KR102483442B1 - A method and platform for intracellular delivery using droplet microfluidics - Google Patents

Abstract

The present invention is an intracellular substance transfer method, comprising the steps of forming a droplet composed of a substance to be delivered and a cell; and delivering the substance to be delivered into the cell by applying strain to the formed droplet.

Description

A method and platform for intracellular delivery using droplet microfluidics}

The present invention relates to a droplet-based microfluidic intracellular mass transfer method and platform, and more particularly, to deliver various substances into various cells uniformly and with high efficiency using droplets and using only a very small amount of a delivery substance. It relates to a droplet-based microfluidic intracellular mass transfer method and platform.

Intracellular mass transfer is one of the most basic experiments in cell engineering, and materials are usually delivered using carriers or by creating nanopores in cell membranes/nuclear membranes.

Virus- or Lipofectamine-based carrier techniques can deliver materials with high efficiency when optimized, but have problems such as safety, slow delivery speed, labor/cost-intensive carrier preparation process, and low reproducibility.

On the other hand, methods of making nanopores by applying energy to cell membranes (e.g., electroporation or microneedle) have the advantage of being able to deliver relatively various materials to various cell lines.

However, low cell viability due to the invasiveness of the method, denaturation of the delivery material, and low throughput have been pointed out as major limitations.

To solve these problems, the use of microfluidic devices that can process a large amount of cells is remarkable. Representatively, there is a platform that creates a bottleneck section in microtubules and creates nanopores in cell membranes through physical deformation of cells when cells pass through the bottleneck section.

However, this approach has major disadvantages such as clogging of the bottleneck itself during the experiment and inconsistent mass transfer efficiency.

Therefore, it is urgent to develop an innovative “next-generation intracellular substance delivery platform” that can deliver various substances uniformly and with high efficiency while maintaining the high processing capabilities of the microfluidic device.

US 2014/0287509 A1 (2014.09.25.)

The present invention has been devised to solve various conventional problems as described above, and a new droplet-based microfluidic intracellular mass transfer method and platform that minimizes the amount of materials to be delivered simultaneously while increasing the efficiency of intracellular mass transfer. want to provide

The present invention is an intracellular substance transfer method, comprising the steps of forming a droplet composed of a substance to be delivered and a cell; and delivering the substance to be delivered into the cell by applying deformation to the formed droplet.

The droplet may be formed by flowing a first-phase fluid containing the cells and substances onto a second-phase fluid that is immiscible with the first-phase fluid.

More specifically, in the method of applying deformation to the droplet, the deformation may be applied to the droplet by narrowing the direct size of the channel through which the diameter flows.

As another aspect, in the method of applying deformation to the droplet, a vortex may be formed in the fluid through which the droplet flows.

In addition, the present invention is an intracellular substance delivery platform, comprising: a channel for flowing a droplet containing a substance to be delivered and a cell; and a transforming means for delivering the material to be delivered into the cells by applying physical strain to the liquid droplets flowing in the channel.

Specifically, the deformation means is a microchannel having a smaller diameter than the channel, and the deformation means may be a narrower microchannel.

1 is a diagram illustrating a droplet changing process using a method for forming a droplet and a bottleneck channel according to an embodiment of the present invention.
2 is a diagram illustrating a droplet change process using a 'T' shaped cell delivery platform according to an embodiment of the present invention.
3 is a diagram illustrating a droplet changing process using a '+' shaped cell delivery platform according to an embodiment of the present invention.

Hereinafter, a preferred embodiment of the inertial-based intracellular delivery microfluidic platform according to the present invention will be described in detail based on the accompanying drawings. For reference, the terms and words used in this specification and claims should not be construed as being limited to their ordinary or dictionary meanings, and the inventors use the concept of terms appropriately to describe their invention in the best way. It should be interpreted as meaning and concept consistent with the technical idea of the present invention based on the principle that it can be defined. In addition, the embodiments described in this specification and the configurations shown in the drawings are only one of the most preferred embodiments of the present invention, and do not represent all of the technical ideas of the present invention. It should be understood that there may be equivalents and variations.

Unlike the cell-squeezing method in which cells and substances to be delivered are compressed into a very narrow channel (bottleneck channel), the present invention uses droplets and applies deformation to the droplets to form micropores in the cells in the droplets. generate and transmit material.

In the present invention, droplets are formed by flowing a fluid (first phase) containing cells and a delivery material and a delivery fluid (second phase) that do not mix with each other through a T-shaped channel. Cells are captured to maximize delivery efficiency while minimizing the amount of delivery material used.

The reason for using droplets in the present invention is, first, that a very small liquid droplet volume (~100 pL/cell) can be used. It has the advantage of being able to use only a very small amount of the transmitter because it is determined.

In addition, due to the very small droplet volume, the captured cells are placed in a high-concentration transport material environment, and the high-concentration transport material is transferred to the cells due to the secondary flow generated in the droplet when passing through a section where deformation is applied to the droplet, such as a bottleneck or a microchannel. is effectively delivered to the inside, which can be achieved with high delivery efficiency. This enables the delivery of macromolecules (eg, larger nanoparticles, plasmid DNA), which was not possible in the prior art.

Furthermore, cell-squeezing, which is a prior art, shows a large pipe blockage at the bottleneck and uses a high flow rate to solve it. In this case, it leads to low cell viability and results in the use of more than the appropriate amount of delivery material due to high flow rate. However, when droplets are used, the clogging of microtubules can be minimized by trapping various debris that clog the microtubules in the droplets, and clogging can also be minimized by the difference in hydrophilicity/hydrophobicity between the droplets and the walls of the microtubules.

Hereinafter, the present invention will be described in more detail through an embodiment of the present invention. However, the scope of the present invention is not limited thereto.

In one embodiment of the present invention, experiments were conducted on the K562 cell line to use the possibility of cancer immunotherapy in the future. 2,000 kDa FITC-dextran, which mimics a large protein, was used as the transport material, and the microfluidic chip was made using general photolithography and soft lithography methods.

The droplet-based microfluidic intracellular mass transfer platform according to an embodiment of the present invention delivers intracellular materials through three steps: 1) droplet formation (cell entrapment) step 2) droplet deformation application (intracellular mass transfer) ) 3) cell recovery. The core of these is droplet formation and droplet deformation of 1) and 2), and the core of the present invention lies therein.

In detail, a method for delivering microfluids into cells based on droplets according to an embodiment of the present invention includes forming droplets composed of a material to be delivered and cells; At least a part of the cell is deformed by applying a physical strain to the formed droplet to generate a nanopore in the cell membrane of the cell, and delivering the substance to be delivered into the cell through the nanopore; and Closing at least a portion of the nanopores generated in the cell membrane; wherein the droplet includes a first phase fluid containing the substance to be delivered and cells, and the first phase fluid comprises an aqueous phase that is non-mixed with the oil phase. It may be characterized by including.
In addition, the droplet-based intracellular mass delivery platform according to an embodiment of the present invention includes a main channel through which a substance to be delivered and cells flow with a first phase fluid; one or more sub-channels branched from and connected to an end of the main channel to transfer the second-phase fluid to the end of the main channel; a droplet forming unit in which an end of the main channel and an end of the sub-channel cross each other and droplets are formed; a first channel connected to the droplet forming unit and through which the droplet formed at the crossing unit flows with the second-phase fluid; and a transforming means for transferring the material to be delivered into the cells by applying physical deformation to the liquid droplets flowing in the first channel, wherein the liquid droplets contain the material to be delivered and the fluid in the first phase containing the cells, It may be formed by flowing the first phase fluid and the immiscible second phase fluid, wherein the first phase fluid includes an oil phase and an immiscible aqueous phase.

1 is a diagram illustrating a droplet changing process using a method for forming a droplet and a bottleneck channel according to an embodiment of the present invention.

Referring to FIG. 1A, a first fluid of cells and a transmitter is produced by flowing a fluid of a first phase including cells and a transmitter and another fluid of a second phase that is not mixed with the first phase at one channel point. 2 Droplets in the fluid. This is usually a flow-focusing method, and other various methods of forming droplets may be used, and the scope of the present invention belongs thereto.
The first phase fluid containing the delivery material and cells is a hydrophilic liquid and may be, for example, an aqueous phase. In addition, the second phase fluid that is not mixed with the first phase fluid is a hydrophobic liquid and may be, for example, an oil phase. Therefore, a substance to be delivered and cells contained in the first-phase fluid, which is the aqueous phase, may meet the second-phase fluid, which is the oil phase, to form droplets, and the droplets containing the first-phase fluid may form droplets in the second phase fluid. It is possible to maintain the shape of a droplet in a fluid.
The first phase fluid including the delivery material and the cells may flow in the main channel and meet the second phase fluid flowing in the sub channel connected to the end of the main channel to form droplets in the droplet forming unit.
Referring to Figure 1b, it can be seen that the delivery material and the cells are uniformly well trapped in the droplet.

delete

Figure 1c is a photograph explaining the occurrence of droplet deformation by passing through a microchannel narrower than the flowing channel.

Referring to FIG. 1C , it can be seen that the droplets are deformed as the cells pass through the microchannel with a diameter that is rapidly narrower than the channel through which the cells flow, and then flow in the opposite direction. At this time, when the cell passes through the bottleneck section or the channel of narrowing diameter shown in FIG. 1B, nanopores are created in the cell membrane according to the cell deformation and enter the cell through strong secondary flow in the droplet and solution exchange inside and outside the cell membrane. The target material enters.

Afterwards, referring to FIG. 1D , the nanopores generated in the cell membrane are all closed within about 1 minute, and through this, the entire intracellular mass transfer process is completed. Thereafter, the cells were recovered through the droplet disruption process, and the delivery efficiency was quantitatively measured using a flow cytometer.

1E to 1G are quantitative analysis results using a flow cytometer, and comparative experimental results of delivery efficiency and cell viability, respectively.

Referring to FIGS. 1E to 1G , it was shown that intracellular substance delivery was successfully possible with a delivery efficiency of up to 95% by processing tens of thousands of FITC-dextran per minute, even though only a very small amount of 2,000 kDa FITC-dextran was used. In future research, stem cells or immune cells can be used, and high-efficiency mass transfer can be realized simultaneously by adjusting and optimizing the size and flow rate of the bottleneck gap in the microtubule, as well as the length and number of bottleneck sections.

When a cell passes through a narrow gap using the platform of the present invention, various materials can be delivered through the nanopores created. In this case, the various materials are not limited to gene editing materials, plasmids, nanoparticles, proteins, nucleic acids, etc., and include all nanobiomaterials that can be put into cells in biology related to cells. Therefore, the microfluidic device of the present invention means that a large amount (tens of thousands or more per minute) is delivered into cells despite not using a vector such as a virus.

In the above-described embodiment, the droplet deformation means has a channel structure (fine channel, bottleneck channel, narrowing diameter channel), but other than that, the droplet deformation means may be an inertial effect such as a vortex.
Specifically, according to an embodiment of the present invention, the transforming means for causing the inertial effect may include second channels connected to both sides of the ends of the first channel; and a barrier rib formed to face the first channel and colliding with the liquid droplet, wherein the droplet collides with the barrier rib and causes deformation.
The barrier rib may further include a protruding groove, and the liquid drop may collide with the protruding groove to cause deformation.
In addition, according to another embodiment of the present invention, the transforming means for causing the inertial effect may include second channels connected to both sides of the ends of the first channel; and a third channel formed to face the first channel and through which the second-phase fluid flows, wherein the second-phase fluid flows in a direction opposite to the flow direction of the fluid in the first channel. It may be characterized by fluidity.

That is, in this case, the droplet colliding with the barrier rib (for example, the channel wall or the fluid wall) causes deformation according to the inertial induction effect, and then the droplet deformation occurs again even in the collapse of the vortex. Through this, the substance in the droplet is delivered into the cell.

2 is a diagram illustrating a droplet change process using a 'T' shaped cell delivery platform according to an embodiment of the present invention.

Referring to FIG. 2 , the cell delivery platform includes a first channel 100 forming a path through which fluid including droplets moves; a second channel 200 vertically extending from the end of the first channel 100 to both sides of the first channel 100; and a cell accelerating unit 300 provided in the first channel 100 to control the velocity of the fluid in the first channel 100 .

According to the present invention, the flow rate and inertia of the fluid passing through the first channel 100 are controlled through the cell accelerating means 300 to induce barrier collision and vortex formation, thereby inducing highly efficient cell delivery.
The cell delivery platform may further include protruding grooves 400 . The protrusion groove 400 may be formed to face the first channel 100 and protrude outward in the same direction as the flow direction of the fluid moving along the first channel 100 .
Since the protruding groove 400 is formed to face the first channel 100, at least a portion of liquid droplets included in the fluid moving along the first channel 100 may collide with the protruding groove 400. there is. A droplet colliding with the protruding groove 400 may be deformed, and after leaving the protruding groove 400, the droplet may be deformed again even when the vortex collapses. Through such droplet deformation, substances in the droplets can be delivered into cells.
More specifically, droplet injection-droplet alignment-collision-cell deformation-mass transfer is induced using only inertia and inertial flow, so that various materials (e.g., : Gene editing materials, plasmids, etc.) can be delivered into cells in large quantities (millions or more per minute) without the use of vectors such as viruses.

delete

3 is a diagram illustrating a droplet changing process using a '+' shaped cell delivery platform according to an embodiment of the present invention.

Referring to FIG. 3 , the '+' shaped cell delivery platform includes a first channel 100 through which a fluid containing droplets flows; a second channel 200 vertically crossing the first channel 100; and a first cell accelerating unit 300 provided on one side of the first channel 100 to control the velocity of the fluid in the first channel 100 in a first direction.

According to one embodiment of the present invention, the fluid in the first channel 100 flows oppositely to the point where it vertically intersects the second channel 200, and the first cell acceleration means 300 is Applying kinetic energy to the cell on the first channel 100 to deform the cell wall to the level of forming nanopores in the cell by the vortex formed at the point where the first channel 100 and the second channel 200 vertically intersect. It is characterized by doing.

In addition, a second cell accelerating unit 300' for controlling the fluid speed in the first channel 100 in a second direction may be further included on the other side of the first channel 100. The fluid whose direction and speed are controlled by the second cell acceleration unit 300' may move along the third channel. The fluid moving along the third channel may move in a second direction, and the second direction may be opposite to the first direction.

In particular, the first and second cell accelerating means (300, 300') controlled in opposite directions in the first channel (100) form a vortex at the point where the first channel (100) and the second channel (200) cross each other perpendicularly. It can be formed, and the cell membrane can be deformed by applying a physical strain to the cell by the inertial force and inertial flow phenomenon generated accordingly.

Like the above-mentioned 'T' shape, the '+' shaped cell delivery platform induces droplet injection - droplet alignment - collision - cell deformation - mass transfer using only inertia and inertial flow, thereby separate active cell delivery Without the use of any means, various substances (eg, gene-cleaving materials, plasmids, etc.) are delivered into cells in large quantities (more than millions per minute) through nanopores without the use of vectors such as viruses.

As described above, the droplet colliding with the channel wall or the fluid wall causes deformation according to the inertial induction effect, and then the droplet deformation occurs again even in the collapse of the vortex. Through this, the substance in the droplet can be delivered into the cell.

The present invention described above is not limited by the above-described embodiment and the accompanying drawings, and various substitutions, modifications, and changes are possible within the scope of the technical spirit of the present invention. It will be clear to those skilled in the art.

Claims (12)

As a method of intracellular substance transfer,
Forming a droplet composed of a material to be delivered and a cell;
By applying a physical strain to the formed droplet, at least a part of the cell is deformed to generate a nanopore in the cell membrane of the cell, and delivering the substance to be delivered into the cell through the nanopore; And
Including; closing at least a portion of the nanopores generated in the cell membrane of the cell,
The intracellular mass transfer method, characterized in that the droplet includes a first phase fluid containing the substance to be delivered and cells, and the first phase fluid includes an aqueous phase that is not mixed with the oil phase. According to claim 1,
The intracellular mass transfer method according to claim 1 , wherein the droplets are formed by flowing the first-phase fluid into a second-phase fluid unmixed with the first-phase fluid. According to claim 1,
The method of applying deformation to the droplet is an intracellular mass transfer method, characterized in that the deformation is applied to the droplet by narrowing the direct size of the channel through which the diameter flows. According to claim 1,
The method of applying deformation to the droplet is an intracellular mass transfer method, characterized in that the deformation is applied to the droplet in such a manner as to form a vortex in the fluid in which the droplet flows. As an intracellular substance delivery platform,
a main channel through which substances and cells to be delivered flow with the first phase fluid;
one or more sub-channels branched from and connected to an end of the main channel to transfer the second-phase fluid to the end of the main channel;
a droplet forming unit in which an end of the main channel and an end of the sub-channel cross each other and droplets are formed;
a first channel connected to the droplet forming unit so that droplets formed in the droplet forming unit flow with the second phase fluid; and
A deformation means for transferring the substance to be delivered into the cells by applying physical deformation to the liquid droplets flowing in the first channel;
The droplet is formed by flowing a first-phase fluid containing the substance to be delivered and cells into a second-phase fluid that is unmixed with the first-phase fluid,
The intracellular mass transfer platform, characterized in that the first phase fluid comprises an aqueous phase that is not mixed with the oil phase. According to claim 5,
The additive transformation means is an intracellular substance delivery platform, characterized in that the microchannel having a narrower diameter than the channel. According to claim 5,
The transforming means is an intracellular substance delivery platform, characterized in that the width is narrowed microchannel. According to claim 5,
The transformation means,
a second channel connected to both ends of the first channel; and
A partition wall formed to face the first channel and colliding with the droplet,
The intracellular mass transfer platform, characterized in that the droplet collides with the partition wall and deformation occurs. According to claim 8,
The partition wall further includes a protruding groove,
Intracellular mass transfer platform, characterized in that the droplet collides with the protruding groove and deformation occurs. According to claim 8,
Intracellular substance delivery platform, characterized in that the transforming means is a T-shaped structure. According to claim 5,
The transformation means,
a second channel connected to both ends of the first channel; and
A third channel formed to face the first channel and through which the second phase fluid flows,
The intracellular mass transfer platform, characterized in that the second phase fluid in the third channel flows in a direction opposite to the flow direction of the fluid in the first channel. According to claim 11,
The transforming means is an intracellular substance delivery platform, characterized in that the + type structure.

Images