Intracellular substance transport platform of droplet base

Abstract

To provide an intracellular substance transport platform of a droplet base which can convey a substance highly efficiently to various cells.SOLUTION: An intracellular substance transfer platform is a device which transmits an intracellular substance and includes: one or more main channels which are extended in a first direction from one end to the other end, and are provided with a passage of fluid for the inside thereof; a first supply part which is connected to one end of the main channel, and injects first fluid containing the cell and a substance; and a second supply part which is connected to one end of the main channel, and injects second fluid which is not mixed with the first fluid, where one or more compression blocks are provided for the inside of the main channel.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a droplet-based intracellular substance transport platform, and more particularly, to a droplet-based intracellular substance transport platform capable of effectively transporting substances into cells with high efficiency without damaging the cells.

Background Art

[0002] Intracellular substance transport is one of the most basic experiments in cell engineering, and usually uses carriers or creates nanopores in the cell membrane / nuclear membrane to transport substances.

[0003] Carrier technologies centered around viruses or Lipofectamine can achieve high-efficiency substance transport when optimized, but there are problems such as safety, slow transport speed, labor / cost-intensive carrier preparation processes, and low reproducibility.

[0004] On the other hand, methods of creating nanopores by applying energy to the cell membrane (e.g., Electroporation or microneedle) have the advantage of being able to transport relatively diverse substances to various cell lines. However, these methods have been pointed out as having major limitations such as low cell viability due to invasiveness, denaturation of transported substances, and low throughput.

[0005] To solve these problems, the use of microfluidic devices capable of processing a large number of cells has been prominent. Typically, there is a platform that creates a bottleneck section in a microtube, and creates nanopores in the cell membrane through physical deformation of the cells when the cells pass through the bottleneck section. However, this approach has major drawbacks such as clogging of the bottleneck section itself during the experiment and non-uniform substance transport efficiency.

[0006] Therefore, various studies have been conducted on methods that can transport various substances to various cells while transporting substances with high efficiency without damaging the cells.

Summary of the Invention

Problems to be Solved by the Invention

[0007] An object of the present invention is to provide a droplet-based intracellular substance transport platform capable of transporting substances to various cells with high efficiency.

[0008] Another object of the present invention is to provide an intracellular substance transport platform that reduces unnecessary loss of the substance to be transported and can continuously process a large number of cells.

Means for Solving the Problems

[0009] According to one aspect of the present invention, an embodiment of the present invention includes an intracellular substance transport platform.

[0010] In one embodiment, as an apparatus for transporting intracellular substances, one or more main channels extending in a first direction from one end to the other end and having a fluid passage therein; a first supply unit connected to one end of the main channel for injecting a first fluid containing the cells and the substance; and a second supply unit connected to one end of the main channel for injecting a second fluid that is not mixed with the first fluid; and an intracellular substance transport platform including one or more compressing blocks provided inside the main channel.

[0011] In one embodiment, the first fluid can be an aqueous phase and the second fluid can be an oil phase.

[0012] In one embodiment, the first supply unit extends parallel to the first direction and is connected to one end of the main channel, and the second supply unit can be connected to the first supply unit at an angle at one end of the main channel.

[0013] In one embodiment, the first fluid is sent from the first supply unit to one end of the main channel, and the second fluid is sent from the second supply unit to one end of the main channel, but is sent at an angle with respect to the flow direction of the first fluid. The first fluid containing cells and substances is formed into droplets, and the droplets can pass through the main channel within the second fluid.

[0014] In one embodiment, the second supply unit includes first and second supply channels, and the first and second supply channels can be connected to one end of the main channel to form a junction.

[0015] In one embodiment, the compression block is provided at a portion separated from one end of the main channel by a first length, and the first length can be 30% to 90% with respect to the total length from one end to the other end of the main channel.

[0016] In one embodiment, the flow rate of the second fluid in the main channel can be 1 mL / h to 70 mL / h.

[0017] In one embodiment, it can further include one or more sub-channels connected to the main channel at an angle and through which the second fluid flows.

[0018] In one embodiment, the sub-channel is connected to the main channel at a portion separated from one end of the main channel by a second length, and includes first and second sub-channels, and the first and second sub-channels can be respectively connected to one side and the other side of the main channel.

[0019] In one embodiment, the inner diameter of the sub-channel is 20% to 150% of a first diameter which is the inner diameter of the main channel, and the second fluid can be sent to the main channel through the sub-channel.

[0020] In one embodiment, the inner diameter of the sub-channel is from 20 μm to 200 μm, the first diameter which is the inner diameter of the main channel is from 20 μm to 1.5 mm, and the second fluid can be sent to the main channel through the sub-channel.

[0021] In one embodiment, the flow rate of the second fluid in the main channel is from 1 mL / h to 45 mL / h, and the flow rate of the second fluid in the sub-channel can be from 1 mL / h to 30 mL / h.

[0022] In one embodiment, the compression block is installed at a portion separated from one end of the main channel by a first length, and the first length can be from 1.1 times to 5 times that of the second length.

[0023] In one embodiment, the first diameter is from 20 μm to 1.5 mm, the first length is from 0.1 mm to 30 mm, and the second length can be from 0.1 mm to 1.5 mm.

[0024] In one embodiment, the length of the compression block in the direction parallel to the first direction can be from 10 μm to 200 μm.

[0025] In one embodiment, the length of the compression block in the direction parallel to the first direction can be from 20 μm to 100 μm.

[0026] In one embodiment, the gap between the compression block and the inner surface of the main channel is provided by a second diameter, and the second diameter can be from 0.1% to 85% of the first diameter which is the inner diameter of the main channel.

[0027] In one embodiment, the second diameter can be from 2 μm to 17 μm.

[0028] In one embodiment, the height of the compression block in the direction perpendicular to the first direction can be from 15% to 99% of the first diameter.

[0029] In one embodiment, the height of the compression block in the direction perpendicular to the first direction can be from 3 μm to 1.5 mm.

[0030] In one embodiment, the main channel includes an inlet portion connected to the first supply portion; a branch portion connected to the inlet portion but branched into a plurality of passages; and an outlet portion connected to the branch portion. The branch portion includes a plurality of passages provided with a diameter smaller than that of the inlet portion and the outlet portion, and links through which the plurality of passages are branched or connected to each other. The compression block can be installed in the branch portion or the outlet portion.

[0031] In one embodiment, droplets formed of the cells, substances, and the first fluid in the main channel are transported from the inlet portion through the branch portion to the outlet portion through the second fluid. The droplets installed in the inlet portion can be installed with an average diameter larger than that of the droplets installed in the branch portion or the outlet portion.

[0032] In one embodiment, the branch portions can be installed symmetrically with respect to each other about a virtual reference line connecting the inlet portion and the outlet portion.

[0033] In one embodiment, the main channel can further include one or more curved passages installed in the inlet portion or the branch portion.

[0034] In one embodiment, the substance can include any one or more of nucleic acids, proteins, transcription factors, vectors, plasmids, gene scissors substances, and nanoparticles.

Advantages of the Invention

[0035] As described above, according to the present invention, it is possible to provide a highly efficient intracellular substance transport platform that can transport substances into cells while preventing damage to the cells for various cells.

[0036] In addition, according to the present invention, a method is provided for transporting substances into cells without any type restrictions using a new method, and a mass-production-friendly intracellular substance transport platform is provided that can transport various substances into a single cell or a single substance into various cells.

Brief Description of Drawings

[0037]

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Embodiments for Carrying Out the Invention

[0038] Specific details of other embodiments are included in the detailed description and the drawings.

[0039] The advantages and features of the present invention, and the methods for achieving them, will become clear by referring to the embodiments described in detail below together with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below and can be realized in various forms. Unless otherwise specified in the following description, all numbers, values, and / or expressions representing the components, reaction conditions, and contents of the components included in the present invention are approximate values reflecting various uncertainties in the measurements that occur when obtaining these values among those that are essentially different from each other, and thus should be understood to be modified by the term "about" in all cases. Also, when a numerical range is disclosed in this description, these ranges are continuous and include all values from the minimum value to the maximum value of these ranges unless otherwise indicated. Further, when these ranges refer to integers, they include all integers from the minimum value to the maximum value unless otherwise indicated.

[0040] Also, when the scope in the present invention is described for a variable, the corresponding variable is understood to include all values within the described scope including the endpoints of the described scope. For example, the range of "from 5 to 10" includes not only the values of 5, 6, 7, 8, 9, and 10, but also any sub-ranges such as from 6 to 10, from 7 to 10, from 6 to 9, from 7 to 9, etc., and also any values between any integers within the category of the described range, such as 5.5, 6.5, 7.5, from 5.5 to 8.5, and from 6.5 to 9. For example, the range of "from 10% to 30%" includes not only the values such as 10%, 11%, 12%, 13%, etc. and all integers up to 30%, but also any sub-ranges such as from 10% to 15%, from 12% to 18%, from 20% to 30%, etc., and also any values between any integers within the category of the described range, such as 10.5%, 15.5%, 25.5%, etc.

[0041] FIG. 1 is a schematic diagram of an intracellular substance transport platform according to an embodiment of the present invention. FIG. 2 is a drawing showing an intracellular substance transport platform according to an embodiment of the present invention. FIG. 3 is a cross-sectional view of FIG. 2.

[0042] One embodiment of the present invention relates to an intracellular substance transport platform (100) for transporting substances into cells, including one or more main channels (110) extending in a first direction (x) from one end to the other end and having a fluid passage therein; a first supply unit (120) connected to one end of the main channel (110) for injecting the cells (10), substances (20), and a first fluid; and a second supply unit (130) connected to one end of the main channel (110) for injecting a second fluid that is not mixed with the first fluid.

[0043] One or more compressing blocks (150) are provided inside the main channel (110), the first fluid can be a water phase, and the second fluid can be an oil phase.

[0044] The intracellular substance transport platform (100) according to this embodiment can form droplets using a first fluid and a second fluid that do not mix with each other, and capture cells (10) and a substance (20) intended to be transported into the cells within the droplets (50). The droplets (50) are formed of the same substance as the first fluid, and the cells (10) and the substance (20) can be moved while maintaining the shape of the droplets (50) within the second fluid.

[0045] Generally, methods for transporting substances into cells, such as using electroporation to damage cell membranes, leave permanent traces on the cells. In addition, methods of directly applying an external force to cells while flowing cells and substances in a fluid to transport the substances into the cells have drawbacks such as causing damage to the cells or low efficiency of the substances to be transported into the cells.

[0046] On the other hand, the intracellular substance transport platform (100) according to this embodiment can effectively transport the substance (20) into the cells while preventing damage to the cells (10) by using the droplets (50). In the intracellular substance transport platform (100) of the present invention, the cells (10) and the substance (20) are protected by the droplets (50) and at the same time can flow efficiently within the fluid. In addition, by maintaining the proximity of the distance between the cells (10) and the substance (20) by the droplets (50), the substance (20) can be efficiently transported into the cells (10) without being lost to the outside.

[0047] The intracellular substance transport platform (100) according to this embodiment can further include a compression block (150). The compression block (150) is provided inside the main channel (110) and can reduce the substantial diameter inside the main channel (110). Specifically, the droplet (50) passes through the main channel (110) at a predetermined speed by the second fluid, but the droplet (50) passes through while being compressed at the portion where the compression block (150) is formed. During the process of the droplet (50) passing through the compression block (150), deformation of the cell (10) occurs, and nanopores for the substance (20) to pass through the cell membrane or nuclear membrane are formed, and through the nanopores, the substance (20) can be effectively transported into the cell (10). Also, the droplet (50) that has passed through the compression block (150) restores its shape, and a vortex is formed inside the droplet (50), and the vortex provides a driving force for the substance (20) to be transported into the cell (10), and the efficiency of intracellular substance transport can be improved. Also, the deformed cell (10) after passing through the compression block (150) restores its original shape and contains the substance (20) inside.

[0048] The droplet (50) can be provided with a very small volume, about 100 pL / cell. Inside the main channel (110), the amount of the substance (20) transported can be determined according to the volume of the droplet (50). Usually, in an intracellular substance transport system, the distance between the cell and the substance to be transported is far, the efficiency of intracellular substance transport decreases, or a large amount of substance is consumed to increase the concentration of the substance around the cell, and other substances are discarded, so an inefficient process has been carried out. However, in the intracellular substance transport platform (100) according to this embodiment, the cell (10) and the substance (20) exist in a small system of the droplet (50), and since the cell (10) and the substance (20) are located close to each other, even a very small amount of the substance (20) can be efficiently transported into the cell (10), and wasteful loss of the substance (20) can be reduced.

[0049] In the intracellular substance transport platform (100) according to this embodiment, the volume of the droplet (50) is small, and the cell (10) can be set in an environment of a high-concentration substance (20) within the droplet (50). When nanopores are formed by the deformation of the cell (10) when the droplet (50) passes through the compression block (150), it is possible to effectively transport substances into the cell (10) even with a very small volume of the droplet (50). Furthermore, the shape of the droplet (50) is maintained even after passing through the compression block (150), and secondary flows such as vortices within the droplet (50) can provide a driving force for more effective transport of the substance (20) into the cell (10).

[0050] The intracellular substance transfer platform (100) in this embodiment is applicable to large macromolecules such as large nanoparticles and plasmid DNA, which were impossible with conventional techniques, and can prevent clogging phenomena caused by the method of passing cells through microtubes or their narrow passages (bottlenecks) in conventional techniques, as well as the problem of reduced cell viability with high-speed flow.

[0051] In the intracellular substance transfer platform (100) according to this embodiment, the inner wall of the main channel (110) may be composed of a hydrophobic material. In the main channel (110), the second fluid that flows the droplet (50) is hydrophobic and can easily flow within the main channel (110). Also, the droplet (50) is hydrophilic, and due to the difference in hydrophilicity and hydrophobicity with the main channel (110), clogging phenomena can be minimized.

[0052] Since the intracellular substance transfer platform (100) according to this embodiment does not cause clogging phenomena, intracellular substance transfer can be performed with high efficiency, and by moving the cells at an appropriate flow rate, high cell viability can be demonstrated.

[0053] The first supply unit (120) extends parallel to the first direction (x) and is connected to one end of the main channel (110). Also, the second supply unit (130) may be connected to one end of the main channel (110) at an angle with respect to the first supply unit (120). For example, the first supply unit (120) and the main channel (110) may be extended and horizontally connected to each other, and the second supply unit (130) may be connected to the main channel (110) at a predetermined angle. More specifically, the second supply unit (130) may be connected perpendicular to the main channel (110).

[0054] One end of the first supply unit (120) is connected to one end of the main channel (110), and the other end of the first supply unit (120) may be connected to the first supply chamber (121). The first supply chamber (121) is provided with a first fluid, cells (10), and a substance (20), and the first fluid, cells (10), and substance (20) can be sent to the first supply unit (120).

[0055] One end of the second supply unit (130) is connected to one end of the main channel (110), and the other end of the second supply unit (130) may be connected to the second supply chamber (131). The second supply chamber (131) is provided with a second fluid, and can be sent to the second supply unit (130) at a predetermined flow rate.

[0056] The first fluid, cells (10), and substance (20) are sent from the first supply unit (120) to one end of the main channel (110), and the second fluid is sent from the second supply unit (130) to one end of the main channel (110) at an angle with respect to the flow direction of the first fluid. As a result, the cells (10) and substance (20) contained in the first fluid may be formed as droplets (50). These droplets (50) can pass through the compression block (150) of the main channel (110) while flowing from one end to the other end of the main channel (110) together with the second fluid.

[0057] By controlling the flow rate of the first fluid in the first supply unit (120) or the flow rate of the second fluid in the second supply unit (130), the size and moving speed of the droplet (50) can be controlled. The intracellular substance transmission platform (100) according to the present embodiment can improve the efficiency of intracellular substance transmission by controlling the size and moving speed of the droplet (50) according to the types of cells (10) of various sizes or substances (20) to be transmitted.

[0058] Specifically, the second supply unit (130) can include first and second supply channels. The first and second supply channels may be a pair of fine channels respectively provided on one side and the other side with respect to the first supply unit (120). The first and second supply channels are connected to one end of the main channel (110) and can form a cross-shaped, Y-shaped, or T-shaped junction (140).

[0059] Specifically, the first supply unit (120) can be extended and connected to the main channel (110). At the portion where the first supply unit (120) and the main channel (110) are connected, the first supply channel may be vertically connected from above, and the second supply channel may be vertically connected from below. The first fluid sent through the first supply unit (120) is formed as a droplet (50) by the second fluid sent from the first and second supply channels at the junction (140), and the second fluid can flow into the main channel (110) together with the droplet (50).

[0060] A compression block (150) may be provided on the inner surface of the main channel (110). The compression block (150) is provided on either the upper, lower, or side surface of the inner surface of the main channel (110) and can reduce the passage of the main channel (110). The surface of the compression block (150) may be formed to be hydrophobic so that the flow of the second fluid is not hindered.

[0061] The compression block (150) may be approximately box-shaped with a height (S1) protruding from the upper part of the inner surface of the main channel (110) and a length (S2) parallel to the first direction (x). For example, the compression block (150) may be provided in a shape with rounded corners, which can prevent the formation of vortices at the corners of the compression block (150).

[0062] The second fluid and the droplet (50) can pass through the compression block (150) inside the main channel (110). When passing through the compression block (150), due to the reduced flow path and increased flow velocity caused by the compression block (150), the droplet (50) may deform, and the cell (10) provided inside the droplet (50) may also deform. Nanopores may be formed in the cell membrane of this cell (10), and the substance (20) can be transmitted into the cell through these nanopores. After the droplet (50) passes through the compression block (150), the shape changed by the compression block (150) can recover before passing through the compression block (150). At this time, the cell (10) inside the droplet (50) can also deform with the droplet (50) and recover to its original shape. During the process of the shapes of the droplet (50) and the cell (10) recovering, secondary vortices may be formed inside the droplet (50). This vortex can promote the movement of the substance (20), thereby potentially promoting the transmission of the substance (20) into the cell (10).

[0063] When the inner diameter of the main channel (110) is the first diameter (a) and the gap between the compression block (150) and the inner surface of the main channel (110) is the second diameter (b), the second diameter (b) may be provided in the range of 0.1% to 85% with respect to the first diameter (a). If the second diameter (b) is less than 0.1% with respect to the first diameter (a), there may be damage to the cell (10) passing through the main channel (110) and the compression block (150). If it exceeds 85%, the droplet (50) may not be sufficiently pressurized by the compression block (150), and the efficiency of intracellular substance transmission may decrease.

[0064] The second diameter (b) may range from 2 μm to 17 μm. By setting the second diameter (b) within the aforementioned range, the droplet (50) can be pressurized within a range that does not damage the cell (10), and by causing physical deformation of the cell (10), the efficiency of intracellular mass transfer can be improved.

[0065] In the compression block (150), the length (S2) in the direction parallel to the first direction (x) may range from 10 μm to 200 μm. When the length (S2) is less than 10 μm, intracellular mass transfer is not effectively carried out, and when it exceeds 200 μm, the contact area with the compression block (150) increases, and there is a possibility of damage to the cell (10). Specifically, in the compression block (150), the length (S2) in the direction parallel to the first direction (x) may range from 20 μm to 100 μm.

[0066] In the compression block (150), the height (S1) in the direction perpendicular to the first direction (x) may be set in the range of 15% to 99% with respect to the first diameter (a). Specifically, in the compression block (150), the height (S1) in the direction perpendicular to the first direction (x) may range from 3 μm to 1.5 mm. By setting the height (S1) within the aforementioned range, phenomena such as clogging by the compression block (150) can be prevented, and intracellular mass transfer can be effectively carried out.

[0067] The compression block (150) may be installed at a distance of the first length (L1) from one end of the main channel (110). Specifically, with respect to the total length (L2) from one end to the other end of the main channel (110), the first length (L1) may range from 30% to 90%. When the first length (L1) is set to less than 30% of the total length (L2), there is a problem that the flow of the droplet and the second fluid before passing through the compression block (150) becomes unstable, and when it exceeds 90%, there is a problem that there is insufficient time for the deformed droplet and cell to recover after passing through the compression block (150).

[0068] The main channel (110), the first and second supply parts (120, 130) may each be provided with a flow rate controller, and this flow rate controller can control the velocity and Reynolds number of the first fluid and the second fluid passing through the main channel (110), the first and second supply parts (120, 130). Specifically, the flow rate of the second fluid in the main channel (110) may be in the range of 1 mL / h to 70 mL / h. When the flow rate of the second fluid in the main channel (110) is less than 1 mL / h, problems such as droplets (50) binding to each other may occur, and when it exceeds 70 mL / h, the droplets (50) cannot be smoothly formed at the junction (140).

[0069] The corresponding substance can include any one or more of nucleic acids, proteins, transcription factors, vectors, plasmids, gene editing substances, nanoparticles, etc.

[0070] Hereinafter, with reference to FIGS. 4 to 10, other embodiments of the present invention will be described. Except for the content described later, since it is similar to the content described in the embodiments described with FIGS. 1 to 3, detailed description will be omitted.

[0071] FIG. 4 is a drawing showing an intracellular substance transmission platform according to another embodiment of the present invention. FIG. 5 is a cross-sectional view of FIG. 4.

[0072] Referring to FIGS. 4 and 5, the intracellular substance transmission platform (200) according to this embodiment includes a main channel (210) including a cell and a substance, one or more droplets including a first fluid, and a second fluid for moving the droplets, a first supply part (220) connected to one end of the main channel (210) and transmitting the first fluid and the cell and the substance, and a second supply part (230) connected to one end of the main channel (210) at an angle and transmitting the second fluid. At the junction (240) where the main channel (210) and the first and second supply parts (220, 230) intersect, droplets are formed and can be transmitted from one end of the main channel (210) to the other end.

[0073] Inside the main channel (210), a compression block (250) is provided, which can pressurize the droplets passing through the inside of the main channel (210). The compression block (250) can induce secondary intracellular material transfer by the vortices generated within the droplets after passing through the compression block (250), along with the primary intracellular material transfer due to the instantaneous cell deformation caused by the pressurization of the droplets.

[0074] The intracellular material transfer platform (200) can further include one or more sub-channels (260) through which a second fluid flows. The sub-channel (260) can be connected at an angle (θ) at a distance of a second length (L4) from one end of the main channel (210). The sub-channel (260) is connected at an angle of about 30° to 75° with respect to the main channel (210), and the flow of the second fluid supplied from the sub-channel (260) to the main channel (210) can prevent the generation of turbulent flow within the main channel (210) and make the flow of the droplets smoother.

[0075] After the droplets are formed, the sub-channel (260) can supply the second fluid to the main channel (210) through which the droplets and the second fluid flow. The sub-channel (260) may be provided with a flow rate controller, which can control the flow rate of the second fluid passing through the sub-channel (260).

[0076] The sub-channel (260) can supply the same second fluid from the outside to the flow of the second fluid flowing in the main channel (210), so that the droplets can pass through the main channel (210) while preventing the generation of friction between the droplets and the inner wall of the main channel (210).

[0077] The sub-channel (260) can include a pair of first and second sub-channels. The first and second sub-channels may be respectively provided on one side and the other side of the main channel (210). Specifically, the first and second sub-channels can be respectively connected to one side and the other side of the main channel (210) at an angle to the main channel (210) at a distance of a second length (L4) from one end of the main channel (210). The first and second sub-channels (260) can be provided as a pair and may be respectively provided at approximately the same position of the main channel (210).

[0078] The inner diameter (c) of the sub-channel (260) can be set in the range of 20% to 150% with respect to the first diameter (a) which is the inner diameter of the main channel (210). By setting the inner diameter (c) of the sub-channel (260) within the above range, the second fluid can be easily moved by droplets without forming unnecessary turbulent flow in the flow of the second fluid in the main channel (210). For example, the inner diameter (c) of the sub-channel (260) is from 20 μm to 200 μm, and the first diameter which is the inner diameter of the main channel may be in the range of 20 μm to 1.5 mm.

[0079] The flow rate of the second fluid supplied to the main channel (210) through the sub-channel (260) can be set in a range that is the same as or different from the flow rate of the second fluid in the main channel (210). Specifically, the flow rate of the second fluid in the main channel (210) is from 1 mL / h to 45 mL / h, and the flow rate of the second fluid in the sub-channel (260) may be in the range of 1 mL / h to 30 mL / h.

[0080] The compression block (250) is installed at a distance of a first length (L3) from one end of the main channel (210), and the first length (L3) may be in the range of 1.1 times to 5 times the second length (L4) at which the sub-channel (260) is installed from one end of the main channel (210). By controlling the first length (L3) and the second length (L4) within the above-mentioned range, the efficiency of intracellular substance transfer by the compression block (250) in the main channel (210) can be improved, and a large number of cells can be effectively processed.

[0081] Specifically, the first diameter (a) is from 20 μm to 1.5 mm, the first length (L3) is from 0.1 mm to 30 mm, and the second length (L4) may be in the range of from 0.1 mm to 1.5 mm.

[0082] FIG. 6 is a drawing showing an intracellular substance transfer platform according to a further embodiment of the present invention.

[0083] Referring to FIG. 6, the intracellular substance transfer platform (300) according to this embodiment includes a main channel (310) in which substances are transferred into cells while droplets (50) move, a first supply unit (320) that transfers cells, substances, and droplets (50) formed of a first fluid to the main channel (310), and a second supply unit (330) that transfers a second fluid to the main channel (310).

[0084] The main channel (310) can include an inlet portion (310a) connected to the first supply unit (320), a branch portion (310b) connected to the inlet portion (310a) and branched into a plurality of passages, and an outlet portion (310c) connected to the branch portion (310b). The branch portion (310b) can include a plurality of passages (311, 312, 313) having a smaller diameter than the inlet portion (310a) and the outlet portion (310c) and links by which these passages (311, 312, 313) branch or connect to each other. The compression block (350) may be installed in the branch portion (310b) or the outlet portion (310c).

[0085] In the main channel (310), droplets (50) formed by cells, substances, and a first fluid can be transmitted from the inlet portion (310a) through the branch portion (310b) to the outlet portion (310c) together with a second fluid. The droplets (50) installed in the inlet portion (310a) may be set to have a larger average diameter than the droplets (50) installed in the branch portion (310b) or the outlet portion (310c).

[0086] The branch portion (310b) may be installed symmetrically with respect to each other about a virtual reference line (SL) that horizontally connects the inlet portion (310a) and the outlet portion (310c). Specifically, the branch portion (310b) includes a plurality of passages (311, 312, 313) having a smaller diameter than the inlet portion (310a) and the outlet portion (310c) and links through which these passages (311, 312, 313) branch or connect to each other, and the compression block (350) may be installed in the branch portion (310b) or the outlet portion (310c). A plurality of passages (311, 312, 313) and branches connected in corresponding forms are formed on one side and the other side of the branch portion (310b) about the virtual reference line (SL), and the droplets (50) flow in through the inlet portion (310a), are divided into one side and the other side of the branch portion (310b), and can pass through the branch portion (310b).

[0087] The branch portion (310b) may be connected from the inlet portion (310a) and may be connected to two primary passages (311) that form one side and the other side of the branch portion (310b). Each primary passage (311) may be branched into two secondary passages (312) through a link. After these two secondary passages (312) are individually extended, they may be connected again through a link to form one tertiary passage (313). One tertiary passage (313) on one side of the branch portion (310b) and one tertiary passage (313) on the other side of the branch portion (310b) may be connected to the outlet portion (310c).

[0088] While passing through the branch portion (310b), the average diameter of the droplet (50) decreases as the average diameter of the branch portion (310b) decreases compared to the inlet portion (310a), and the droplet (50) can pass through the branch portion (310b). The average diameter of the droplet (50) may be approximately the same as or 20% or less than the average diameter of the branch portion (310b). When the droplet (50) passes through the branch portion (310b), the droplets (50) flow in a line, thereby preventing cell damage and effectively controlling intracellular substance transport.

[0089] In addition, one or more compression blocks (350) may be installed in either or both of the branch portion (310b) and the outlet portion (310c). The droplet (50) is pressurized while passing through the compression block (350), temporarily deforming the shape of the cell and forming nanopores in the cell membrane or nuclear membrane. Also, the shape of the droplet is deformed, reducing the distance between the substance and the cell. Due to the deformation of the flow of the first fluid within the droplet, substances may be effectively transported into the cell through the nanopores.

[0090] FIG. 7 is a drawing showing an intracellular substance transport platform according to another embodiment of the present invention. FIG. 8 is an enlarged view of the main channel of FIG. 7, and FIG. 9 is a drawing showing the branch portion of FIG. 7.

[0091] Referring to FIGS. 7 to 9, the intracellular substance transport platform (400) according to this embodiment includes a first supply unit (420) to which a first fluid containing cells and substances is supplied, a main channel (410) connected to the first supply unit (420), and a second supply unit (430) that supplies a second fluid to a portion where the first supply unit (420) and the main channel (410) are connected. At a junction (440) such as a cross shape, a Y shape, or a T shape where the first supply unit (420), the second supply unit (430), and the main channel (410) intersect, the first fluid containing cells and substances may be formed into droplets (50) by the second fluid. These droplets can move within the main channel (410) together with the second fluid.

[0092] The main channel (410) can include an inlet section (410a, 410b, 410c), a branch section (410d, 410e), and an outlet section (410f) that are sequentially connected. The main channel (410) can further include one or more curved passages (410b) installed in the inlet section (410a, 410b, 410c) or the branch section (410d, 410e). Specifically, in this embodiment, the curved passage (410b) may be included in the inlet section (410a, 410b, 410c), and this curved passage (410b) is installed in one or more curved shapes, capable of controlling the flow velocity of the second fluid and droplets (50) in the main channel (410) and generating a vortex within the droplets. This vortex can improve the mass transfer efficiency within the droplets and, at the same time, play a role in mixing the cells contained within the droplets.

[0093] When the cells within the droplets are uniformly mixed by the flow within the droplets generated by the curved passage (410b), the number of cells within the droplets can be evenly divided at the portion where the flow of the second fluid branches. For example, when the droplets are divided into two at the branching portion, the cells within the droplets can also be divided at a ratio of approximately 50:50, enabling control to obtain a uniform effect for each batch.

[0094] The intracellular material transmission platform (400) can include a portion (1) where droplets (50) are formed, a portion (2) where the flow of the second fluid containing the droplets branches, and a portion (3) where the droplets are pressurized by a compression block (450) and the cells are mechanically perforated by mechanoporation. The intracellular material transmission platform (400) uses a first fluid and a second fluid that do not mix with each other to form droplets (50) composed of cells, substances, and the first fluid, and moves the droplets (50) through the second fluid. However, the average diameter, moving speed, and moving form of the droplets (50) can be controlled by factors such as the average diameter of the main channel (410) and the number of channels. In addition, the intracellular material transmission platform (400) further includes a compression block (450), and can form reversible nanopores in the cells by mechanoporation without damaging the cells or the droplets (50). When the nanopores are opened, substances are transmitted into the cells, and then the nanopores are immediately closed so that the substances are not released outside the cells, and the substances can be transmitted into the cells without damaging the cells.

[0095] Specifically, in the intracellular material transmission system of FIG. 7, the portion (1) where droplets (50) are formed, the portion (2) where the flow of the second fluid containing the droplets branches, and the portion (3) where the droplets are pressurized by a compression block (450) and the cells are mechanically perforated by mechanoporation are the drawings obtained by conducting experiments in the following manner.

[0096] Specifically, as the cells, substance, and first fluid, 40 million Jurkat cells (ATCC, TIB-152) per mL were included, the culture medium used was the substance obtained using ThermoFisher's Opti-mem, and fluorocarbon oil was used as the second fluid. The droplets composed of cells, substance, and the first fluid decrease in size to that of smaller droplets (50) when passing through the branch portions (410d, 410e), and a relatively small number of cells may be captured in the small-sized droplets (50). These droplets (50) are pressurized by the compression block (450), and the substance in the droplets may be transmitted into the cells at the portion (3) where mechanoporation occurs.

[0097] The inlet portions (410a, 410b, 410c) are connected to the junction (440) and can include a first portion (410a) into which the droplets (50) flow, a second portion (410c) connected to the branch portions (410d, 410e), and a curved passage (410b) connected between the first portion (410a) and the second portion (410c).

[0098] The branch portions (410d, 410e) may be installed so that the shape formed by connecting a plurality of passages and links is symmetric with respect to a virtual reference line (SL) that vertically connects the inlet portions (410a, 410b, 410c) and the outlet portion (410f). Specifically, one (410d) and the other (410e) of the branch portions may be respectively connected to a pair of first passages (411) via a first link (D1) connected from the second portion (410c) around the virtual reference line (SL). For example, sandwiching the first link (D1), the second portion (410c) and the pair of first passages (411) can be connected at an angle to form a T-shaped or Y-shaped passage.

[0099] The average diameter (L6) of the first passage (411) may be set smaller than the average diameter (L5) of the end of the inlet portion (410a, 410b, 410c). Specifically, the average diameter (L6) of the first passage (411) may be set in the range of 40% to 80% with respect to the average diameter (L5) of the end of the inlet portion (410a, 410b, 410c).

[0100] The first passage (411) is further connected to a pair of second passages (412, 413) through a second link (D2), and each second passage (412, 413) may be connected to a third link (D3). The third passages (414, 415) connected through the third link (D3) are branched as a pair, and at the ends of each third passage (414, 415), they may be connected to a fifth passage (417) that is further connected to one passage through a fourth passage (416) having a circular or polygonal shape. The fifth passage (417) is connected through a fourth link (D4) to a sixth passage (418), and may further be connected to a seventh passage (419) through a fifth link (D5).

[0101] Each seventh passage (419) connected from one (410d) and the other (410e) of the branch portions may be connected to an outlet portion (410f) through a sixth link (D6). The outlet portion (410f) is installed in parallel with the inlet portions (410a, 410b, 410c) and may include one or more compression blocks (450) inside.

[0102] The branch portions (410d, 410e) or the outlet portion (410f) may include one or more compression blocks (450). The compression block (450) can physically pressurize the droplets (50) and promote intracellular substance transfer by mechanoporation. The compression block (450) may be installed in the branch portions (410d, 410e) or the fourth passage (416).

[0103] FIG. 10 is a drawing showing an intracellular substance transfer platform according to another embodiment of the present invention.

[0104] Referring to FIG. 10, the intracellular substance transmission platform (500) according to this embodiment may include a first supply unit (520) that supplies cells, substances, and a first fluid, and a main channel (510) connected to an end of the first supply unit (520) through which substances are transmitted into cells while droplets (50) move.

[0105] The main channel (510) may include an inlet part (510a, 510b, 510c) connected to the first supply unit (520), a branch part (510d, 510e, 510f) connected to the inlet part (510a, 510b, 510c) and composed of a plurality of passages, and an outlet part (510g) connected to an end of the branch part (510d, 510e, 510f) and having a compression block (550) inside.

[0106] The branch parts (510d, 510e, 510f) may be installed symmetrically with respect to the center of the branch parts (510d, 510e, 510f). The branch parts (510d, 510e, 510f) are configured by connecting a plurality of passages and a plurality of links connecting these passages, and may be installed in a size smaller than the average diameter of the inlet part (510a, 510b, 510c) or the outlet part (510g).

[0107] The inlet parts (510a, 510b, 510c) or branch parts (510d, 510e, 510f) are installed in a channel shape for providing a passage through which droplets and the second fluid pass, at least a part of which is linear and at least a part of which may be installed to include bent passages (510b, 510f) having a bent shape. The bent passages (510b, 510f) are installed in a curved shape and can generate a vortex within the droplet. This vortex can improve the mass transfer efficiency within the droplet and at the same time can play a role in mixing the cells contained within the droplet. The cells within the droplet are uniformly mixed in the bent passages (510b, 510f), and the number of cells within the droplet can be made equal. Also, when passing through the bent passages (510b, 510f), the droplets (50) can move in an aligned form. The bent passages (510b, 510f) are installed in the inlet parts (510a, 510b, 510c) and can include a first bent passage (510b) having an average diameter corresponding to the inlet parts (510a, 510b, 510c) and a second bent passage (510f) installed in the branch parts (510d, 510e, 510f) and having an average diameter corresponding to the branch parts (510d, 510e, 510f).

[0108] The inlet parts (510a, 510b, 510c) include a first part (510a) connected to the first supply part (520) and a second part (510c) connected to the branch parts (510d, 510e, 510f). There may be a first bending passage (510b) installed between the first part (510a) and the second part (510c). The first bending passage (510b) is installed in a curved shape so that the cells in the droplet (50) formed in the first part (510a) can be evenly distributed. The branch parts (510d, 510e, 510f) can include one (510d) of a pair of branch parts and the other (510e) in which passages are formed in a pattern. Also, there may be a second bending passage (510f) in a curved shape connected to one or more of either one (510d) or the other (510e) of the branch parts installed in the branch parts (510d, 510e, 510f). One (510d) and the other (510e) of the branch parts may be installed with a passage shape having the same shape pattern or a different shape pattern.

[0109] The second part (510c) may be branched into two passages and connected to one (510d) of the branch parts having one or more second bending passages (510f) and the other (510e) of the branch parts having one or more second bending passages (510f), respectively. One (510d) and the other (510e) of the branch parts may be installed in a symmetric shape with respect to the central part.

[0110] The second bending passage (510f) is installed at the start of one (510d) and the other (510e) of the branch parts, respectively, to align the second fluid flowing into the branch parts and the droplet (50) in a row, and the cells contained in the droplet (50) can be evenly distributed.

[0111] Hereinafter, embodiments and comparative examples of the present invention will be described. However, the following embodiments are merely preferred embodiments of the present invention, and the scope of rights of the present invention is not limited by the following embodiments.

[0112] Figures 11 to 16 show the results of confirming intracellular substance transmission using the intracellular substance transmission platform according to the examples and comparing the efficiency of substance transmission with the prior art.

[0113] Figure 11 is a drawing showing the confirmation of intracellular substance transmission using the intracellular substance transmission platform according to this example.

[0114] Referring to Figure 11, the intracellular substance transmission platform was fabricated using PDMS (polydimethylsiloxane) (Dow, Sylgard 184) and a slide glass (Marienfeld Superior, HSU - 1000612). A master mold with a microfluidic pattern etched through photolithography and etching (DRIE) processes on a silicon wafer was fabricated, and the microfluidic pattern was replicated onto the PDMS surface through a soft lithography process of PDMS. The PDMS with the microfluidic pattern and the slide glass were bonded by oxygen plasma using a plasma cleaner (Femtoscience, CUTE) to manufacture the intracellular substance transmission platform. To generate stable droplets, 10 μL of surface coating oil (RAN Biotechnologies, 909 - FluoroCoat) was used to hydrophobize the inside of the microchannels of the intracellular substance transmission platform, and O / N bonding and drying were performed at 75 °C in a forced convection oven (Jeio Tech, OF4 - S).

[0115] The intracellular substance transmission platform manufactured as described above has a main channel with a total length of 3.1538 mm where droplets are formed and move, and the average inner diameter of the main channel is 80 μm. The compression block is installed at a distance of 2.0538 mm from one end of the main channel, with a height of 4.8 μm and a length of 100 μm. Also, the average inner diameter of the sub - channels is 49 μm, and a pair is connected at an angle of about 58 degrees at a distance of 1 mm from one end of the main channel.

[0116] As materials for the intracellular substance transfer experiment using the above intracellular substance transfer platform, a cell suspension (ATCC, CCL-243) and a fluorocarbon oil (Bio Rad, Droplet Generation Oil) were used. The cell suspension used in the experiment contained 15 million K562 cells per mL. After cell preparation, a cell suspension was made with a culture medium (Corning, RPMI), and impurities in the oil were removed with a 0.2 μm syringe filter (Advantec, 13HP020AN / 25HP020AN). The fluorocarbon oil and the cell suspension were each injected into a disposable syringe (BD, Luer-lok Tip Syringe), and the syringe and the microfluidic platform were connected with a capillary tube (IDEX, 1 / 32´´ OD PEEK Tubing). Using a syringe pump (Harvard Apparatus, 11 Elite Microfluidic Syringe Pump), the cell suspension and the fluorocarbon oil were each injected into the intracellular substance transfer platform at a constant flow rate of 0.5 mL / h and 2.0 mL / h, respectively. The separately injected cell suspension and fluorocarbon oil met at a flow focusing junction, and cells were captured inside the droplets while the droplets were generated. The corresponding droplets passed through the main channel while maintaining the droplet state together with the fluorocarbon oil. At this time, the fluorocarbon oil flowed in from the sub-channel connected to the main channel at a rate of 16 mL / h, and the flow of the droplets in the main channel was accelerated. Also, by additionally supplying the fluorocarbon oil through the sub-channel, the droplets in the main channel were wrapped by the fluorocarbon oil and moved.

[0117] Next, when the droplets passed through a compression block installed in the main channel, deformation of the cells inside the droplets was induced to form nanopores in the cell membrane. It was confirmed that the substances coexisting inside the droplets were transferred into the cells through these nanopores.

[0118] Figure 12 shows the result of confirming the intracellular substance transmission efficiency according to the size of the compression block in the intracellular substance transmission platform according to FIG. 11.

[0119] Figure 12 uses an intracellular substance transmission platform with the same shape as that in FIG. 11, and only changes the compression block installed inside to confirm the intracellular substance transmission efficiency. In the cell suspension used in this experiment, 2,000 kDa FITC-conjugated dextran (Sigma Aldrich, FD2000S) as a transmission substance was diluted at a concentration of 0.3 mg / mL and repeated 3 times for each condition. After culturing the cells treated through the intracellular substance transmission platform for 18 hours, the change in transmission efficiency and mean fluorescence intensity fold change compared with the control group were analyzed using a flow cytometer (Merck, Guava EasyCyte).

[0120] In the part where the compression block was installed in the intracellular substance transmission platform of FIG. 12, the gap height between the inner surface of the main channel and the compression block was set to three different values of 6.3 μm, 4.8 μm, and 3.6 μm respectively. As a result, it was confirmed that the intracellular substance transmission efficiency increased in the order of 6.3 μm, 4.8 μm, and 3.6 μm, and the highest result was shown at 3.6 μm. In addition, as the squeezing length of the compression block was set to different values of 40 μm, 70 μm, and 100 μm respectively to confirm the efficiency of substance transmission, it was confirmed that 100 μm was the most excellent. The intracellular substance transmission efficiency by the compression block in this experiment is shown in Tables 1 and 2 below.

[0121]

Table 1

[0122]

Table 2

[0123] Figure 13 shows the results of confirming the intracellular substance transport efficiency according to the type of transported substance in the intracellular substance transport platform based on Figure 11.

[0124] In Figure 13, experiments were conducted using a sample in which 2,000 kDa FITC-conjugated dextran (Sigma Aldrich, FD2000S) was diluted at a concentration of 0.3 mg / mL as the transported substance in the cell suspension, and a sample in which green fluorescent protein-expressing mRNA (EGFP mRNA; TriLink, L-7601) was diluted at a concentration of 20 μg / mL. In the cells treated through the intracellular substance transport platform, for the transport of 2,000 kDa FITC-Dextran, the transport efficiency was analyzed by comparing with the control using a flow cytometer (Merck, Guava EasyCyte) after culturing for 18 hours, and for the transport of EGFP mRNA, after culturing for 24 hours. Bright field and GFP images of the control and the substance transport sample were acquired using an optical microscope (Zeiss, Axio Observer 7) and a camera (ZEISS, Axiocam 305 mono), and the degree of fluorescence expression was qualitatively compared.

[0125] Here, the control is a cell exposed to FITC-dextran or EGFP-mRNA at the same concentration as in the example for the same time. Substance transport was performed in a cell suspension state using FITC-dextran or EGFP-mRNA, without using the intracellular substance transport platform corresponding to the example, and the endocytosis effect was confirmed.

[0126] In the case of the control example without using the droplet and intracellular substance transport platform, it was confirmed that almost no substance transport into cells occurred on both sides of 2,000 kDa FITC-conjugated dextran and fluorescent protein-expressing mRNA. On the other hand, when the intracellular substance transport platform according to the present invention was used, it was confirmed that substance transport into cells was carried out with high efficiency. Also, as in this example, regardless of the type of substance transported into cells, it was confirmed that high efficiency was shown when using the intracellular substance transport platform according to the examples of the present invention. The intracellular substance transport efficiency by the compression block in this experiment is shown in Table 3 below.

[0127] [Table 3]

[0128] Figure 14 is a drawing showing the results of intracellular gene transfer using a conventional general-purpose device. Figure 15 is the result of comparing the EMX1 target gene editing efficiency with a conventional general-purpose device. Figure 14 is the intracellular substance transfer data published by MaxCyte (2021), and Figure 15 compares the intracellular substance transfer platform (droplet) of the present invention with a conventional general-purpose device, an electroporation device (EP) and liposome nanoparticles (LNP), and confirms the EMX1 target gene editing efficiency of K562 cells.

[0129] In the experiment of Fig. 15, the experimental method of Fig. 11 was adopted, and 500 pmol each of sgRNA (Single guide RNA) targeting the EMX1 expression gene of K562 cells and Cas9 protein without endotoxin were added into the cell suspension. After culturing the cells treated through the intracellular substance transmission platform for 48 hours, gDNA (Genomic DNA) was extracted using a DNA extraction kit (Intronbiotechnology, G-spin (trademark) Total DNA Extraction Mini Kit). The gDNA was amplified by causing a polymerase chain reaction (PCR) using a thermal cycler (Bio-Rad, T100). Quantification was performed using a micro-spectrophotometer (ThermoFisher, NanoDrop One), and after treating 200 ng of the PCR product with 10 U of T7 endonuclease 1 (New England BioLabs, M0302S) at 37°C for 15 minutes, the gene editing efficiency was analyzed through electrophoresis (Bio-Rad, BR164-0302). The same was true for K562 cells treated with liposome nanoparticles (Invitrogen; Lipofectamine 3000) and an electroporation device (ThemoFisher, Neon Transfection System).

[0130] The intracellular substance transmission platform (droplet) according to the present invention was shown to have a gene editing efficiency that was extremely excellent compared to an electroporation device (EP) and liposome nanoparticles (LNP), and it was confirmed that high-efficiency editing of Multiplexing editing (editing of two or more targets) was possible. In addition, the intracellular substance transmission platform of this example enabled high-efficiency editing of HDR (Homology Directed Repair). That is, it was confirmed that the intracellular substance transmission platform according to the present invention can transmit substances into cells with high efficiency compared to the prior art without damaging the cells, and can be applied to various cells without restrictions.

[0131] Figure 16 shows the result of confirming intracellular substance transfer using a droplet separation type intracellular substance transfer platform. Figure 16 uses the droplet separation type intracellular substance transfer platform based on Figure 7.

[0132] The manufacturing method of the droplet separation type intracellular substance transfer platform used in Figure 16 was manufactured in the same way as the intracellular substance transfer platform based on Figure 11 described above.

[0133] The intracellular substance transfer platform of Figure 16 is an inlet where droplets are formed in the main channel where droplets are formed and move. The length of the straight passage is 1.4 mm and the average inner diameter is 0.4 mm. The length of the subsequent curved passage is 2.51 mm and the average inner diameter is 0.2 mm. The length of the further subsequent straight passage is 1 mm and the average inner diameter is 0.2 mm. Here, a pair of branch parts is connected, and the passages of the branch parts are connected with an average inner diameter of 0.09 mm. Here, the height of the press block is 8 μm and the length is 70 μm.

[0134] The cell suspension used in this experiment contained K562 cells (ATCC, CCL-243), and the transport substance, 3 kDa FITC-conjugated dextran (Sigma Aldrich, FD4), was diluted to a concentration of 0.3 mg / mL. After the operation of the intracellular substance transmission platform, the droplets containing the cells were separated through a demulsification process using a PFO solution (Sigma Aldrich, 1H,1H,2H,2H-Perfluoro-1-octanol). The viability of the corresponding cells was measured with an automatic cell counter (Logos biosystems, Luna-FX7), and a cell viability of around 80% was confirmed. Also, after culturing the corresponding cells for 18 hours, they were analyzed with a flow cytometer (BD, FACSLyric). Results of a transmission efficiency of 90% or more and a mean fluorescence intensity fold change of 100-fold or more were confirmed compared to the control. Using an optical microscope (Zeiss, Axio Observer 7) and a camera (ZEISS, Axiocam 305 mono), Bright field and GFP images of the control and the substance transmission samples were obtained, and the results of qualitatively comparing the degree of fluorescence expression also confirmed that a higher substance transmission efficiency was shown when using the intracellular substance transmission platform according to this example. Here, the control was obtained by performing substance transmission in a cell suspension state using cells exposed to the transport substance at the same concentration as in the example for the same time, without using the intracellular substance transmission platform corresponding to the example, and is the result of confirming the substance transmission effect due to the influence of endocytosis.

[0135] Those with ordinary knowledge in the technical field to which the present invention pertains will be able to understand that the present invention can be implemented in other specific forms without changing its technical idea and essential features. Therefore, it should be understood that the above-described embodiments are illustrative in all aspects and not restrictive. The scope of the present invention is indicated by the claims described later rather than the above detailed description, and all changes or modified forms derived from the meaning and scope of the claims and based thereon should be construed as being included within the scope of the present invention.

Explanation of Reference Numerals

[0136] 100, 200, 300, 400, 500: Intracellular signal transduction platform 110, 210, 310, 410, 510: Main channel 120, 220, 320, 420, 520: First supply unit 130, 230, 330, 430: Second supply unit

Claims

1. An intracellular substance delivery platform comprising: one or more main channels extending in a first direction from one end to the other end and having a fluid passage therein; a first supply part connected to one end of the main channels and for injecting a first fluid containing the cells and the substance; and a second supply part connected to one end of the main channels and for injecting a second fluid that is not mixed with the first fluid; and wherein the main channels are provided with one or more compressing blocks.

2. The intracellular substance transfer platform according to claim 1 , wherein the first fluid is a water phase and the second fluid is an oil phase.

3. The intracellular substance transfer platform of claim 1 , wherein the first supply portion extends parallel to the first direction and is connected to one end of the main channel, and the second supply portion is connected at an angle to the first supply portion at one end of the main channel.

4. The intracellular substance transfer platform of claim 3, wherein the first fluid is transferred from the first supply to one end of the main channel, and the second fluid is transferred from the second supply to one end of the main channel, but at an angle to the flow direction of the first fluid, and the first fluid containing the cells and substances is formed as droplets, and the droplets pass through the main channel within the second fluid.

5. The intracellular substance transfer platform according to claim 3 , wherein the second supply section includes first and second supply channels, the first and second supply channels being connected to one end of the main channel to form a junction.

6. The intracellular substance transfer platform of claim 1 , wherein the compression block is provided at a portion spaced a first length from one end of the main channel, and the first length is 30% to 90% of the entire length from one end to the other end of the main channel.

7. The intracellular substance transfer platform according to claim 1 , wherein the flow rate of the second fluid in the main channel is from 1 mL / h to 70 mL / h.

8. The intracellular substance transfer platform according to claim 1 , further comprising one or more subchannels connected at an angle to the main channel, through which the second fluid flows.

9. The intracellular substance transfer platform of claim 8, wherein the subchannel is connected to the main channel at a portion spaced a second length from one end of the main channel and includes first and second subchannels, the first and second subchannels being connected to one and the other sides of the main channel, respectively.

10. The intracellular substance transfer platform of claim 9 , wherein the inner diameter of the sub-channel is 20% to 150% of a first diameter which is an inner diameter of the main channel, and the second fluid is transferred to the main channel through the sub-channel.

11. The intracellular substance transfer platform of claim 10, wherein the inner diameter of the sub-channel is 20 μm to 200 μm, the first diameter which is the inner diameter of the main channel is 20 μm to 1.5 mm, and the second fluid is transferred to the main channel through the sub-channel.

12. The intracellular substance transfer platform of claim 9 , wherein the flow rate of the second fluid in the main channel is 1 mL / h to 45 mL / h, and the flow rate of the second fluid in the sub-channel is 1 mL / h to 30 mL / h.

13. The intracellular substance transfer platform of claim 8 , wherein the compression block is provided at a portion spaced from one end of the main channel by a first length, and the first length is 1.1 to 5 times the second length.

14. The intracellular substance transfer platform of claim 13, wherein the first diameter is between 20 μm and 1.5 mm, the first length is between 0.1 mm and 30 mm, and the second length is between 0.1 mm and 1.5 mm.

15. The intracellular substance transfer platform according to claim 1 , wherein the compression block has a length in a direction parallel to the first direction of 10 μm to 200 μm.

16. The intracellular substance transfer platform according to claim 15 , wherein the compression block has a length in a direction parallel to the first direction of 20 μm to 100 μm.

17. The intracellular substance transfer platform of claim 1, wherein a gap between the compression block and an inner surface of the main channel is provided with a second diameter, the second diameter being 0.1% to 85% of a first diameter, which is an inner diameter of the main channel.

18. The intracellular substance transfer platform according to claim 17 , wherein the second diameter is between 2 μm and 17 μm.

19. The intracellular substance transfer platform according to claim 1 , wherein the height of the compression block in a direction perpendicular to the first direction is 15% to 99% of the first diameter.

20. The intracellular substance transfer platform according to claim 19, wherein the height of the compression block in a direction perpendicular to the first direction is between 3 μm and 1.5 mm.

21. The intracellular substance transfer platform of claim 1, wherein the main channel includes an inlet portion connected to the first supply portion; a branch portion connected to the inlet portion but branching into multiple passages; and an outlet portion connected to the branch portion; the branch portion includes multiple passages having diameters smaller than the inlet portion and the outlet portion, and links at which the multiple passages branch off or connect to each other, and the compression block is provided in the branch portion or the outlet portion.

22. The intracellular substance transfer platform of claim 21, wherein droplets formed from the cells, substance and first fluid in the main channel are transferred from the inlet portion through the branch portion to the outlet portion via the second fluid, and the droplets provided in the inlet portion have an average diameter larger than that of the droplets provided in the branch portion or the outlet portion.

23. The intracellular substance transfer platform according to claim 21 , wherein the branch portions are provided in shapes symmetrical to each other about a virtual reference line connecting the inlet portion and the outlet portion.

24. The intracellular substance transfer platform of claim 21 , wherein the main channel further comprises one or more bend passages provided in the inlet or branch portion.

25. The intracellular substance transfer platform according to claim 1 , wherein the substance comprises at least one of a nucleic acid, a protein, a transcription factor, a vector, a plasmid, a genetic scissors substance, and a nanoparticle.

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