstacles are formed using an etching process.

51. The particle separation device of any one of the preceding claims, wherein the array is configured to process the sample at a rate equal to or greater than 300 mL/hr.

52. The particle separation device of any one of the preceding claims, wherein the array is configured to process the sample at a flow pressure of at least 1 psi.

53. The particle separation device of any one of the preceding claims, wherein the array is configured to process the sample at a flow pressure of at least 10 psi.

54. The particle separation device of any one of the preceding claims, wherein the array is configured to process the sample to generate a product that is enriched with at least 70% recovery of the target particles.

55. The particle separation device of any one of the preceding claims, wherein the sample is a human sample.

56. The particle separation device of any one of the preceding claims, wherein the sample comprises a blood-related product.

57. The particle separation device of claim 56, wherein the blood-related product comprises an apheresis product.

58. The particle separation device of claim 57, wherein the apheresis product is a leukapheresis product.

59. The particle separation device of any one of the preceding claims, wherein the target particles that have been separated from the sample comprise cells.

60. The particle separation device of claim 59, wherein the cells are human cells.

61. The particle separation device of claims 59 or 60, wherein the cells are greater than 90% viable upon recovery.

62. The particle separation device of any one of claims 59-61, wherein the cells comprise peripheral blood mononuclear cells.

63. The particle separation device of any one of claims 59-61, wherein the cells comprise CD3+ T cells.

64. The particle separation device of claim 63, wherein the T cells exhibit a naive or central memory phenotype.

65. A method for enriching target particles or target cells of a predetermined size from contaminants in a sample, the method comprising: a) obtaining a sample comprising the target particles or target cells and the contaminants; b) separating the target particles or target cells from the contaminants by: i) applying the sample to one or more sample inlets on the microfluidic particle separation device of any one of claims 1 to 64; ii) flowing the sample to the outlets on the particle separation device of any one of claims 1 to 64; and iii) obtaining a product enriched in target particles or target cells from one or more or outlets while removing the contaminants.

66. The method of claim 65, wherein the target particles or target cells have a size larger than a critical size of the array of obstacles and at least some contaminants have sizes smaller than the critical size of the array of obstacles and wherein target cells or target particles flow to the one or more product outlets where a product enriched in target cells or target particles is obtained and contaminants with a size smaller than the critical size of the array of obstacles flow to one more waste outlets.

67. The method of claim 65 or 66, wherein the flow rate of the cartridge is about 400 mL per hour.

68. The method of claim 65 or 66, wherein the flow rate of the cartridge is at least about 100 mL per hour or greater.

69. The method of claim 65 or 66, wherein the flow rate of the cartridge is at least about 300 mL per hour or greater.

70. The method of claim 65 or 66, wherein the flow rate of the cartridge is about 1000 mL per hour.

71. The method of claim 65 or 66, wherein the internal pressure of the cartridge is at least about 1.5 pounds per square inch or greater.

72. The method of claim 65 or 66, wherein the internal pressure of the cartridge is about 15 pounds per square inch.

73. The method of claim 65 or 66, wherein the internal pressure of the cartridge is about 50 pounds per square inch or less.

74. The method of claim 65 or 66, wherein the internal pressure of the cartridge is from about 10 pounds per square inch to about 20 pounds per square inch.

75. The method of any one of claims 65 to 74, wherein the sample is blood or a blood related product.

76. The method of any one of claims 65 to 74, wherein the sample is an apheresis or leukapheresis sample.

77. The method of any one of claims 65 to 76, wherein the sample comprises platelets as contaminants.

78. The method of claim 77, wherein the method results in the removal of at least 80% of the platelets from the sample.

79. The method of claim 77, wherein the method results in the removal of at least 90% of the platelets from the sample.

80. The method of claim 77, wherein the method results in the removal of at least 95% of the platelets from the sample.

81. The method of any one of claims 65 to 80, wherein the enriched target cells comprise leukocytes.

82. The method of any one of claims 65 to 80, wherein the enriched target cells comprise stem cells.

83. The method of any one of claims 65 to 80, wherein the enriched target cells comprise peripheral blood mononuclear cells.

84. The method of claim 83, wherein the peripheral blood mononuclear cells comprise CD3+ cells.

85. The method of any one of claims 65 to 84, further comprising genetically engineering the enriched target cells, to obtain genetically engineered target cells.

86. The method of claim 85, wherein said genetic engineering comprises transfecting or transducing the target cells with a recombinant nucleic acid.

87. The method of claim 85 or 86, wherein the enriched target cells or genetically engineered target cells are expanded by culturing them in vitro.