Transformation cartridge, transformation device, transformation system including the same, and transformation method using the same

Abstract

The transformation cartridge according to the present invention comprises a main body portion in which a cell flow path, a material flow path and a resultant flow path are formed adjacent to each other, and an electric field forming portion including electrodes coupled to the main body portion to generate an electric field in the resultant flow path, wherein the cell flow path and the material flow path are formed in a form converging into the resultant flow path, and the resultant flow path includes a mixing flow path extending from a point connected to the cell flow path and the material flow path, and the thickness of the mixing flow path is smaller than the thickness of the cell flow path.

Description

[Technical Field]

[0001] The present invention relates to a transformation cartridge, a transformation device, a transformation system including the same, and a transformation method using the same. [Background technology]

[0002] Well-known gene transfer techniques for transformation include viral vector-based transfer methods that use viruses as carriers and nonviral delivery techniques that use synthetic phospholipids or synthetic cationic polymers.

[0003] Viruses, by their very nature, live by parasitizing other cells with their own DNA inserted into their nuclei, and are therefore considered to be highly efficient DNA delivery systems. In other words, in viral DNA delivery systems, the gene to be delivered is inserted into a virus that is unable to replicate and then used. This technique has the advantage of being highly efficient for transformation. However, this technique has several drawbacks. Essentially, these include the possibility of the emergence of recombination competent viruses (RCVs), even though inactivated viruses are used; the difficulty of repeated use due to immune responses; the limitation of substances that can be delivered due to their inherent characteristics; and the difficulty of injecting a predetermined amount of substance into cells.

[0004] To avoid using viral vectors, a method called electroporation can be used, in which an electric shock is applied to cells with a substance located around them to open the phospholipid bilayer. However, due to the nature of the substance to be delivered, in general electroporation, cells must be placed in a special buffer (e.g., RNase-free buffer) rather than in culture medium. This requires pretreatment, such as cell washing, in which the culture medium is wiped off the cells using methods such as centrifugation.

[0005] When using such a conventional electroporation method, there are problems such as a significant decrease in cell viability and a large amount of cell loss during the pretreatment process, the special buffer used is very expensive and uneconomical, and it is difficult to perform pretreatment and other operations in a closed environment, making automation difficult and maintaining a hygienic working environment difficult. Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention has been developed to solve these problems, and provides a transformation cartridge, a transformation device, a transformation system including the same, and a transformation method using the same, which enable transformation by electroporation simply by adding a cell solution and a material solution. [Means for solving the problem]

[0007] A transformation cartridge according to an embodiment of the present invention includes a main body portion in which a cell flow path, a material flow path, and a resultant flow path are formed, and an electric field forming portion including electrodes coupled to the main body portion to generate an electric field in the resultant flow path, wherein the cell flow path and the material flow path are formed in a shape converging into the resultant flow path, and the resultant flow path includes a mixing flow path extending from a point connected to the cell flow path and the material flow path, and the thickness of the mixing flow path is smaller than the thickness of the cell flow path.

[0008] A transformation cartridge according to an embodiment of the present invention includes a main body portion in which a cell channel, a material channel, and a resultant channel are formed, and an electric field forming portion connected to the main body portion and including electrodes disposed at the inlets of the material channels and the outlets of the resultant channel, wherein the cell channel and the material channel are formed to converge at the resultant channel, and the resultant channel includes a mixing channel extending from a point connected to the cell channel and the material channel, and the value obtained by dividing the thickness of the cell channel by the sum of the thickness of the cell channel and the thickness of the material channel and multiplying it by the thickness of the mixing channel is smaller than the diameter of cells flowing in the cell channel.

[0009] A transformation device according to an embodiment of the present invention includes a pump unit configured to pump a cell solution and a material solution into the cell flow path and material flow path of the transformation cartridge, respectively; a power application unit configured to be connected to terminals of the transformation cartridge to apply power to electrodes of the transformation cartridge; and a processor electrically connected to the power application unit and the pump unit, wherein the processor controls the pump unit based on information about the transformation cartridge, a preset flow rate ratio, and a preset exposure time.

[0010] A transformation system according to an embodiment of the present invention includes a main body portion in which a cell flow path, a material flow path, and a resultant flow path are formed, the cells being adjacent to each other; an electric field forming portion connected to the main body portion and including electrodes disposed at the inlets of the material flow paths and the outlets of the resultant flow paths; a pump portion configured to pump a cell solution and a material solution into the cell flow paths and the material flow paths, respectively; and a processor electrically connected to the pump portion, wherein the cell flow paths and the material flow paths are formed to converge into the resultant flow path, and the resultant flow path includes a mixing flow path extending from a point connected to the cell flow path and the material flow path; and the processor controls the pump portion so that the thickness of the solution flowing into the mixing flow path via the cell flow path is smaller than the diameter of the cells contained in the cell solution.

[0011] A transformation method according to an embodiment of the present invention includes the steps of preparing a cartridge having a cell flow path, a material flow path, and a mixing flow path that are adjacent to each other, injecting a cell solution containing cells into the cell flow path, and injecting a material solution containing a material for transforming the cells into the material flow path, wherein the ratio of the flow rate of the injected cell solution to the flow rate of the material solution is a value that allows the cells and the material to come into contact in the mixing flow path and undergo transformation. [Effects of the Invention]

[0012] This allows transformation by electroporation simply by adding a cell solution and a substance solution.

[0013] Transformation can be performed without mixing the substance solution and the cell solution.

[0014] Pretreatment processes such as cell washing can be omitted for cells and transformation substances, the cell survival rate during the process is increased, and cell and substance losses can be significantly reduced.

[0015] Since no additional special buffers need to be used, an economical transformation process can be constructed.

[0016] Since no pretreatment is required and the transformation process can be carried out simply by injecting the solution into a sealed and controlled environment, a hygienic working environment can be maintained, which is advantageous in terms of various regulations.

[0017] Since the entire process required for transformation is simply to inject a culture medium containing cells and a substance solution and then apply an electric field, the process can be easily automated. [Brief explanation of the drawings]

[0018] [Figure 1] FIG. 1 is a perspective view of a transformation cartridge according to a first embodiment of the present invention.

[0019] [Figure 2] FIG. 1 is an exploded perspective view of a transformation cartridge according to a first embodiment of the present invention.

[0020] [Figure 3] FIG. 1 is a plan view of a transformation cartridge according to a first embodiment of the present invention.

[0021] [Figure 4] FIG. 1 is a conceptual diagram of a main body of a transformation cartridge according to a first embodiment of the present invention.

[0022] [Figure 5] FIG. 10 is a conceptual diagram of a main body of a transformation cartridge according to a second embodiment of the present invention.

[0023] [Figure 6] FIG. 10 is a conceptual diagram of a main body of a transformation cartridge according to a third embodiment of the present invention.

[0024] [Figure 7] FIG. 10 is a perspective view of a transformation cartridge according to a fourth embodiment of the present invention.

[0025] [Figure 8] FIG. 10 is an exploded perspective view of a transformation cartridge according to a fourth embodiment of the present invention.

[0026] [Figure 9] FIG. 1 is a diagram showing a transformation system including a transformation cartridge and a transformation device according to a first embodiment of the present invention.

[0027] [Figure 10] FIG. 10 is a diagram showing a transformation system including a transformation cartridge and a transformation device according to a fourth embodiment of the present invention.

[0028] [Figure 11] FIG. 2 is a diagram illustrating cell sedimentation performed in the transformation cartridge according to the first embodiment of the present invention.

[0029] [Figure 12] FIG. 10 is a diagram illustrating cell sedimentation performed in a transformation cartridge according to a third embodiment of the present invention.

[0030] [Figure 13] FIG. 1 shows the results of an experiment confirming the transformation efficiency of mRNA using the Neon™ Transfection System (ThermoFisher) and the transformation efficiency of mRNA using the transformation cartridge of the present invention.

[0031] [Figure 14] FIG. 11 shows the results of an experiment confirming the effect of the flow rate ratio between the two solutions in the mixing channel on the transfer efficiency of substances such as mRNA during transformation by separately supplying a cell solution and a substance solution using the transformation cartridge of the present invention.

[0032] [Figure 15] This figure shows the results of an experiment conducted three times to confirm whether the transformation cartridge of the present invention can deliver substances with high efficiency to cells (primary cells) taken from the human body.

[0033] [Figure 16] FIG. 1 shows the results of an experiment confirming the production efficiency (transduction efficiency) of CAR-NK cells (A) produced using the transformation cartridge of the present invention and the degree of killing of cancer cells (B). DETAILED DESCRIPTION OF THE INVENTION

[0034] Hereinafter, some embodiments of the present invention will be described in detail with reference to the exemplary drawings. When assigning reference numerals to components in each drawing, it should be noted that the same reference numerals are used for the same components even if they are depicted in different drawings. Furthermore, when describing the embodiments of the present invention, if it is determined that a detailed description of related known structures or functions would hinder understanding of the embodiments of the present invention, the detailed description will be omitted.

[0035] Furthermore, when describing components of an embodiment of the present invention, terms such as first, second, A, B, (a), (b), etc. may be used. These terms are used to distinguish the component from other components, and do not limit the nature, order, or sequence of the components. When a component is described as being "coupled," "coupled," or "connected" to another component, it should be understood that the component may be directly coupled or connected to the other component, but that there may also be other components "coupled," "coupled," or "connected" between the components.

[0036] First embodiment

[0037] Fig. 1 is a perspective view of a transformation cartridge 1 according to a first embodiment of the present invention. Fig. 2 is an exploded perspective view of the transformation cartridge 1 according to the first embodiment of the present invention. Fig. 3 is a plan view of the transformation cartridge 1 according to the first embodiment of the present invention.

[0038] The transformation cartridge 1 according to the first embodiment of the present invention includes a main body 10 and an electric field generating unit 20. In the present specification, the directions of up (UP), down (DOWN), left (LEFT), right (RIGHT), front (FRONT), and back (BACK) are mutually orthogonal directions used for convenience of explanation, and are relative directions that may change depending on the arrangement state of the transformation cartridge 1.

[0039] The electric field forming unit 20 is a component that forms an electric field in the main body 10. The electric field forming unit 20 is coupled to the main body 10 and includes electrodes that are arranged at the inlet 130 of the material flow path 13 and the outlet 140 of the resultant flow path 14 so as to generate an electric field in the resultant flow path 14. The electric field forming unit 20 may be, but is not limited to, a printed circuit board.

[0040] The electric field generating unit 20 may include a plate-shaped electric field generating body 21. The electric field generating body 21 may be made of a multi-layered epoxy resin, but the material is not limited thereto. Electrodes may be formed on the electric field generating body 21, and each electrode may be formed in a ring shape and surround each hole formed in the electric field generating unit 20 to correspond to each flow path of the main body 10.

[0041] The electrodes 22, 23, and 24 may be made of a conductor such as a metal. The electrodes may be formed by coating copper on the electric field generating body 21. The electrodes of the electrode electric field generating unit 20 may include a cell electrode 22, a material electrode 23, and a result electrode 24. The electric field generating unit 20 may include terminals 26 and 27 electrically connected to the electrodes 22, 23, and 24 and connectable to a power source. The terminals 26 and 27 may be made of the same material as the electrodes 22, 23, and 24. The transformation cartridge 1 according to the first embodiment of the present invention may include a material terminal 26 electrically connected to the material electrode 23 and a result terminal 27 electrically connected to the result electrode 24. Each terminal 26 and 27 may be disposed on the outside of the electric field generating body 21 so as to be easily connected to a power source.

[0042] The electric field generating unit may include electrodes disposed inside the main body so as to surround each flow path and coupled to the main body. Such electrodes may also be disposed in the mixing flow path.

[0043] The electric field generating unit 20 can generate an electric field using a simple DC power supply, but it can also generate an electric field using a pulsed power supply. A processor 201 of the transformation device (2 in FIG. 9 ) described below can control the electric field generating unit 20 using a power applying unit 203 electrically connected thereto in a PWM control manner. The pulse frequency can be 1 kHz to 100 kHz, and the duty ratio can be 10% to 90%. The electric field generating unit 20 can generate an electric field while varying its size. The size of the electric field can be varied by decreasing it to 50% of the maximum size of the electric field generated by the electric field generating unit 20.

[0044] The transformation cartridge 1 according to the first embodiment of the present invention may include a sealing sheet 30. The sealing sheet 30 is located between the main body 10 and a lower mount 63 (described later) and prevents shock from being immediately transmitted from the lower mount 63 to the main body 10, thereby stably supporting the main body 10. The sealing sheet 30 may be made of a material containing silicone, but the material is not limited thereto. Holes are formed on the sealing sheet 30 at positions corresponding to the lower sides of the inlets of each of the channels 12, 13, and 14, allowing the status of each of the channels 12, 13, and 14 to be checked from below to above.

[0045] The transformation cartridge 1 according to the first embodiment of the present invention may include an O-ring unit 40. The O-ring unit 40 is composed of elastic annular O-rings 42, 43, and 44 and is positioned between the electric field generating unit 20 and the guide unit 50 (described later). The O-ring unit 40 maintains a watertight seal between the electric field generating unit 20 and the guide unit 50 and allows a needle inserted into the guide unit 50 to be inserted stably into the solution. The O-ring unit 40 may be made of a material including, but not limited to, silicon. The O-ring unit 40 may include a cell O-ring 42 positioned between the cell electrode 22 and the cell guide 52, a material O-ring 43 positioned between the material electrode 23 and the material guide 53, and a resultant O-ring 44 positioned between the resultant electrode 24 and the resultant guide 54.

[0046] The transformation cartridge 1 according to the first embodiment of the present invention may include a guide unit 50. The guide unit 50 may serve as an inlet and an outlet for a solution. The guide unit 50 may include a plate-shaped guide body 51 and a cell guide 52, a substance guide 53, and a product guide 54 connected to the guide body 51. Each guide 52, 53, and 54 may be formed as a pipe extending upward from the guide body 51. Each guide 52, 53, and 54 has a vertically penetrating shape and is disposed at a position corresponding to each electrode 22, 23, and 24 and each channel 12, 13, and 14. Therefore, when a needle is inserted into the guide 52, 53, and 54 and a liquid is discharged by, for example, connecting it to a pump, the discharged liquid can be delivered to each channel via each electrode. A needle can be stably inserted into each guide 52, 53, and 54, enabling liquid to be discharged at the appropriate position. O-rings 42, 43, and 44 may be disposed under the guides 52, 53, and 54, respectively, and the electric field generating unit 20 may be located under the guide body 51. The guide unit 50 may be made of a material including polycarbonate, but the material is not limited thereto.

[0047] The transformation cartridge 1 according to the first embodiment of the present invention may include an upper mount 61 and a lower mount 63. The upper mount 61 may be located above the guide unit 50, and the lower mount 63 may be located below the sealing sheet 30. The upper mount 61 and the lower mount 63 may be fastened to each other with fasteners such as bolts, thereby fixing the sealing sheet 30, the main body 10, the electric field generating unit 20, the O-ring unit 40, and the guide unit 50 located therebetween in a stacked state. The upper mount 61 and the lower mount 63 apply pressure to the components therebetween, thereby enabling effective sealing. The upper mount 61 may have a hole through which the guide can pass, and the lower mount 63 may have a hole at a position corresponding to the hole in the sealing sheet 30. The upper mount 61 and the lower mount 63 may be made of a material containing aluminum, which may be anodized aluminum, but the material is not limited thereto.

[0048] The transformation cartridge 1 according to the first embodiment of the present invention may include a body mount 62. The body mount 62 has a hole in the center into which the body unit 10 is inserted, allowing the body unit 10 to be aligned with other components when positioned appropriately. The body mount 62 is located between the upper mount 61 and the lower mount 63, and may be further connected via a fastener. The body mount 62 may be made of a material containing aluminum, and such aluminum may be anodized aluminum, but the material is not limited thereto.

[0049] FIG. 4 is a conceptual diagram of the main body 10 of the transformation cartridge 1 according to the first embodiment of the present invention.

[0050] The main body 10 has a cell channel 12, a material channel 13, and a product channel 14 that are adjacent to each other. The channels 12, 13, and 14 may be formed by penetrating the main body 11. The main body 11 may be made of a material including glass, and the glass may be borosilicate glass, but the material is not limited thereto.

[0051] The inlets 120, 130 or outlets 140 of the channels 12, 13, and 14 may be arranged on the top surface of the main body 11. The inlet 130 of the material channel 13, the inlet 120 of the cell channel 12, and the outlet 140 of the resultant channel 14 may be arranged in this order, moving forward as a reference direction. A cell solution SC may be introduced into the inlet 120 of the cell channel 12, a substance solution SM may be introduced into the inlet 130 of the material channel 13, and the resultant solution may be discharged from the outlet 140 of the resultant channel 14.

[0052] The cell flow channel 12 is a channel through which the cell solution SC is introduced and flows. The cell solution SC may be composed of a culture medium or electrolyte containing cells CO. The cells CO contained in the cell solution SC may be blood cells, which are somatic cells, and may be immune cells in particular. The immune cells may be any cells capable of inducing immunity and eliciting the desired therapeutic effect, and may be, but are not limited to, any one selected from the group consisting of natural killer cells (NK cells), T cells, natural killer T cells (NKT cells), cytokine-induced killer cells (CIK), macrophages, and dendritic cells. The cell flow channel 12 may include a cell inlet channel 121 extending from an inlet 120 of the cell flow channel 12. The cell inlet channel 121 may extend downward from the inlet 120 of the cell flow channel 12.

[0053] The material flow channel 13 is a channel through which a material solution SM is introduced and flows. The material solution SM may be water or a buffer containing a transforming material M. The transforming material M is introduced into the cell C0, for example, into the cytoplasm or nucleus, to perform its function in the cell C0. The transforming material M may be in any form, such as a protein, peptide, or nucleic acid, as long as it can perform its function in the cell C0. In particular, the transforming material M may be a nucleic acid, such as mRNA. The material flow channel 13 may include a material inlet flow channel 131 extending from an inlet 130 of the material flow channel 13. The material inlet flow channel 131 may extend downward from the inlet 130 of the material flow channel 13. The material flow channel 13 may include a material transfer channel 132 extending from a lower end of the material inlet flow channel 131 toward the resultant flow channel 14. The material transfer channel 132 may extend forward, i.e., horizontally. Therefore, the material flow channel 13 can bend at the location where the material inlet channel 131 and the material transfer channel 132 meet, and the angle of the bend may be 90 degrees.

[0054] The cell channel 12 and the material channel 13 can be in contact with each other with the cell channel 12 positioned above the material transfer channel 13. The lower end of the cell inlet channel 121 can be positioned above the front end of the material transfer channel 132. Therefore, at the point where the material channel 13 and the cell channel 12 are in contact with each other, the cell solution SC can flow into the resultant channel 14 while being positioned above the material solution SM.

[0055] The resultant flow channel 14 is a channel through which a resultant solution containing transformed cells C1 flows. The resultant solution can be discharged through an outlet 140 of the resultant flow channel 14. The cell flow channel 12 and the material flow channel 13 are formed to converge at the resultant flow channel 14. Therefore, the cell solution SC and the material solution SM are combined to form a resultant solution.

[0056] The resultant flow channel 14 includes a mixing channel 141 extending from a point where it is connected to the cell flow channel 12 and the material flow channel 13. The mixing channel 141 may extend horizontally forward from the point. The resultant flow channel 14 may include a resultant discharge channel 142 connecting the end of the mixing channel 141 to an outlet 140 of the resultant flow channel 14. The resultant discharge channel 142 may extend upward from the front end of the mixing channel 141.

[0057] Because the cell channel 12 contacts the substance transfer channel 132 and the mixing channel 141 in a non-parallel manner, the flow of the cell solution SC bends at the contact point. The cell solution SC can bend 90 degrees. Because the direction of the flow of the cell solution SC changes at the point where the channels contact, the resulting centrifugal acceleration causes the cells C0 to settle downward, allowing them to easily come into contact with the substance M.

[0058] The electric field forming unit 20 can form an electric field along the solutions flowing in the channels 12, 13, and 14 using the electrodes 22, 23, and 24. The electric field forming unit 20 can form an electric field in the material channel 13 and the result channel 14 using the material electrode 23 and the result electrode 24. The electric field forming unit 20 can form an electric field in the cell channel 12 and the result channel 14 using the cell electrode 22 and the result electrode 24. The electric field formed in the direction from the cell channel 12 or the material channel 13 to the result channel 14 is represented by dotted lines in the drawing. By forming an electric field, the flow direction of the solutions SC and SM in the channels 12, 13, and 14 can be regulated, and transformation by electroporation can be performed in the mixing channel 141, which will be described later.

[0059] The transforming substance M in the substance solution SM can enter the interior of the cells C0 in the cell solution SC from the mixing channel 141. The electric field applied to the mixing channel 141 locally opens the phospholipid bilayer membrane of the cells C0, allowing the transforming substance M to enter the cells C0.

[0060] The thickness T11 + T21 of the mixing channel 141 may be smaller than the thickness T10 of the cell channel 12. Here, the thickness of the channels 12, 13, and 14 refers to the distance between the boundaries of the channels 12, 13, and 14 in a direction perpendicular to the flow direction of the solutions SC and SM in the channels 12, 13, and 14, as seen in a cross section of the channels 12, 13, and 14 cut along a plane perpendicular to the left-right direction as shown in FIG. 4. When the channels 12, 13, and 14 are formed as pipes extending from a cylinder, the thickness of the channels 12, 13, and 14 may be the inner diameter of the channels 12, 13, and 14. The thickness T11 + T21 of the mixing channel 141 may be smaller than the thickness T20 of the material channel 13. The thickness of the mixing channel 141 may be 6 μm or more and 400 μm or less.

[0061] The thickness T11 + T21 of the mixing channel 141 may be smaller than the thickness T10 of the cell channel 12 or the thickness T20 of the material channel 13, which converge into the resultant channel 14. The thickness T11 of the cell solution SC in the mixing channel 141 may be smaller than the thickness T10 of the cell channel 12, which is the thickness of the cell solution SC in the cell channel 12. Similarly, the thickness T21 of the material solution SM in the mixing channel 141 may be smaller than the thickness T20 of the material channel 13, which is the thickness of the material solution SM in the material channel 13. Here, if the thickness T11 of the cell solution SC in the mixing channel 141 is smaller than the diameter of the cell C0, a portion of the cell C0 may be exposed to the material solution SM and come into contact with the transforming substance M. An electric field is formed in the mixing channel 141, allowing the transforming substance M to enter the cell C0 and form a transformed cell C1. Because a portion of the cell C0 is exposed to the material solution SM, transformation can occur even if the material solution SM and the cell solution SC are not mixed.

[0062] To enable the above-described flow, the value obtained by dividing the thickness of the cell flow path 12 by the sum of the thickness of the cell flow path 12 and the thickness of the material flow path 13 and multiplying the result by the thickness of the mixing flow path 141 can be smaller than the diameter of the cell C0 flowing in the cell flow path 12.

[0063] The flow rate at which each solution is injected may be controlled to determine the thickness T11 of the cell solution SC flowing within the mixing channel 141. The processor 201 can control the pump unit 202 so that the thickness T11 of the cell solution SC, which is a solution flowing into the mixing channel 141 via the cell channel 12, within the mixing channel 141 is smaller than the diameter of the cells contained in the cell solution SC. With other conditions remaining the same, if the pressure or flow rate at which the pump unit 202 pumps the cell solution SC increases, the thickness T11 of the cell solution SC within the mixing channel 141 can increase.

[0064] In order to allow the cells C0 to be easily exposed to the substance solution SM in the mixing channel 141, the above-mentioned cell channel 12 can be placed above the substance channel 13 and come into contact with each other. With this channel arrangement, the cell solution SC is placed above the substance solution SM in the mixing channel 141, so that the cells C0 carried by the cell solution SC are moved by gravity toward the substance solution SM below, allowing them to be better exposed to the substance solution SM.

[0065] The structure of the main body 10 described above allows transformation to occur without any pretreatment of the cells or transforming substance M by simply injecting them into the main body 10 and applying an electric field, yielding transformed cells C1. This eliminates the need for pretreatment processes such as cell washing, significantly reducing cell and substance loss during the process. Furthermore, since no special buffers are required, the process can be configured economically. Furthermore, since pretreatment is not required and the process can be configured simply by injecting the solution into a sealed, controlled environment, a hygienic working environment can be maintained, which is advantageous in terms of various regulations. Simply injecting the cell-containing culture medium and substance solution and applying an electric field is the entire process required for transformation, making the process easily automatable.

[0066] Second embodiment

[0067] FIG. 5 is a conceptual diagram of the main body 10b of the transformation cartridge according to the second embodiment of the present invention.

[0068] The transformation cartridge according to the second embodiment of the present invention is the same as the transformation cartridge 1 according to the first embodiment except for the additional second material flow path 1302b. Therefore, only the differences will be described below, and the description of the transformation cartridge 1 according to the first embodiment is applicable to the remaining components. The electric field generating unit, guide, O-ring, etc. of the transformation cartridge according to the second embodiment are formed with portions for the second material flow path 1302b.

[0069] The material flow path 13b formed in the main body 11b includes a first material flow path 1301b through which a first material solution SM1 containing a first transforming substance M1 flows and a second material flow path 1302b through which a second material solution SM2 containing a second transforming substance M2 flows. The first transforming substance M1 and the second transforming substance M2 may be different from each other. The first material flow path 1301b may be the same as the material flow path 13 according to the first embodiment. Therefore, the first material flow path 1301b is considered to be the same as the material flow path 13 of the first embodiment, and the second material flow path 1302b will be further described.

[0070] A first material flow path 1301b contacts the cell flow path 12b on one side of the cell flow path 12b, and a second material flow path 1302b contacts the cell flow path 12b on the other side of the cell flow path 12b. The first material flow path 1301b contacts the cell flow path 12b on the lower side of the cell flow path 12b, and the second material flow path 1302b contacts the cell flow path 12b on the upper side of the cell flow path 12b. Therefore, in the resultant flow path 14b, as described in the first embodiment, the first material solution SM1 flows above the cell solution SC and the second material solution SM2 flows below, exposing the cells to the first material solution SM1 and the second material solution SM2. The cells exposed to each material solution SM are released by the electric field, allowing the first transforming substance M1 and the second transforming substance M2 to enter the cells and undergo transformation.

[0071] Therefore, according to the second embodiment, a plurality of different transforming substances M1 and M2 can be simultaneously injected into cells using one transformation cartridge to form transformed cells C2.

[0072] Third embodiment

[0073] FIG. 6 is a conceptual diagram of a main body 10c of a transformation cartridge according to a third embodiment of the present invention.

[0074] The transformation cartridge according to the third embodiment of the present invention is identical to the transformation cartridge 1 according to the first embodiment except for the shape of the flow paths 12c, 13c, and 14c formed in the main body portion 10c. Therefore, only the differences will be further described, and the description of the transformation cartridge 1 according to the first embodiment can be applied to the remaining components.

[0075] The appearance of the main body 11c according to the third embodiment may be the same as the appearance of the main body 11 according to the first embodiment. The material channel 13c according to the third embodiment may have a downwardly convex arc shape. The material channel 13c and the resultant channel 14c may have a downwardly convex continuous arc shape. That is, the material channel 13c and the resultant channel 14c may be connected while drawing a downwardly convex continuous arc. Although the cell channel 12c is illustrated as having a curved profile with an arc shape, the cell channel 12c may also have a downwardly extending shape. When the cell channel 12c has an arc shape, the radius of curvature of the cell channel 12c may be smaller than the radius of curvature of the material channel 13c.

[0076] Since the substance flow path 13c has an arc shape, centrifugal acceleration is applied to the transforming substance introduced into the substance flow path 13c, making it easier for the transforming substance to come into contact with the cells.

[0077] The arc-shaped flow path as in the third embodiment can also be applied to the first material flow path 1301b and the second material flow path 1302b in the second embodiment.

[0078] Fourth embodiment

[0079] Fig. 7 is a perspective view of a transformation cartridge 1d according to a fourth embodiment of the present invention, and Fig. 8 is an exploded perspective view of the transformation cartridge 1d according to the fourth embodiment of the present invention.

[0080] The transformation cartridge 1d according to the fourth embodiment of the present invention has the same components as the main body portion 10, O-ring portion 40 and sealing sheet 30 of the transformation cartridge 1 according to the first embodiment, and since there are some differences in shape in the remaining components, the differences will be further described, and the description of the transformation cartridge 1 according to the first embodiment can be applied as is to the corresponding components.

[0081] The guide unit 50d of the transformation cartridge 1d according to the fourth embodiment can receive and mix multiple solutions and deliver them to the material flow paths of the main body 10d. To this end, the guide unit 50d can include multiple material guides 53d. Different material solutions are injected into the respective material guides 53d, and while the different material solutions are delivered to the inlets of the material flow paths through the guide unit 50d, they are integrated into a single flow path and delivered, allowing them to be mixed during the flow process.

[0082] The guide unit 50d can discharge the resultant solution into multiple guides. Therefore, the resultant guide 54d can include a main resultant guide 541d and an auxiliary resultant guide 542d. The main resultant guide 541d can have a pipe shape that extends upward and then curves to the left. The auxiliary resultant guide 542d can also have a pipe shape that extends upward. The resultant solution discharged from the resultant flow path can branch from the guide unit 50d to the main resultant guide 541d and the auxiliary resultant guide 542d and be discharged therefrom.

[0083] The guide unit 50d may include a power supply guide 55d. A plurality of power supply guides 55d may be formed. The power supply guides 55d allow the terminals 25d, 26d, and 27d of the electric field generating unit 20d located below the guide body 51d to be exposed to the outside of the guide unit 50d, and connectors for connecting to the terminals 25d, 26d, and 27d may be stably contacted and fixed to the terminals 25d, 26d, and 27d. The power supply guide 55d may also be formed in a pipe shape extending upward from the guide body 51d.

[0084] The electric field generating unit 2d0 may include a material terminal 26d electrically connected to the material electrode 23d, a resultant terminal 27d electrically connected to the resultant electrode 24d, and a cell terminal 25d electrically connected to the cell electrode 22d. Each of the terminals 25d, 26d, and 27d may be disposed on the electric field generating body 21d at a position corresponding to the power supply guide 55d. The electric field generating unit 20d may be coupled to the guide unit 50d via a fastener.

[0085] The body mount 62d includes a hole in the center into which the body 10d is inserted, allowing the body 10d to be aligned with other components when positioned appropriately. However, a step is formed in the hole of the body mount 62d, preventing the body 10d and the sealing sheet 30d from slipping downwards even when inserted. The body mount 62d can be connected to the electric field forming unit 20d via a fastener.

[0086] Fig. 9 is a diagram showing a transformation system 100 including a transformation cartridge 1 and a transformation device 2 according to a first embodiment of the present invention. Fig. 10 is a diagram showing a transformation system 100d including a transformation cartridge 1d and a transformation device 2d according to a fourth embodiment of the present invention.

[0087] Referring to the drawings, transformation systems 100 and 100d according to the first and fourth embodiments of the present invention include a transformation cartridge 1 and a transformation device 2 and a transformation device 2 and a transformation device 2. The transformation systems 100 and 100d may include a syringe 3. Since the only differences between the first and fourth embodiments are the number of syringes 3, the number of pumps, the number of guides, etc., the cell transformation method of the present invention will be described with reference to FIG. 9 as a representative example.

[0088] Transformation can be performed by injecting each solution into the transformation cartridge 1 using the transformation device 2. A transformation cartridge 1 having a cell channel 12, a material channel 13, and a resultant channel 14 formed therein is prepared. That is, the transformation method includes the step of preparing the transformation cartridge 1. Syringes 3 containing the cell solution and the material solution, respectively, can be inserted into the guide portion 50 of the transformation cartridge 1. The transformation cartridge 1 with the syringes 3 inserted therein can be inserted into the transformation device 2.

[0089] The transformation device 2 according to the first embodiment of the present invention includes a pump unit 202 and a processor 201. The transformation device 2 may include an input unit 204 and a power application unit 203.

[0090] The pump unit 202 is provided to pump the cell solution SC and the substance solution SM to the cell flow path 12 and the substance flow path 13, respectively. Therefore, the pump unit 202 may include multiple pumps for pumping each solution. The pumps in the pump unit can transfer the solutions from storage tanks containing the respective solutions to the respective flow paths of the main body unit 10, which will be described later, via the respective piping.

[0091] Each pump may be a syringe pump that can contact the syringe 3 and push the piston of the syringe 3 at a desired speed. Therefore, when the transformation cartridge 1 with the syringe 3 inserted therein enters the transformation device 2, the pump approaches and contacts the piston of the syringe 3, and as it starts to operate, it can pump fluid by pushing the syringe 3. However, the pump is not limited to a syringe pump, and the pump may be configured in such a way that piping is directly connected to each flow path of the cartridge to pump fluid.

[0092] The pump unit 202 can bring a pressure member for pressing the piston into contact with the piston when the transformation cartridge 1 enters the transformation device 2. Here, the pump unit 202 can measure the distance traveled by the pressure member, and the processor 201 can calculate the volume of the solution stored in the syringe 3.

[0093] The processor 201 is electrically connected to the pump unit 202 and can control at least one of the pressure or flow rate at which the pump unit 202 pumps each solution. The processor 201 is a component including elements capable of performing logical operations to execute control commands, and may include a central processing unit (CPU). The processor 201 is connected to various components of the transformation device 2 according to one embodiment of the present invention and can transmit signals according to control commands to each component, and can be connected to various sensors or acquisition units and receive acquired information in the form of signals. The processor 201 can be electrically connected to each component and can communicate with each other via wires or a communication module capable of wireless communication.

[0094] The transformation device 2 may further include a storage medium, and control instructions executed by the processor may be stored in the storage medium and utilized. The storage medium may be a device such as a hard disk drive (HDD), a solid state drive (SSD), a server, a volatile medium, or a non-volatile medium, but is not limited thereto. In addition, the storage medium may further store data required for the processor 201 to perform its operations.

[0095] The transformation device 2 may receive necessary information via the input unit 204. The input unit 204 may receive information via a button, switch, touch screen, or other means, or may receive information by scanning a code, such as a barcode, QR code, or RFID scanner. Information regarding the transformation cartridge 1 inserted into the transformation device 2 may be input via the input unit 204. The information input to the input unit 204 is transmitted to the processor 201.

[0096] The processor 201 can use input information and pre-set and stored information to determine the flow rate of each solution delivered by the pump unit to the transformation cartridge 1. The processor 201 can determine the flow rate of each solution so that the cells C0 and the substance M come into contact in the mixing channel 141 and transformation occurs. Once the processor 201 knows which substance solution SM and which cell solution SC are used, it can derive the optimal flow rate ratio corresponding to the substance solution SM and the cell solution SC from stored data. The processor 201 can also derive the optimal exposure time for the transformation process from stored data. The processor 201 can determine the flow rate of the cell solution SC and the substance solution SM by combining the derived flow rate ratio and exposure time with the input information about the transformation cartridge 1 (such as the cartridge channel length and channel shape), and can operate the pump unit 202 to push the syringe 3 according to the predetermined flow rate. Here, the flow rate ratio determines the proportion of the substance solution SM and the cell solution SC in the thickness of the entire mixing channel 141. The optimal exposure time means a time sufficient for transformation to occur as the cells C0 and substance M pass through the mixing channel 141.

[0097] The flow rate ratio may be a value that allows cell C0 and substance M to come into contact in mixing channel 141 and undergo transformation. The flow rate ratio may be a value that allows the thickness T11 of the solution flowing into mixing channel 141 via cell channel 11 to be three times or less the diameter of cell C0, and preferably a value that allows the thickness T11 to be smaller than the diameter of cell C0.

[0098] The transformation method may include a step in which the pump unit 202 operates according to the determined flow rate ratio and flow rate value to inject the cell solution SC into the cell flow path 12 and the substance solution SM into the substance flow path 13.

[0099] The power application unit 203 is electrically connected to the processor 201 and may be connected to terminals 26 and 27 to apply power to the electrodes 22, 23, and 24 included in the electric field generation unit 20 of the transformation cartridge 1. When the transformation cartridge 1 enters the transformation device 2, the connecting arms of the power application unit 203 approach and contact the terminals 26 and 27, thereby establishing electrical connection. When the pump unit 202 is used to inject the cell solution SC into the cell channel 12 and the material solution SM into the material channel 13, the power application unit 203 may apply power to the electric field generation unit 20 to generate an electric field. When the solutions are supplied and an electric field is generated, transformation occurs in the mixing channel 141, and the solution containing the transformed cells C1 may be discharged through the outlet 140 of the resultant channel 14.

[0100] FIG. 11 is a diagram illustrating the sedimentation of cells C0 performed in the transformation cartridge 1 according to the first embodiment of the present invention.

[0101] 11, the flow rate of the solution that can be provided by the transformation device 2 when the transformation cartridge 1 according to the first embodiment of the present invention is provided to the transformation device 2 will be described. When the cell channel 12 and the mass transfer channel 132 come into contact with each other and connect to the mixing channel 141, the flow of the cell solution SC flowing in the cell channel 12 can bend at a right angle, and the flow of the cell solution SC can be approximated as moving circularly around a corner. Here, SS, which is the distance that the cells C0 sediment downward in the mixing channel 141 due to the centrifugal force of the circular motion, can be expressed as follows using an equation related to centrifugation: SS may be 1 μm or more and 20 μm or less.

[0102] [Mathematical formula 1]

[0103] TIFF2025540903000002.tif9167

[0104] where Vs is the downward velocity of cell C0 as shown, ts is the time it takes to pass through the corner when the corner is approximated as a quadrant, r is the radius of the approximated quadrant, d is the diameter of cell C0, μ is the viscosity of the cell solution SC, Δρ is the difference in density between cell C0 and the cell solution SC, ω is the angular velocity of cell C0, and Vc is the horizontal velocity of cell C0 in the mixing channel 141.

[0105] The transformation device 2 can provide the cell solution SC and the substance solution SM to the transformation cartridge 1 at a flow rate ratio that makes the thickness of the cell solution SC smaller than the above-mentioned SS in the mixing channel 141. Each solution is provided at such a flow rate ratio, and the cells CO can come into contact with the substance M in the mixing channel 141 and undergo transformation.

[0106] FIG. 12 is a diagram illustrating cell sedimentation performed in a transformation cartridge according to a third embodiment of the present invention.

[0107] 12, the flow rate of the solution that can be provided by the transformation device 2 when the transformation cartridge according to the third embodiment of the present invention is provided to the transformation device 2 will be described. The mixing channel, which is part of the product channel 14c, can have an arc shape as shown, and the flow of the cell solution can be approximated as moving circularly. Here, SS, which is the distance that the cells sediment downward in the mixing channel due to the centrifugal force of the circular motion, can be expressed as follows: SS may be 2 μm or more and 30 μm or less.

[0108] [Mathematical formula 2]

[0109] TIFF2025540903000003.tif9167

[0110] where LE is the length of the mixing channel and R is the radius of curvature of the mixing channel.

[0111] The transformation device 2 can provide the cell solution and the substance solution to the transformation cartridge at a flow rate ratio that makes the thickness of the cell solution in the mixing channel smaller than the SS described above. By providing each solution at such a flow rate ratio, the cells can come into contact with the substances in the mixing channel and undergo transformation.

[0112] Experimental Example 1

[0113] First, the transformation efficiency of mRNA using the transformation cartridge of the present invention was confirmed.

[0114] To this end, using the above-described transformation cartridge of the present invention, 1 ml of a cell solution containing NK-92 cells (ATCC CAT#CRL-2407) at a concentration of 4 x 107 cells / mL in cell culture medium was injected into the cell flow channel, and a substance solution containing eGFP mRNA (RiboPro, CAT#RB-079) at a concentration of 200 μg / mL was injected into the substance flow channel. An electric field of 1200 V was then applied between the substance flow channel inlet and the resultant discharge channel, and the cell solution and substance solution passed through a mixing channel (depth 30 μm, width 3 mm, length 24 mm) to which an electric field was applied at a flow rate ratio of 1:2. This resulted in the production of transformants in which the eGFP mRNA had been introduced into the NK-92 cells, which were used as the experimental group.

[0115] Separately, using the Neon™ Transfection System (ThermoFisher), a material solution containing the same eGFP mRNA as used in the preparation of the transfectants for the experimental group was mixed with a cell solution containing NK-92 cells in the same cell culture medium as used in the preparation of the transfectants for the experimental group, rather than the R-buffer, a buffer solution exclusive to the Neon™ Transfection System. The remaining steps were carried out according to the manufacturer's instructions to obtain transfectants, which served as the positive control group. A cell solution containing NK-92 cells without mRNA transfection was used as the negative control group.

[0116] Flow cytometry was performed on each of the experimental group, positive and negative control groups obtained as described above. Cells with stronger fluorescence than the top 1% signal value of the negative control group were classified as successful expression cells and analyzed for expression efficiency (eTX). Cell viability was measured using Texas Red fluorescent staining.

[0117] As a result, the negative control group and the experimental group prepared using the transformation cartridge of the present invention exhibited high cell viability rates of approximately 95%, and the positive control group, which was prepared using cells in culture medium without pretreatment and the Neon™ Transfection System, also exhibited high cell viability rates of approximately 90%. However, as shown in Figure 13, the expression efficiency (eTX) of the positive control group was 0.78%, indicating that there were almost no transformants expressing GFP fluorescence (left graph of Figure 13), whereas the expression efficiency (eTX) of the experimental group prepared using the transformation cartridge of the present invention was 98.3%, indicating that virtually all cells were transformants expressing GFP fluorescence (right graph of Figure 13).

[0118] Because mRNA is easily destroyed in cell culture medium containing various components necessary for cell growth, existing mixed-transfection techniques such as the Neon™ Transfection System require that target cells be washed, transferred to a dedicated buffer (R-buffer in the case of the Neon™ Transfection System), and mixed with mRNA in that state. However, in the positive control group, NK-92 cells were used in cell culture medium without the R-buffer, the dedicated buffer solution for the Neon™ Transfection System. This resulted in the destruction of eGFP mRNA in the mixture of eGFP mRNA and NK-92 cells, and therefore the production of transfectants in which eGFP mRNA was introduced into NK-92 cells was not confirmed. These results demonstrate that, with existing mixed-transfection techniques such as the Neon™ Transfection System, it is virtually impossible to directly deliver mRNA to cells in culture medium without pretreatment such as washing the cells or using a dedicated buffer solution.

[0119] In contrast, when using the above-described transformation cartridge of the present invention, NK-92 cells are used in cell culture medium, but the cell solution and the material solution containing eGFP mRNA are supplied through separate channels, allowing the eGFP mRNA to be delivered to the NK-92 cells without being destroyed by components in the cell culture medium. As a result, eGFP mRNA is introduced into almost all NK-92 cells, resulting in highly efficient production of transformants. These results demonstrate that the transformation cartridge of the present invention can deliver mRNA directly to cells in culture medium. Therefore, this embodiment clearly demonstrates that the use of the transformation cartridge of the present invention eliminates the need for pretreatment, such as washing cells or using specialized buffer solutions. This eliminates cell loss and damage during the cell washing process, obviates the need for expensive consumables such as specialized buffer solutions, and significantly simplifies the transformation process itself.

[0120] In particular, compared to the existing mixed transfection technology, which involves a cell washing process that causes cell loss or damage, followed by transfer with a dedicated buffer solution, transformation using the transformation cartridge of the present invention is found to be significantly advantageous not only in terms of expression efficiency and cell viability, which are two important factors in intracellular substance transfer, but also in terms of process automation rate, process closure rate, and production yield, which are the three most important factors in the process of producing cell therapeutic agents.

[0121] Experimental Example 2

[0122] Next, in the process of performing transformation by separately supplying cell solution and substance solution using the transformation cartridge of the present invention, the effect of the flow rate ratio between the two solutions in the mixing channel on the transfer efficiency of substances such as mRNA was confirmed.

[0123] Fluid flow within a mixing channel is laminar and has a parabolic velocity profile that follows a quadratic function depending on the depth, with the velocity being greatest at the center of the channel and zero at the top and bottom. Therefore, the flow velocity profile changes depending on the flow rate ratio of the cell solution and the material solution, which can affect the material transfer efficiency. Furthermore, from a geometric perspective, the smaller the flow rate for a fixed mixing channel height, the smaller the flow height. In the case of cell flow, adjusting the flow rate can adjust the thickness of the cell flow fluid, which can be adjusted according to the size of the cells. The basic trend is that the thinner the cell flow fluid, the more effectively the cells interact with the material flow, resulting in higher efficiency.

[0124] To assess this, the delivery and expression efficiency of eGFP mRNA was measured at flow rate ratios (substance flow rate / cell flow rate) of 0.5, 1, and 2. Specifically, using the above-described transformation cartridge of the present invention, 1 mL of a cell solution containing NK-92 cells (ATCC CAT#CRL-2407) at a concentration of 4 x 107 cells / mL in cell culture medium was injected into the cell channel, and a substance solution containing eGFP mRNA (RiboPro, CAT#RB-079) at a concentration of 200 μg / mL was injected into the substance channel. An electric field of 1200 V was applied between the substance channel inlet and the product outlet channel, and the cell solution and substance solution passed through a mixing channel (30 μm deep, 3 mm wide, 24 mm long) to which an electric field was applied at flow rate ratios (substance flow rate / cell flow rate) of 2, 1, and 0.5, thereby obtaining transformants in which the eGFP mRNA had been introduced into NK-92 cells. These were designated transformants #1, #2, and #3, respectively.

[0125] Flow cytometry was performed on each of the transformants #1, #2, and #3 obtained as described above, using the same method as in Example 1, to analyze transduction and expression efficiencies. As shown in Figure 14, the lower the cell flow rate, the higher the transduction efficiency. For NK-92 cells, whose average size is approximately 15 μm, the cell flow thickness was analyzed to be approximately 10 μm, 15 μm, and 20 μm at each mixing ratio. When the thickness was 10 μm, which is smaller than the average cell diameter, an expression rate of over 90%, which is the level at which almost all cells were expressed, was observed. When the cell solution flow thickness was similar to the cell diameter, a high expression rate of approximately 75% was maintained, but when it was thicker than the cell diameter, the expression efficiency decreased to 40%.

[0126] These results suggest that the mass transfer process in the mixing channel of the transformation cartridge of the present invention allows cells in the cell solution to effectively move toward and come into contact with the substance solution, enabling mass transfer even when the two fluids are not effectively mixed. That is, in the case of laminar fluid flow, the fluid mixing effect due to inertial forces that occurs in turbulent flow regions is absent, preventing two or more separate fluid flows from effectively mixing in a short period of time. However, even under laminar flow conditions where the cell solution and the substance solution cannot directly mix, as in the transformation cartridge of the present invention, cells in the cell solution can move directly toward and immediately come into contact with the substance solution, enabling effective intracellular mass transfer and transformation. Furthermore, this experimental example confirmed that almost all cells can be transformed when the cell fluid flow thickness is less than the cell diameter. It was also found that when the cell solution flow thickness is thicker than the cell thickness, only a portion of the cells were transformed, and the percentage of transformed cells decreased as the thickness increased.

[0127] Experimental Example 3

[0128] Furthermore, it was confirmed whether the transformation cartridge of the present invention could be used to efficiently deliver substances to cells (primary cells) extracted from the human body.

[0129] While existing mixed transfection technologies, such as the Neon™ Transfection System, inevitably result in cell loss and damage during the process of washing cells to remove cell culture medium, cell viability is known to be good for established cell lines capable of indefinite proliferation and for use in laboratory culture. However, compared to the aforementioned cell lines, cells collected from human blood or tissues (primary cells) are generally more difficult to grow through culture and suffer greater cell damage during the cell washing process, resulting in a significant decrease in yield during the process of producing transformants used in cell therapeutics. However, as confirmed in Example 1, when transformation is performed using the transformation cartridge of the present invention, substances can be directly delivered to cells in cell culture medium without pretreatments such as cell washing or the use of a dedicated buffer solution, and therefore, it is expected that substances can be delivered with high efficiency even to cells collected from the human body (primary cells).

[0130] To confirm this, using the above-mentioned transformation cartridge of the present invention, 1 mL of a cell solution containing human peripheral blood monoclonal cells (hPBMCs) (Lonza, CAT# CC-2702) collected from the human body at a concentration of 5 x 107 cells / mL in cell culture medium was injected into the cell flow channel, and a material solution containing eGFP mRNA (RiboPro, CAT# RB-079) at a concentration of 200 μg / mL was injected into the material flow channel. An electric field of 1800 V was then applied between the material flow channel inlet and the resulting discharge channel, and the cell solution and material solution passed through a mixing channel (depth 30 μm, width 3 mm, length 24 mm) subjected to an electric field at a flow rate ratio of 2:1. Transformants in which the eGFP mRNA had been introduced into the hPBMCs were obtained, and these were used as the experimental group.

[0131] The eGFP expression efficiency (eTX) and cell viability of the transformants obtained as described above were measured using the same method as in Example 1. As a result, as shown in Figure 15, compared to the negative control group where no transfection was performed, the transformants of the experimental group (hPBMCs into which eGFP mRNA was introduced using the transfection cartridge of the present invention) not only exhibited a similar level of cell viability, but also demonstrated such high cell viability, and GFP expression was maintained for more than one week, with the expression efficiency reaching a maximum level of 98% or more.

[0132] These results demonstrate that transformation using the transformation cartridge of the present invention allows direct delivery of substances to cells in cell culture media without pretreatments such as cell washing or the use of specialized buffer solutions. This allows for effective intracellular substance delivery without causing cell damage that could lead to cell death. These advantages also enable highly efficient substance delivery to primary cells derived from the human body. Furthermore, the transformed cells derived from the human body thus produced exhibited maximum expression efficiency after approximately one week, and when used in cell therapy, their efficacy can be sustained for more than one week. This is an outstanding effect not achieved with existing mixed-transfection technologies such as the Neon™ Transfection System. These experimental examples demonstrate the potential for the transformation technology using the transformation cartridge of the present invention to be highly applicable to processes utilizing human-derived cells, where the number of available cells is extremely limited.

[0133] Experimental Example 4

[0134] Based on the excellent transformation efficiency for various cells, including cell lines and human-derived cells, confirmed in the above embodiments, the transformation cartridge of the present invention was used to transduce CAR (chimeric antigen receptor) mRNA into NK-92 cells (ATCC CAT#CRL-2407) to produce CAR-NK cells.

[0135] NK cells are one of the most difficult cells to transform using various transformation techniques currently used in cell manipulation, posing significant challenges for the generation of CAR-NK. NK cells account for less than 5% of all blood cells, and the absolute amount that can be obtained from human cells is very low. Furthermore, the transformation efficiency via viral infection is known to be less than 1%. Therefore, when transformants are generated from human-derived NK cells using existing mixed transfection techniques, such as the Neon™ Transfection System, the production yield is known to be significantly low due to cell loss and cell damage that occurs during the cell washing process. However, as confirmed in the above embodiment, the use of the transformation cartridge of the present invention minimizes cell loss and enables the delivery of substances with high viability and efficiency, even to human-derived cells. Therefore, by confirming the expression of CARs, which have proven anti-cancer therapeutic effects, it is expected that CAR-NK therapeutics can also be generated with high yields.

[0136] To confirm this, using the above-mentioned transformation cartridge of the present invention, 1 mL of a cell solution containing NK-92 cells (ATCC CAT#CRL-2407) at a concentration of 1.0 x 107 cells / mL in cell culture medium was injected into the cell flow channel, and a material solution containing CD19 CAR mRNA (RiboPro, CAT#RB-073-3) at a concentration of 500 μg / mL was injected into the material flow channel. Then, while applying an electric field of 1200 V between the material flow channel inlet and the resultant discharge flow channel, the cell solution and material solution passed through a mixing flow channel (depth 30 μm, width 3 mm, length 24 mm) to which an electric field was applied at a flow rate ratio of 2:1, thereby obtaining a transformant in which the CD19 CAR mRNA had been introduced into NK-92 cells, which was designated NKL-CD19-500.

[0137] The expression efficiency (eTX) of the CD19 CAR in the transformants obtained as described above was measured using anti-FMC63-PE antibody, and the transformants were mixed with Nalm6 (ATCC CAT#CRL-3273), a CD19-overexpressing cancer cell line, at a 2:1 ratio (CAR-NK cells:Nalm6 cells) and cultured to measure the cytotoxicity of Nalm6 cancer cells.

[0138] As a result, as shown in Figure 16, it was confirmed that CD19 CAR was expressed in more than 90% of the cells (Figure 16 (A)), and more than 80% of Nalm6 cancer cells were killed within one day (Figure 16 (B)).

[0139] These results demonstrate that when transformation is performed using the transformation cartridge of the present invention, substances can be directly delivered to cells in cell culture medium without pretreatments such as cell washing or the use of dedicated buffer solutions, thereby enabling the efficient production of transformants using human-derived NK cells, which was virtually impossible using existing mixed-transfection techniques such as the Neon™ Transfection System. This is an outstanding effect not achieved with existing mixed-transfection techniques such as the Neon™ Transfection System, and this experimental example confirms the great potential for the development and production of CAR-NK anti-cancer immune cell therapeutics, which are expected to bring about a revolutionary innovation in the treatment of solid cancers. This high production performance, which has not been reported with existing NK cell transformation techniques, clearly demonstrates that the transformation technique using the transformation cartridge of the present invention may be very useful in the field of human-derived NK cell-based anti-cancer immune cell therapeutics.

[0140] Although it has been described above that all components constituting the embodiments of the present invention are combined or operate in combination, the present invention is not necessarily limited to such embodiments. That is, all components may operate in selective combination with one or more other components within the scope of the present invention. Furthermore, unless specifically stated to the contrary, the terms "comprise," "comprise," "have," and the like used above mean that the component in question may be inherent, and should be interpreted as including other components rather than excluding other components. All terms, including technical and scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the present invention pertains, unless otherwise defined. Commonly used terms, such as dictionary-defined terms, should be interpreted in accordance with the context of the relevant art and should not be interpreted in an idealized or overly formal sense unless expressly defined in the present invention.

[0141] The above description merely exemplifies the technical concept of the present invention, and various modifications and variations are possible within the scope of the essential characteristics of the present invention, as long as they are not deviated from the essential characteristics of the present invention. Therefore, the embodiments disclosed in the present invention are intended to illustrate, rather than limit, the technical concept of the present invention, and the scope of the technical concept of the present invention is not limited by such embodiments. The scope of protection of the present invention should be interpreted by the following claims, and all technical concepts within the scope equivalent thereto should be interpreted as being included in the scope of the present invention. [Explanation of symbols]

[0142] 1. 1d Transformation Cartridge

[0143] 2. 2d Transformation Device

[0144] 3 syringes

[0145] 10, 10b, 10c, 10d Main body

[0146] 11, 11b, 11c Main body

[0147] 12, 12b, 12c Cell flow path

[0148] 13, 13b, 13c Material flow path

[0149] 14, 14b, 14c Results distribution path

[0150] 20, 20d Electric field forming part

[0151] 21, 21d Electric field forming body

[0152] 22, 22d cell electrode

[0153] 23, 23d material electrode

[0154] 24, 24d resultant electrode

[0155] 25d cell terminal

[0156] 26, 26d material terminal

[0157] 27, 27d Resultant terminal

[0158] 30, 30d ceiling sheet

[0159] 40, 40d O-ring part

[0160] 42 Cell O-ring

[0161] 43 Material O-ring

[0162] 44 Result O-ring

[0163] 50, 50d Guide section

[0164] 51, 51d Guide body

[0165] 52, 52d Cell Guide

[0166] 53, 53d Material Guide

[0167] 54, 54d Result Guide

[0168] 55d Power Supply Guide

[0169] 61 Upper Mount

[0170] 62, 62d body mount

[0171] 63 Lower Mount

[0172] 100, 100d transformation system

[0173] 120 Cell channel inlet

[0174] 121 Cell inflow channel

[0175] 130 Entrance of material flow path

[0176] 131 Material inflow channel

[0177] 132 Mass transfer channel

[0178] 140 Outlet of the product flow path

[0179] 141 Mixing channel

[0180] 142 Resultant discharge channel

[0181] 201 processor

[0182] 202 Pump section

[0183] 203 Power application unit

[0184] 204 Input section

[0185] 541d Main Results Guide

[0186] 542d Auxiliary Product Guide

[0187] 1301b First material flow path

[0188] 1302b Second material flow path

[0189] C0 cells before transformation

[0190] C1, C2 transformed cells

[0191] M Transforming Agent

[0192] M1 First transforming substance

[0193] M2 second transforming substance

[0194] SC cell solution

[0195] SM substance solution

[0196] SM1 First substance solution

[0197] SM2 Second substance solution

Claims

1. a main body portion in which a cell flow path, a substance flow path, and a resultant flow path are formed, the flow paths being in contact with each other; an electric field generating unit including electrodes coupled to the body to generate an electric field in the resultant flow path; The cell channel and the material channel are formed to converge into the resultant channel; the product flow path includes a mixing flow path extending from a point where the mixing flow path is connected to the cell flow path and the material flow path; A transformation cartridge, wherein the thickness of the mixing channel is smaller than the thickness of the cell channel.

2. The transformation cartridge according to claim 1 , wherein the cell flow path and the substance flow path are in contact with each other with the cell flow path positioned above the substance flow path.

3. The transformation cartridge according to claim 1 , wherein the cell flow path includes a cell inlet flow path extending downward from the inlet.

4. The transformation cartridge according to claim 1 , wherein the mixing channel extends horizontally in one direction from a point where the mixing channel is connected to the cell channel and the substance channel.

5. The transformation cartridge according to claim 1 , wherein the substance flow path has a downwardly convex arc shape.

6. The transformation cartridge according to claim 5 , wherein the material flow path and the resultant flow path have a shape of a continuous arc that is convex downward.

7. The transformation cartridge according to claim 1 , wherein the material flow path includes a material inlet flow path extending downward from its inlet, and a material transfer flow path extending horizontally from a lower end of the material inlet flow path toward the resultant flow path.

8. The transformation cartridge of claim 1 , wherein the inlet of the material flow path, the inlet of the cell flow path, and the outlet of the resultant flow path are arranged in the order of the inlet of the material flow path, the inlet of the cell flow path, and the outlet of the resultant flow path according to a reference direction.

9. The material flow path is a first material flow path that contacts the cell flow path at one side of the cell flow path; The transformation cartridge according to claim 1 , further comprising a second substance flow path that contacts the cell flow path on the other side of the cell flow path.

10. the first substance flow path is in contact with the cell flow path below the cell flow path; The transformation cartridge according to claim 9 , wherein the second substance flow path contacts the cell flow path above the cell flow path.

11. The transformation cartridge according to claim 1 , wherein the electric field generating unit is configured to generate an electric field using a pulsed power source.

12. The resultant flow path is The transformation cartridge according to claim 1 , further comprising a product discharge channel connecting an end of the mixing channel to an outlet of the product channel.

13. 2. The transformation cartridge according to claim 1, wherein the thickness of the mixing channel is 6 μm or more and 400 μm or less.

14. a pump unit provided to pump a cell solution and a substance solution into the cell flow path and the substance flow path of the transformation cartridge, respectively; a power applying unit connected to a terminal of the transformation cartridge to apply power to an electrode of the transformation cartridge; a processor electrically connected to the power applying unit and the pump unit, The processor: A transformation apparatus that controls the pump unit based on information from the transformation cartridge, a preset flow rate ratio, and a preset exposure time.

15. a main body portion in which a cell flow path, a substance flow path, and a resultant flow path are formed, the flow paths being in contact with each other; an electric field generating unit coupled to the main body and including electrodes disposed at the inlet of the material flow path and the outlet of the resultant flow path; a pump unit provided to pump a cell solution and a substance solution into the cell flow path and the substance flow path, respectively; a processor electrically connected to the pump unit, The cell channel and the material channel are formed to converge into the resultant channel; the product flow path includes a mixing flow path extending from a point where the mixing flow path is connected to the cell flow path and the material flow path; The processor: A transformation system that controls the pump unit so that the thickness of the solution flowing into the mixing channel via the cell channel is smaller than the diameter of the cells contained in the cell solution.

16. preparing a cartridge having a cell flow path, a substance flow path, and a mixing flow path formed therein, the cell flow path, and the substance flow path being in contact with each other; Injecting a cell solution containing cells into the cell flow path; and injecting a substance solution containing a substance for transforming the cell into the substance flow path; A transformation method, wherein the ratio of the flow rate of the injected cell solution to the flow rate of the injected substance solution is a value that allows the cells and the substance to come into contact in the mixing channel and undergo transformation.

17. The transformation method according to claim 16, wherein the flow rate ratio is a value that makes the thickness of the solution flowing into the mixing channel via the cell channel within the mixing channel smaller than three times the diameter of the cell.

18. The transformation method according to claim 17, wherein the flow rate ratio is a value that makes the thickness of the solution flowing into the mixing channel via the cell channel within the mixing channel smaller than the diameter of the cell.

19. The transformation method according to claim 17, wherein the flow rate ratio is a value that makes the thickness of the solution flowing into the mixing channel via the cell channel within the mixing channel smaller than the distance the cells sediment downward in the mixing channel.

Images