Droplet-based intracellular material transport platform

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 that can effectively transport substances into cells with high efficiency without damaging the cells. [Background technology]

[0002] Intracellular transport of substances is one of the most fundamental experiments in cell engineering, and substances are usually transported using carriers or by creating nanopores in the cell membrane / nuclear membrane.

[0003] Viral or Lipofectamine-based carrier technologies, when optimized, can deliver highly efficient materials; however, they have drawbacks such as safety, slow delivery rate, labor- and cost-intensive carrier preparation process, and low reproducibility.

[0004] In contrast, methods that create nanopores by applying energy to the cell membrane (e.g., electroporation or microneedles) have the advantage of being able to deliver a relatively wide range of substances to various cell lines. However, these methods have been identified as having major limitations, such as low cell viability due to their invasiveness, denaturation of the transported substances, and low throughput.

[0005] To solve these problems, the use of microfluidic devices capable of processing large amounts of cells has become prominent. A typical platform involves creating a bottle-neck section in a microtube, and creating a nanopore in the cell membrane through physical deformation of the cell as it passes through the bottle-neck section. However, this approach has major drawbacks, such as clogging of the bottle-neck section itself during the experiment and uneven mass transport efficiency.

[0006] Therefore, various studies have been conducted on methods for delivering various substances to various cells with high efficiency without damaging the cells. Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a droplet-based intracellular substance delivery platform that can deliver substances to various cells with high efficiency.

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

[0009] In accordance with one aspect of the present invention, an embodiment of the present invention includes an intracellular mass transport platform.

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

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

[0012] In one embodiment, 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 can be connected at an angle to the first supply portion at one end of the main channel.

[0013] In one embodiment, the first fluid is delivered from the first supply to one end of the main channel, and the second fluid is delivered from the second supply to one end of the main channel, but at an angle to the flow direction of the first fluid, so that the first fluid containing the cells or substances forms droplets that can pass through the main channel within the second fluid.

[0014] In one embodiment, the second supply section includes first and second supply channels, which 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 spaced a first length from one end of the main channel, and the first length can be 30% to 90% of the total length of the main channel from one end to the other.

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

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

[0018] In one embodiment, the sub-channel 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 sub-channels, which can be connected to one side and the other side of the main channel, respectively.

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

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

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

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

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

[0024] In one embodiment, the compressed block can have a length parallel to the first direction of 10 μm to 200 μm.

[0025] In one embodiment, the compressed block may have a length in a direction parallel to the first direction of 20 μm to 100 μm.

[0026] In one embodiment, the gap between the compression block and the inner surface of the main channel is defined by a second diameter, which can be 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 compressed block in a direction perpendicular to the first direction can be 15% to 99% of the first diameter.

[0029] In one embodiment, the height of the compressed block in a direction perpendicular to the first direction can be 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 branching into multiple passages; and an outlet portion connected to the branch portion; the branch portion includes multiple passages with smaller diameters 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 can be installed in the branch portion or the outlet portion.

[0031] In one embodiment, droplets formed from the cells, substance, and first fluid are transported from the inlet portion through the branch portion to the outlet portion within the main channel via the second fluid, and the droplets placed in the inlet portion may have a larger average diameter than the droplets placed in the branch portion or the outlet portion.

[0032] In one embodiment, the branch sections may be arranged symmetrically with respect to each other about an imaginary reference line connecting the inlet section and the outlet section.

[0033] In one embodiment, the main channel may further include one or more curved passages located at the inlet or branch portions.

[0034] In one embodiment, the substance may include one or more of a nucleic acid, a protein, a transcription factor, a vector, a plasmid, a genetic scissors substance, and a nanoparticle. [Effects of the Invention]

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

[0036] Furthermore, the present invention provides an intracellular substance transport platform that can be easily mass-produced and that can transport any type of substance into cells using a new method, and can transport various substances into a single cell or one substance into various cells. [Brief explanation of the drawings]

[0037] [Figure 1] FIG. 1 is a schematic diagram of an intracellular material transport platform according to one embodiment of the present invention. [Figure 2] FIG. 2 is a diagram showing an intracellular substance transport platform according to one embodiment of the present invention. [Figure 3] FIG. 3 is a cross-sectional view of FIG. [Figure 4] FIG. 4 is a diagram showing an intracellular substance transport platform according to another embodiment of the present invention. [Figure 5] FIG. 5 is a cross-sectional view of FIG. [Figure 6] FIG. 6 is a diagram showing an intracellular substance transport platform according to yet another embodiment of the present invention. [Figure 7] FIG. 7 is a diagram showing an intracellular substance transport platform according to another embodiment of the present invention. [Figure 8] FIG. 8 is an enlarged view of the main channel of FIG. [Figure 9] FIG. 9 is a diagram showing the branch portion of FIG. [Figure 10] FIG. 10 is a diagram showing an intracellular substance transport platform according to another embodiment of the present invention. [Figure 11] FIG. 11 shows the intracellular substance transport confirmed using the intracellular substance transport platform according to this embodiment. [Figure 12] FIG. 12 shows the results of confirming the intracellular substance transport efficiency depending on the size of the compressed block in the intracellular substance transport platform shown in FIG. [Figure 13] FIG. 13 shows the results of confirming the intracellular substance transport efficiency depending on the type of transport substance in the intracellular substance transport platform shown in FIG. [Figure 14] FIG. 14 shows the results of intracellular gene delivery using a conventional general-purpose device. [Figure 15] Figure 15 shows the results of comparing the efficiency of EMX1 targeted gene editing with that of a conventional general-purpose device. [Figure 16] Figure 16 shows the results of intracellular material transport confirmed using the droplet separation-type intracellular material transport platform. DETAILED DESCRIPTION OF THE INVENTION

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

[0039] The advantages and features of the present invention, as well as methods for achieving them, will become clearer with reference to the following detailed examples, along with the accompanying drawings. However, the present invention is not limited to the examples disclosed below and can be embodied in various forms. Unless otherwise specified in the following description, all numbers, values, and / or expressions expressing components, reaction conditions, and component contents included in the present invention are approximations that reflect various uncertainties in measurement that arise when obtaining these values ​​from inherently different sources, and should be understood in all cases to be modified by the term "about." Furthermore, when numerical ranges are disclosed in this description, these ranges are continuous and include all values ​​from the minimum value to the maximum value, inclusive, unless otherwise specified. Furthermore, when these ranges refer to integers, they include all integers from the minimum value to the maximum value, inclusive, unless otherwise specified.

[0040] Furthermore, when a range is described for a variable herein, it is understood that the variable in question includes all values ​​within the described range, including the endpoints of the described range. For example, the range "5 to 10" is understood to include not only the values ​​5, 6, 7, 8, 9, and 10, but also any subranges such as 6 to 10, 7 to 10, 6 to 9, and 7 to 9, as well as any value between any integer within the described range category, such as 5.5, 6.5, 7.5, 5.5 to 8.5, and 6.5 to 9. For example, the range "10% to 30%" is understood to include values ​​such as 10%, 11%, 12%, 13%, etc., and all integers up to 30%, as well as any subranges such as 10% to 15%, 12% to 18%, 20% to 30%, and any value between any integer within the described range category, 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 diagram showing an intracellular substance transport platform according to an embodiment of the present invention, and 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, and includes one or more main channels (110) extending from one end to the other in a first direction (x) and having a fluid passage therein; a first supply section (120) connected to one end of the main channel (110) for injecting the cells (10), a substance (20) and a first fluid; and a second supply section (130) connected to one end of the main channel (110) for injecting a second fluid that is immiscible with the first fluid.

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

[0044] The intracellular material transport platform 100 according to this embodiment forms droplets using a first fluid and a second fluid that are immiscible with each other, and can capture a cell 10 and a substance 20 to be transported into the cell within the droplet 50. The droplet 50 is formed of the same material as the first fluid, and can move the cell 10 and substance 20 within the second fluid while maintaining the droplet 50 shape.

[0045] Conventional methods for transporting substances into cells leave permanent marks on the cells, such as by damaging the cell membrane using electroporation, while methods for transporting substances into cells by directly applying an external force to the cells while the cells and substances are flowing through a fluid have the disadvantages of causing damage to the cells or low efficiency of transporting substances into the cells.

[0046] Meanwhile, the intracellular material transport platform 100 according to this embodiment utilizes droplets 50 to effectively transport a substance 20 into a cell while preventing damage to the cell 10. In the intracellular material transport platform 100 of the present invention, the droplets 50 protect the cell 10 and the substance 20 while allowing them to flow efficiently within the fluid. Furthermore, the droplets 50 maintain a close distance between the cell 10 and the substance 20, allowing the substance 20 to be efficiently transported into the cell 10 without being lost to the outside.

[0047] The intracellular material transport platform 100 according to this embodiment may further include a compression block 150. The compression block 150 is disposed within the main channel 110 and can reduce the effective diameter of the main channel 110. Specifically, the droplets 50 pass through the main channel 110 at a predetermined speed due to the second fluid, and the droplets 50 are compressed as they pass through the compression block 150. As the droplets 50 pass through the compression block 150, deformation of the cells 10 occurs, forming nanopores in the cell membrane or nuclear membrane through which the material 20 can pass, and the material 20 can be effectively transported into the cells 10 through the nanopores. Furthermore, the droplets (50) that have passed through the compression block (150) recover their shape, forming vortices within the droplets (50), which provide the driving force for transporting the substance (20) into the cells (10), improving the efficiency of intracellular substance transport. Furthermore, the cells (10) that were deformed after passing through the compression block (150) recover their original shape, containing the substance (20) inside.

[0048] The droplets (50) can be provided with a very small volume, approximately 100 pL per cell. The volume of the droplets (50) determines the amount of substance (20) delivered within the main channel (110). Conventional intracellular substance delivery systems involve long distances between cells and the substance being delivered, resulting in inefficient processes, either by reducing the efficiency of intracellular substance delivery or by consuming a large amount of substance to increase the concentration of the substance around the cells, while the remaining substance is discarded. However, in the intracellular substance delivery platform (100) of this embodiment, the cells (10) and substance (20) exist within a small system called a droplet (50), and the cells (10) and substance (20) are positioned in close proximity to each other. This allows even a very small amount of substance (20) to be delivered efficiently into the cells (10), thereby reducing wasteful loss of the substance (20).

[0049] In the intracellular substance transport platform 100 according to this embodiment, the droplet 50 has a small volume, and within the droplet 50, the cell 10 can be placed in an environment with a high concentration of the substance 20. If a nanopore is formed due to deformation of the cell 10 as the droplet 50 passes through the compression block 150, it is possible to effectively transport a substance into the cell 10 even with a droplet 50 of a very small volume. 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) of this embodiment can be applied to large molecules such as large nanoparticles and plasmid DNA, which was not possible with conventional technology, and can prevent the clogging phenomenon that occurred in conventional methods of passing cells through microtubes or their narrow passages (bottlenecks), as well as the problem of reduced cell viability due to 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 made of a hydrophobic material. The second fluid that drives the droplets 50 in the main channel 110 is hydrophobic and can easily flow within the main channel 110. In addition, the droplets 50 are hydrophilic, and the difference in hydrophilicity and hydrophobicity between the droplets 50 and the main channel 110 can minimize clogging.

[0052] The intracellular substance transfer platform (100) according to this embodiment does not cause clogging, allowing for highly efficient intracellular substance transfer, and by moving cells at an appropriate flow rate, it is possible to achieve high cell survival rates.

[0053] The first supply unit (120) extends parallel to the first direction (x) and is connected to one end of the main channel (110). The second supply unit (130) may be connected from one end of the main channel (110) at an angle to the first supply unit (120). For example, the first supply unit (120) and the main channel (110) may extend from each other and be connected horizontally, 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 part (120) may be connected to one end of the main channel (110), and the other end of the first supply part (120) may be connected to the first supply chamber (121). The first supply chamber (121) is provided with a first fluid, cells (10), and substances (20), and can deliver the first fluid, cells (10), and substances (20) to the first supply part (120).

[0055] One end of the second supply part (130) may be connected to one end of the main channel (110), and the other end of the second supply part (130) may be connected to a second supply chamber (131). The second supply chamber (131) contains a second fluid, which can be delivered to the second supply part (130) at a predetermined flow rate.

[0056] A first fluid, cells (10), and substances (20) are delivered from a first supply unit (120) to one end of the main chamber (110), and a second fluid is delivered from a second supply unit (130) to one end of the main chamber (110) at an angle to the flow direction of the first fluid, so that the cells (10) and substances (20) contained in the first fluid may form droplets (50). These droplets (50) can pass through a compression block (150) of the main channel (110) while flowing together with the second fluid from one end to the other of the main channel (110).

[0057] The size and movement speed of the droplets 50 can be controlled 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 intracellular substance transfer platform 100 according to this embodiment can improve the efficiency of intracellular substance transfer by controlling the size and movement speed of the droplets 50 according to the size of cells 10 or the type of substance 20 to be transferred.

[0058] Specifically, the second supply section (130) may include first and second supply channels. The first and second supply channels may be a pair of microchannels, one on each side of the first supply section (120). The first and second supply channels may be connected to one end of the main channel (110) to form a cross-, Y-, or T-shaped junction (140).

[0059] Specifically, the first supply unit 120 may be extended and connected to the main channel 110. At the connection between the first supply unit 120 and the main channel 110, the first supply channel may be connected vertically from above, and the second supply channel may be connected vertically from below. The first fluid delivered through the first supply unit 120 may be formed into droplets 50 at the junction 140 by the second fluid delivered from the first and second supply channels, and the second fluid may flow into the main channel 110 together with the droplets 50.

[0060] The inner surface of the main channel 110 may be provided with a compression block 150. The compression block 150 may be provided on any one of the upper, lower, and side surfaces of the inner surface of the main channel 110, and may reduce the passage of the main channel 110. The compression block 150 may be formed with a hydrophobic surface so that the flow of the second fluid is not impeded.

[0061] The compression block (150) may be provided in an approximately box-like shape having a height (S1) protruding from the upper 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 to prevent vortex formation at the corners of the compression block (150).

[0062] The second fluid and droplets 50 may pass through a compression block 150 within the main channel 110. As the droplets 50 pass through the compression block 150, the reduced flow path and increased flow rate caused by the compression block 150 may cause the droplets 50 to deform, potentially deforming the cells 10 contained within the droplets 50. Nanopores may be formed in the cell membrane of the cells 10, allowing the substance 20 to be delivered into the cells through the nanopores. After the droplets 50 pass through the compression block 150, the shape changed by the compression block 150 may be restored to its original shape before passing through the compression block 150. At this time, the cells 10 within the droplets 50 may also deform along with the droplets 50 and restore their original shape. As the droplets 50 and cells 10 recover their shape, secondary vortices may be formed within the droplets 50. The vortex may facilitate the movement of the substance (20), thereby facilitating the transfer of the substance (20) into the cell (10).

[0063] When the inner diameter of the main channel (110) is a first diameter (a) and the gap between the compression block (150) and the inner surface of the main channel (110) is a second diameter (b), the second diameter (b) may be in the range of 0.1% to 85% of the first diameter (a). If the second diameter (b) is less than 0.1% of the first diameter (a), damage may occur to the cells (10) passing through the main channel (110) and the compression block (150). If the second diameter (b) is more than 85%, the droplets (50) may not be sufficiently pressurized by the compression block (150), reducing the efficiency of intracellular substance transfer.

[0064] The second diameter (b) may be in the range of 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 cells (10), causing physical deformation of the cells (10), thereby improving the efficiency of intracellular substance transfer.

[0065] The length (S2) of the compression block (150) in the direction parallel to the first direction (x) may range from 10 μm to 200 μm. If the length (S2) is less than 10 μm, material transfer within the cell (10) may be ineffective, and if it is greater than 200 μm, the contact area with the compression block (150) may increase, potentially damaging the cell (10). Specifically, the length (S2) of the compression block (150) in the direction parallel to the first direction (x) may range from 20 μm to 100 μm.

[0066] The height (S1) of the compression block (150) in a direction perpendicular to the first direction (x) may be set in a range of 15% to 99% of the first diameter (a). Specifically, the height (S1) of the compression block (150) in a direction perpendicular to the first direction (x) may be set in a range of 3 μm to 1.5 mm. Setting the height (S1) within the above range prevents clogging caused by the compression block (150) and allows for effective intracellular substance transfer.

[0067] The compression block (150) may be positioned at a first length (L1) from one end of the main channel (110). Specifically, the first length (L1) may be in the range of 30% to 90% of the total length (L2) from one end of the main channel (110) to the other. If the first length (L1) is set to less than 30% of the total length (L2), the flow of the droplets and the second fluid may become unstable before passing through the compression block (150). If the first length (L1) is set to more than 90%, the droplets and cells deformed after passing through the compression block (150) may not have enough time to recover.

[0068] The main channel (110) and the first and second supply portions (120, 130) may each be provided with a flow rate controller, which can control the velocity and Reynolds number of the first and second fluids passing through the main channel (110) and the first and second supply portions (120, 130). Specifically, the flow rate of the second fluid through the main channel (110) may range from 1 mL / h to 70 mL / h. If the flow rate of the second fluid through the main channel (110) is less than 1 mL / h, problems such as droplets (50) joining together may occur, and if it exceeds 70 mL / h, droplets (50) may not form smoothly at the junction (140).

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

[0070] Another embodiment of the present invention will be described below with reference to Figures 4 to 10. Except for the following content, the content is similar to that described in the embodiment described with reference to Figures 1 to 3, so detailed description will be omitted.

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

[0072] 4 and 5, the intracellular substance transfer platform (200) according to this embodiment may include a main channel (210) containing one or more droplets containing cells, substances, and a first fluid, and a second fluid for transporting the droplets, a first supply part (220) connected to one end of the main channel (210) for transferring the first fluid, cells, and substances, and a second supply part (230) connected at an angle to one end of the main channel (210) for transferring the second fluid. Droplets are formed at a junction (240) where the main channel (210) and the first and second supply parts (220, 230) intersect, and can be transferred from one end of the main channel (210) to the other end.

[0073] A compression block (250) is provided inside the main channel (210) and can apply pressure to droplets passing through the main channel (210). The compression block (250) can induce primary intracellular mass transfer due to instantaneous cell deformation caused by the pressure applied to the droplets, as well as secondary intracellular mass transfer due to vortices generated within the droplets after they pass through the compression block (250).

[0074] The intracellular substance transfer platform 200 may further include one or more subchannels 260 through which a second fluid flows. The subchannels 260 may be connected at an angle θ at a distance of a second length L4 from one end of the main channel 210. The subchannels 260 may be connected at an angle of approximately 30° to 75° relative to the main channel 210, so that the flow of the second fluid supplied from the subchannels 260 to the main channel 210 can prevent turbulence in the main channel 210 and ensure smooth droplet flow.

[0075] The sub-channel (260) can supply the second fluid to the main channel (210) through which the droplets and the second fluid flow after droplets are formed. The sub-channel (260) can be equipped with a flow rate controller that can control the rate at which the second fluid flows through the sub-channel (260).

[0076] The sub-channel (260) supplies the same second fluid from the outside to the flow of the second fluid flowing in the main channel (210), allowing the droplets to pass through the main channel (210) while preventing friction with the inner walls of the main channel (210).

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

[0078] The inner diameter (c) of the sub-channel (260) may be set in the range of 20% to 150% of 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 aforementioned range, the second fluid can easily move droplets without creating unnecessary turbulence in the flow of the second fluid within the main channel (210). For example, the inner diameter (c) of the sub-channel (260) may be 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 may be set to be 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 may be in the range of 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 disposed 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 to 5 times the second length (L4) from one end of the main channel (210) to the location of the subchannel (260). By controlling the first length (L3) and the second length (L4) within the aforementioned range, the efficiency of intracellular substance transfer by the compression block (250) within the main channel (210) can be improved, and even large amounts of cells can be effectively processed.

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

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

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

[0084] The main channel (310) may include an inlet portion (310a) connected to the first supply portion (320), a branch portion (310b) connected to the inlet portion (310a) and branching into multiple passages, and an outlet portion (310c) connected to the branch portion (310b). The branch portion (310b) may include multiple passages (311, 312, 313) with diameters smaller than those of the inlet portion (310a) and outlet portion (310c), and links where these passages (311, 312, 313) branch or connect to each other. A 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 from cells, substances, and a first fluid can be transferred together with a second fluid from the inlet 310a through the branch 310b to the outlet 310c. The droplets 50 placed in the inlet 310a may have a larger average diameter than the droplets 50 placed in the branch 310b or the outlet 310c.

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

[0087] The branch section (310b) may be connected from the inlet section (310a) to two primary passages (311) that form one and the other of the branch section (310b). Each primary passage (311) may branch into two secondary passages (312) through a link. These two secondary passages (312) may be extended separately and then reconnected through a link to form one tertiary passage (313). One tertiary passage (313) of the branch section (310b) and the other tertiary passage (313) of the branch section (310b) may be connected to the outlet section (310c).

[0088] While passing through branch portion 310b, droplets 50 can pass through branch portion 310b with their average diameter decreasing as the average diameter of branch portion 310b decreases compared to inlet portion 310a. The average diameter of droplets 50 can be approximately the same as or 20% or less than the average diameter of branch portion 310b. As droplets 50 pass through branch portion 310b, they flow in a single file, which prevents cell damage and effectively controls intracellular substance transfer.

[0089] One or more compression blocks (350) may be installed in one or more of the branch portion (310b) and the outlet portion (310c). The droplet (50) is pressurized as it passes through the compression block (350), temporarily deforming the shape of the cell and forming a nanopore in the cell membrane or nuclear membrane. The shape of the droplet also deforms, shortening the distance between the substance and the cell. The deformation of the first fluid flow within the droplet may allow the substance to be effectively delivered to the interior of the cell through the nanopore.

[0090] Fig. 7 is a view showing an intracellular substance transfer 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 view showing the branch portion of Fig. 7.

[0091] 7 to 9, the intracellular substance transfer platform (400) according to this embodiment may include 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) to supply a second fluid to the portion where the first supply unit (420) and the main channel (410) are connected. At a junction (440) such as a cross, Y, or T-shaped junction 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. The droplets can move within the main channel (410) together with the second fluid.

[0092] The main channel 410 may include inlets 410a, 410b, and 410c, branch sections 410d and 410e, and an outlet 410f, which are connected in sequence. The main channel 410 may further include one or more curved passages 410b disposed in the inlets 410a, 410b, and 410c or the branch sections 410d and 410e. Specifically, in this embodiment, the curved passages 410b may be disposed in the inlets 410a, 410b, and 410c. The curved passages 410b may be disposed in one or more curved shapes to control the flow speed of the second fluid and droplets 50 within the main channel 410 and generate vortices within the droplets. These vortices may improve the efficiency of mass transfer within the droplets and simultaneously mix the cells contained within the droplets.

[0093] When the cells in the droplets are uniformly mixed by the flow within the droplets generated by the curved passage (410b), the number of cells in the droplets can be evenly divided at the point where the second fluid flow branches. For example, when a droplet splits into two at the branching point, the cells in the droplets can also be split at an approximately 50:50 ratio, allowing for control to achieve uniform results for each batch.

[0094] The intracellular substance transfer platform (400) may include a section (1) where droplets (50) are formed, a section (2) where a second fluid flow containing the droplets is branched, and a section (3) where cells are mechanically perforated by mechanoporation, in which the droplets are pressurized by a compression block (450). The intracellular substance transfer platform (400) uses immiscible first and second fluids to form droplets (50) composed of cells, a substance, and the first fluid, and then moves the droplets (50) through the second fluid. The average diameter, movement speed, and movement pattern of the droplets (50) can be controlled by the average diameter of the main channel (410) and the number of passages. The intracellular substance transfer platform (400) further includes a compression block (450), which allows for the formation of reversible nanopores in cells by mechanoporation without damaging the cells or droplets (50). When the nanopore opens, the substance is delivered into the cell, and then it closes immediately afterwards to prevent the substance from being released outside the cell, allowing the substance to be delivered into the cell without damaging the cell.

[0095] Specifically, in the intracellular substance transfer system of Figure 7, the part (1) where droplets (50) are formed, the part (2) where the flow of the second fluid containing the droplets is branched, and the part (3) where the droplets are pressurized by the compression block (450) and the cells are mechanically perforated by mechanoporation are shown in the drawings obtained by conducting an experiment using the following method.

[0096] Specifically, the cells, material, and first fluid contained 40 million Jurkat cells (ATCC, TIB-152) per mL, the culture medium was a material obtained using ThermoFisher's Opti-mem, and the second fluid was fluorocarbon oil. Droplets composed of the cells, material, and first fluid were reduced in size to smaller droplets (50) as they passed through the branches (410d, 410e), and the smaller droplets (50) may contain relatively small numbers of cells. These droplets (50) were pressurized by the compression block (450), and the material in the droplets could be transferred to the interior of the cells in the mechanoporation section (3).

[0097] The inlet portions (410a, 410b, 410c) are connected to the junction (440) and may 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 the multiple passages and links is symmetrical with respect to an imaginary reference line (SL) that vertically connects the inlet portions (410a, 410b, 410c) and the outlet portion (410f). Specifically, one branch portion (410d) and the other branch portion (410e) may be connected to a pair of first passages (411) via a first link (D1) connected from the second portion (410c) about the imaginary reference line (SL). For example, the second portion (410c) and the pair of first passages (411) may be connected at an angle across the first link (D1) 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), and specifically, the average diameter (L6) of the first passage (411) may be set in the range of 40% to 80% of 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 of the second passages (412, 413) may be connected to a third link (D3). The third passages (414, 415) connected through the third link (D3) branch into a pair, and the end of each third passage (414, 415) may be connected to a fifth passage (417) that is further connected to another passage through a circular or polygonal fourth passage (416). The fifth passage (417) is connected through a fourth link (D4) to a sixth passage (418), and may be further connected to a seventh passage (419) through a fifth link (D5).

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

[0102] The branch portions 410d, 410e or the outlet portion 410f may include one or more compression blocks 450. The compression blocks 450 may physically compress the droplets 50 to facilitate intracellular substance transfer by mechanoporation. The compression blocks 450 may be installed in the branch portions 410d, 410e or the fourth passage 416.

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

[0104] Referring to Figure 10, the intracellular substance transfer platform (500) according to this embodiment may include a first supply section (520) that supplies cells, substances, and a first fluid, and a main channel (510) connected to the end of the first supply section (520) and through which substances are transferred into the cells as the droplets (50) move.

[0105] The main channel (510) may include inlet sections (510a, 510b, 510c) connected to a first supply section (520), branch sections (510d, 510e, 510f) connected to the inlet sections (510a, 510b, 510c) and consisting of multiple passages, and an outlet section (510g) connected to the ends of the branch sections (510d, 510e, 510f) and having a compression block (550) therein.

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

[0107] The inlet sections (510a, 510b, 510c) or branch sections (510d, 510e, 510f) are configured in a channel shape to provide a path through which the droplets and the second fluid pass, and may be configured to include a curved path (510b, 510f) that is at least partially straight and at least partially curved. The curved path (510b, 510f) is configured in a curved shape to generate a vortex within the droplet. This vortex can improve the efficiency of mass transfer within the droplet and also serve to mix the cells contained in the droplet. The curved path (510b, 510f) allows the cells within the droplet to be uniformly mixed, ensuring an equal number of cells within the droplet. Furthermore, the droplets (50) can move in an aligned manner as they pass through the curved path (510b, 510f). The curved passages (510b, 510f) may include a first curved passage (510b) disposed in the inlet portion (510a, 510b, 510c) and having an average diameter corresponding to the inlet portion (510a, 510b, 510c), and a second curved passage (510f) disposed in the branch portion (510d, 510e, 510f) and having an average diameter corresponding to the branch portion (510d, 510e, 510f).

[0108] The inlet portions (510a, 510b, 510c) include a first portion (510a) connected to the first supply portion (520) and a second portion (510c) connected to the branch portions (510d, 510e, 510f), and a first curved passage (510b) may be provided between the first portion (510a) and the second portion (510c). The first curved passage (510b) may be provided in a curved shape to allow cells in the droplets (50) formed in the first portion (510a) to be evenly distributed. The branch portions (510d, 510e, 510f) may include a pair of branch portions (510d) and (510e) having a passage formed in a pattern. In addition, the branch portions 510d, 510e, and 510f may have a curved second bent passage 510f connected to at least one of the branch portions 510d and 510e. The branch portions 510d and 510e may have passage shapes with the same pattern or different patterns.

[0109] The second portion (510c) may be branched into two passages, each connected to one branch portion (510d) having one or more second bend passages (510f) and the other branch portion (510e) having one or more second bend passages (510f). The one branch portion (510d) and the other branch portion (510e) may be arranged symmetrically with respect to the center.

[0110] The second curved passage (510f) is located at the beginning of one of the branch sections (510d) and the other of the branch sections (510e), respectively, and can align the second fluid flowing into the branch sections with the droplets (50) and evenly distribute the cells contained in the droplets (50).

[0111] Examples of the present invention and comparative examples are described below. However, the following examples are merely preferred examples of the present invention, and the scope of the present invention is not limited to the following examples.

[0112] 11 to 16 show the results of confirming intracellular substance transfer using the intracellular substance transfer platform according to the embodiment and comparing the efficiency of substance transfer with that of the prior art.

[0113] FIG. 11 shows the results of intracellular substance transfer confirmed using the intracellular substance transfer platform according to this embodiment.

[0114] As shown in Figure 11, the intracellular mass transfer platform was fabricated using polydimethylsiloxane (PDMS) (Dow, Sylgard 184) and a glass slide (Marienfeld Superior, HSU-1000612). A master mold with a microfluidic pattern was fabricated using silicon wafer photolithography and etching (DRIE). The microfluidic pattern was then replicated on the PDMS surface using PDMS soft lithography. The intracellular mass transfer platform was fabricated by oxygen plasma bonding of the PDMS with the microfluidic pattern to the glass slide using a plasma cleaner (Femtoscience, CUTE). To ensure stable droplet generation, the interior of the microchannels of the intracellular mass transfer platform was hydrophobized using 10 μL of surface coating oil (RAN Biotechnologies, 909-FluoroCoat). Bonding and drying were performed overnight at 75°C in a forced convection oven (Jeio Tech, OF4-S).

[0115] The intracellular mass transfer platform fabricated as described above has a main channel where droplets form and move, with a total length of 3.1538 mm and an average inner diameter of 80 μm. The compression block is placed 2.0538 mm from one end of the main channel, with a height of 4.8 μm and a length of 100 μm. The average inner diameter of the subchannel is 49 μm, and a pair of subchannels are connected at an angle of approximately 58 degrees, 1 mm from one end of the main channel.

[0116] Cell suspensions (ATCC, CCL-243) and fluorocarbon oil (Bio-Rad, Droplet Generation Oil) were used for intracellular substance transfer experiments using the intracellular substance transfer platform described above. The cell suspension used for the experiments contained 15 million K562 cells per mL. After cell preparation, a cell suspension was prepared in culture medium (Corning, RPMI) and impurities in the oil were removed using a 0.2 μm syringe filter (Advantec, 13HP020AN / 25HP020AN). The fluorocarbon oil and cell suspension were each injected into disposable syringes (BD, Luer-lok Tip Syringes), and the syringes and the microfluidic platform were connected with coupler tubing (IDEX, 1 / 32" OD PEEK Tubing). Using a syringe pump (Harvard Apparatus, 11 Elite Microfluidic Syringe Pump), the cell suspension and fluorocarbon oil were injected into the intracellular mass transfer platform at constant flow rates of 0.5 mL / h and 2.0 mL / h, respectively. The separately injected cell suspension and fluorocarbon oil met at the flow focusing junction, generating droplets within which the cells were trapped. The droplets, along with the fluorocarbon oil, passed through the main channel, maintaining their liquid state. At this time, fluorocarbon oil flowed in from a sub-channel connected to the main channel at a rate of 16 mL / h, accelerating the flow of droplets within the main channel. Furthermore, as additional fluorocarbon oil was supplied through the sub-channel, the droplets within the main channel were enveloped in the fluorocarbon oil and moved.

[0117] Next, as the droplet passed through a compression block placed in the main channel, it induced deformation of the cells within the droplet, forming nanopores in the cell membrane, and it was confirmed that substances coexisting within the droplet were delivered into the cells through these nanopores.

[0118] FIG. 12 shows the results of confirming the intracellular mass transfer efficiency depending on the size of the compressed block in the intracellular mass transfer platform according to FIG.

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

[0120] In the intracellular mass transfer platform shown in Figure 12, where the compression block was installed, the gap height between the inner surface of the main channel and the compression block was set to three different heights: 6.3 μm, 4.8 μm, and 3.6 μm. The intracellular mass transfer efficiency increased in the order of 6.3 μm, 4.8 μm, and 3.6 μm, with 3.6 μm showing the highest results. The mass transfer efficiency was also measured with the compression block length set to 40 μm, 70 μm, and 100 μm, with 100 μm showing the highest results. The intracellular mass transfer efficiency of the compression block from this experiment is shown in Tables 1 and 2.

[0121] [Table 1]

[0122] [Table 2]

[0123] FIG. 13 shows the results of confirming the intracellular substance transport efficiency according to the type of substance to be transported using the intracellular substance transport platform based on FIG.

[0124] In Figure 13, experiments were performed using a sample of 2,000 kDa FITC-conjugated dextran (Sigma Aldrich, FD2000S) diluted to a concentration of 0.3 mg / mL and a sample of green fluorescent protein-expressing mRNA (EGFP mRNA; TriLink, L-7601) diluted to a concentration of 20 μg / mL as the transport substance in a cell suspension. After 18 hours of incubation for 2,000 kDa FITC-Dextran transport and 24 hours for EGFP mRNA transport, the transport efficiency was analyzed compared to a control using a flow cytometer (Merck, Guava EasyCyte). Bright-field and GFP images of the control and transported samples were acquired using a light 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 example was cells exposed to the same concentrations of FITC-dextran or EGFP-mRNA as in the examples for the same period of time. The substance transport was performed in a cell suspension state using FITC-dextran or EGFP-mRNA, and the endocytosis effect was confirmed without using the intracellular substance transport platform corresponding to the examples.

[0126] In the control example, in which droplets and the intracellular material delivery platform were not used, it was confirmed that almost no material was delivered into the cells for both 2,000 kDa FITC-conjugated dextran and fluorescent protein-expressing mRNA. In contrast, when the intracellular material delivery platform according to the present invention was used, it was confirmed that material was delivered into the cells with high efficiency. Furthermore, as in this example, it was confirmed that high efficiency was achieved when the intracellular material delivery platform according to the present invention was used, regardless of the type of material being delivered into the cells. The intracellular material delivery efficiency using the compression block in this experiment is shown in Table 3 below.

[0127] [Table 3]

[0128] Figure 14 shows the results of intracellular gene transfer using a conventional general-purpose device. Figure 15 shows the results of comparing the efficiency of EMX1-targeted gene editing with that of a conventional general-purpose device. Figure 14 shows intracellular substance transfer data published in MaxCyte (2021), and Figure 15 shows the results of comparing the intracellular substance transfer platform (droplet) of the present invention with conventional general-purpose devices, such as an electroporation device (EP) and liposome nanoparticles (LNP), to confirm the efficiency of EMX1-targeted gene editing in K562 cells.

[0129] In the experiment shown in Figure 15, the experimental method shown in Figure 11 was used. 500 pmol of single guide RNA (sgRNA) targeting the EMX1 gene in K562 cells and endotoxin-free Cas9 protein were added to the cell suspension. After culturing the cells treated with the intracellular delivery platform for 48 hours, genomic DNA (gDNA) was extracted using a DNA extraction kit (Intronbiotechnology, G-spin™ Total DNA Extraction Mini Kit). The gDNA was amplified by polymerase chain reaction (PCR) using a thermal cycler (Bio-Rad, T100). The gene editing efficiency was analyzed using a microspectrophotometer (ThermoFisher, NanoDrop One) after treatment of 200 ng of PCR product with 10 U of T7 endonuclease 1 (New England BioLabs, M0302S) at 37°C for 15 minutes, followed by 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 transfer platform (droplet) according to the present invention was shown to have gene editing efficiency far superior to that of electroporation devices (EP) and liposome nanoparticles (LNP), confirming that it is capable of highly efficient multiplexing editing (editing of two or more targets). Furthermore, the intracellular substance transfer platform of this example was capable of highly efficient editing by Homology Directed Repair (HDR). In other words, it was confirmed that the intracellular substance transfer platform according to the present invention can deliver substances into cells more efficiently than conventional technologies without damaging the cells, and can be applied to a variety of cells without restriction.

[0131] Figure 16 shows the results of intracellular substance transfer confirmed using the droplet separation-type intracellular substance transfer platform. Figure 16 shows the results of using the droplet separation-type intracellular substance transfer platform based on Figure 7.

[0132] The droplet separation-type intracellular substance transfer platform used in FIG. 16 was manufactured by the same method as the intracellular substance transfer platform based on FIG. 11 described above.

[0133] The intracellular mass transfer platform shown in Figure 16 has a main channel where droplets are formed and transported. The inlet, where droplets are formed, has a straight passageway length of 1.4 mm and an average inner diameter of 0.4 mm. The following curved passageway is 2.51 mm long and has an average inner diameter of 0.2 mm. The following straight passageway is 1 mm long and has an average inner diameter of 0.2 mm. A pair of branch sections are connected to this, and the branch section passages are connected with an average inner diameter of 0.09 mm. The press block is 8 μm high and 70 μm long.

[0134] The cell suspension used in this experiment contained K562 cells (ATCC, CCL-243) diluted with the transporter, 3kDa FITC-conjugated dextran (Sigma Aldrich, FD4), at a concentration of 0.3 mg / mL. After operating the intracellular transport platform, the cells were separated from the trapped droplets by demulsification using PFO solution (Sigma Aldrich, 1H,1H,2H,2H-Perfluoro-1-octanol). Cell viability was measured using an automated cell counter (Logos biosystems, Luna-FX7) and confirmed to be approximately 80%. After culturing the cells for 18 hours, they were analyzed using a flow cytometer (BD, FACSLyric). Compared to the control, the transduction efficiency was over 90% and the mean fluorescence intensity fold change was over 100-fold. Bright field and GFP images of the control and substance transfer samples were obtained using an optical microscope (Zeiss, Axio Observer 7) and a camera (ZEISS, Axiocam 305 mono), and a qualitative comparison of the level of fluorescence expression confirmed that the use of the intracellular substance transfer platform according to the present invention showed higher substance transfer efficiency. Here, the control was performed in a cell suspension state using cells exposed to the same concentration of transport substance as in the examples for the same period of time, and the substance transfer effect due to the influence of endocytosis was confirmed without using the intracellular substance transfer platform according to the examples.

[0135] Those skilled in the art will understand that the present invention can be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. Therefore, the above-described embodiments should be understood to be illustrative in all respects and not limiting. The scope of the present invention is defined by the claims that follow rather than the above detailed description, and it is intended that the meaning and scope of the claims and all modifications and variations derived therefrom are included within the scope of the present invention. [Explanation of symbols]

[0136] 100, 200, 300, 400, 500: Intracellular substance transfer platform 110, 210, 310, 410, 510: Main channels 120, 220, 320, 420, 520: 1st supply section 130, 230, 330, 430: 2nd supply section

Claims

1. An intracellular substance transfer 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 unit connected to one end of the main channels and injecting a first fluid containing the cells and a substance; and a second supply unit connected to one end of the main channels and injecting a second fluid immiscible with the first fluid; wherein the main channels include one or more compressing blocks; an inner diameter of the main channel in a region where the compression block is located is defined as a first diameter; The main channel includes: an inlet portion connected to the first supply portion; a branch portion connected to the inlet portion and branching into a plurality of passages; and an outlet portion connected to the branch portion; An intracellular substance transfer platform comprising:

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. 4. The intracellular substance transfer platform of claim 3, wherein the first fluid is transferred from the first supply unit to one end of the main channel, and the second fluid is transferred from the second supply unit 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, and the first and second supply channels are 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 1 mL / h to 70 mL / h.

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

9. The intracellular substance transfer platform of claim 8, wherein the subchannels include first and second subchannels, which are connected to the main channel at a portion spaced a second length from one end of the main channel, and the first and second subchannels are connected to one side and the other side 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 the first diameter, which is the 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 9, 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 1.1 to 5 times the second length.

14. The intracellular substance transfer platform according to 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 of 10 μm to 200 μm in a direction parallel to the first direction.

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

17. The intracellular substance transfer platform of claim 1, wherein the gap between the compression block and the inner surface of the main channel is a second diameter, and the second diameter is 0.1% to 85% of the first diameter, which is the 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. 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 3 μm to 1.5 mm.

21. The intracellular substance transfer platform of claim 1, wherein the branch section includes a plurality of passages each having a diameter smaller than that of the inlet section and the outlet section, and links at which the plurality of passages branch or connect to each other, and the compression block is provided in the branch section or the outlet section.

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

24. The intracellular substance transfer platform according to claim 21 , wherein the main channel further comprises one or more curved passages provided in the inlet section or the branch section.

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, and a nanoparticle.

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