Conversion box, conversion device, conversion system including same, and conversion method using same

Abstract

The cell transformation cassette according to the present invention comprises: a body part in which a cell flow path, a substance flow path, and a result flow path are formed that merge with each other; and an electric field forming part including an electrode connected to the body part to generate an electric field in the result flow path, in which the cell flow path and the substance flow path are formed in a shape converging to the result flow path, in which the result flow path includes a mixing flow path extending from a position connected to the cell flow path and the substance flow path, the thickness of the mixing flow path is smaller than that of the cell flow path.

Description

Technical Field

[0001] The present invention relates to a cell transformation cassette, a cell transformation device, a cell transformation system comprising the same, and a cell transformation method using the same. Background Art

[0002] As gene delivery techniques for transformation, viral vector-based transfer methods using viruses as transporters and non-viral delivery techniques using synthetic phospholipids, synthetic cationic polymers, and the like are widely known.

[0003] Viruses can be considered to be very effective DNA delivery systems because they survive as parasites in a state where their DNA is inserted into the nuclei of other cells due to their own life mechanisms. That is, in a DNA delivery system using viruses, the gene to be delivered is inserted into a non-replicable virus to be used. The advantage of this technology is that it is very effective for transformation. However, this technology has several disadvantages: from a basic point of view, there is a problem of producing a virus (RCV) with recombinant ability even if an inactivated virus is used, and a problem of difficulty in using the virus multiple times due to the immune response caused by the virus; the problem that the material that can be delivered by the virus is limited by its inherent properties; the problem of difficulty in injecting a certain amount of material into the cell, etc.

[0004] In order to avoid using viral vectors, a method (electroporation) can be used to open the phospholipid bilayer membrane by electric shock in the presence of substances around cells. However, in general electroporation processes, due to the nature of the substances to be delivered, there may be cases where cells should be placed in a special buffer (e.g., a ribonuclease-free buffer) instead of a culture medium, and for this purpose, the culture fluid needs to be pretreated, which is a process of washing cells by centrifugation or the like.

[0005] When using this typical electroporation method, cell viability may be significantly reduced, and there is a problem that a large amount of cell loss may occur during the pretreatment process. In addition, there is a problem that the special buffer used is very expensive and uneconomical. In addition, there is a problem that it is difficult to perform tasks such as pretreatment in a closed environment; therefore, it is difficult to perform automation and difficult to control the working environment hygienically. Summary of the invention

[0006] Technical issues

[0007] The present invention is designed to solve the above-mentioned problems and provides a cell transformation cassette, a cell transformation device, a cell transformation system including the same, and a cell transformation method using the same, which can transform a cell only by adding a cell solution and a substance solution and performing electroporation.

[0008] Technical Solution

[0009] According to an embodiment of the present invention, a cell transformation box includes: a main body portion, in which a cell flow path, a material flow path, and a result flow path that merge with each other are formed; and an electric field forming portion, including an electrode connected to the main body portion to generate an electric field in the result flow path, wherein the cell flow path and the material flow path are formed in a shape that converges to the result flow path, wherein the result flow path includes a mixing flow path extending from a position connected to the cell flow path and the material flow path, and wherein the thickness of the mixing flow path is less than the thickness of the cell flow path.

[0010] According to an embodiment of the present invention, a cell transformation box includes: a main body portion, in which a cell flow path, a material flow path, and a result flow path that merge with each other are formed; and an electric field forming portion, including electrodes connected to the main body portion and placed at an inlet of the material flow path and an outlet of the result flow path; wherein the cell flow path and the material flow path are formed in a shape that converges to the result flow path, wherein the result flow path includes a mixing flow path extending from a position connected to the cell flow path and the material flow path, and wherein a value obtained by multiplying (the thickness of the mixing flow path) by (a value obtained by dividing the cell flow path by the sum of the thickness of the cell flow path and the thickness of the material flow path) is smaller than the diameter of a cell flowing in the cell flow path.

[0011] According to an embodiment of the present invention, a cell transformation device includes: a pump unit, configured to pressurize and supply a cell solution and a substance solution to a cell flow path and a substance flow path of a cell transformation box, respectively; a power application unit, configured to be connected to a terminal of the cell transformation box to apply power to an electrode of the cell transformation box; and a processor, electrically connected to the power application unit and the pump unit, wherein the processor controls the pump unit based on information of the cell transformation box, a preset flow ratio, and a preset exposure time.

[0012] According to an embodiment of the present invention, a cell transformation system includes: a main body portion, in which a cell flow path, a material flow path, and a result flow path that merge with each other are formed; and an electric field forming portion, including electrodes connected to the main body portion and placed at the inlet of the material flow path and the outlet of the result flow path; a pump portion, configured to supply a cell solution and a material solution to the cell flow path and the material flow path, respectively, under pressure; and a processor, electrically connected to the pump portion, wherein the cell flow path and the material flow path are formed in a shape that converges to the result flow path, wherein the result flow path includes a mixed flow path extending from a position connected to the cell flow path and the material flow path, and wherein the processor controls the pump portion so that the thickness of a solution in the mixed flow path that flows into the mixed flow path through the cell flow path is smaller than the diameter of cells contained in the cell solution.

[0013] According to an embodiment of the present invention, the cell transformation method includes: preparing a box in which a cell flow path, a material flow path, and a mixing flow path that merge with each other are formed; injecting a cell solution including cells into the cell flow path; and injecting a material solution including a material for transforming the cells into the material flow path, wherein the ratio of the flow rate of the cell solution to the flow rate of the injected material solution is a value that allows transformation to occur when the cells and the material merge in the mixing flow path.

[0014] Beneficial Effects

[0015] Thus, transformation can occur simply by injecting a solution of cells and substances and by electroporation.

[0016] The transformation can occur even if the cell solution and the substance solution are not mixed.

[0017] Since pretreatment of cells or transformation factors, such as cell washing, can be omitted, cell viability can be increased and cell and material losses can be significantly reduced in the corresponding process.

[0018] This constitutes an economical transformation method since no separate special buffer needs to be used.

[0019] Since no pretreatment is required, the conversion process can be constituted only by injecting the solution into a closed and controlled environment; therefore, a hygienic working environment can be maintained and it is advantageous according to various regulations.

[0020] Since all that is necessary for transformation is the injection of a medium containing the cells and the substance solution and the application of an electric field, the process can be easily automated. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is a perspective view of a cell transformation cassette according to a first embodiment of the present invention.

[0022] Figure 2 is an exploded perspective view of a cell transformation cassette according to a first embodiment of the present invention.

[0023] Figure 3 is a plan view of a cell transformation cassette according to a first embodiment of the present invention.

[0024] Figure 4 1 is a conceptual diagram of the main body of the cell transformation cassette according to the first embodiment of the present invention.

[0025] Figure 5 1 is a conceptual diagram of a main body of a cell transformation cassette according to a second embodiment of the present invention.

[0026] Figure 6 1 is a conceptual diagram of a main body of a cell transformation cassette according to a third embodiment of the present invention.

[0027] Figure 7 is a perspective view of a cell transformation cassette according to a fourth embodiment of the present invention.

[0028] Figure 8 is an exploded perspective view of a cell transformation cassette according to a fourth embodiment of the present invention.

[0029] Fig. 9 1 is a diagram showing a cell transformation system including a cell transformation cassette and a cell transformation device according to a first embodiment of the present invention.

[0030] Fig.10 1 is a view showing a cell transformation system including a cell transformation cassette and a cell transformation apparatus according to a fourth embodiment of the present invention.

[0031] Fig.11 It is a view showing cell sedimentation occurring in the cell transformation cassette according to the first embodiment of the present invention.

[0032] Fig.12 It is a diagram showing cell sedimentation occurring in the cell transformation cassette according to the third embodiment of the present invention.

[0033] Fig.13 Shows confirmation of using Neon TM Experimental results of mRNA conversion efficiency of the transfection system (ThermoFisher) and mRNA conversion efficiency using the cell transformation cassette of the present invention.

[0034] Fig.14 The experimental results in the process of using the cell transformation cassette of the present invention to separately provide a cell solution and a substance solution for transformation are shown, confirming the effect of the flow ratio of the two solutions in the mixing flow path on the delivery efficiency of substances such as mRNA.

[0035] Fig.15 The results of experiments using the cell transformation cassette of the present invention are shown, and the experiment was repeated three times to determine whether a substance can be efficiently delivered to cells collected from the human body (primary cells).

[0036] Fig.16 The results of experiments to confirm the construction efficiency (transduction efficiency) (A) of CAR-NK cells constructed using the cell transformation cassette of the present invention and the degree of killing of cancer cells (B) are shown. DETAILED DESCRIPTION

[0037] Exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings. When adding reference numerals to the components of each of the accompanying drawings, it should be noted that the same reference numerals are given to the same components as much as possible, even if they are indicated on different drawings. In addition, when describing an embodiment of the present invention, if it is determined that a detailed description of a related known configuration or function hinders the understanding of the embodiment of the present invention, the detailed description will be omitted.

[0038] In addition, when describing the components of the embodiments of the present invention, terms such as first, second, A, B, (a) and (b) may be used. These terms are only used to distinguish a component from other components, and the nature, order, sequence, etc. of the components are not limited by these terms. When a component is described as being "connected", "coupled" or "conjugated" to another element, the component may be directly connected or conjugated to the other component, but it should be understood that there may be another component "connected", "coupled" or "conjugated" between the components.

[0039] First embodiment

[0040] Figure 1 is a perspective view of a cell transformation cassette (1) according to a first embodiment of the present invention. Figure 2 1 is an exploded perspective view of a cell transformation cassette (1) according to a first embodiment of the present invention. Figure 3 1 is a plan view of a cell transformation cassette (1) according to a first embodiment of the present invention.

[0041] The cell transformation box (1) according to the first embodiment of the present invention comprises a main body (10) and an electric field forming unit (20). In the specification of the present invention, for the sake of convenience, the up-down, left-right, and front-back directions are orthogonal to each other and are relative directions that can be changed according to the configuration state of the cell transformation box (1).

[0042] The electric field forming part (20) is a component that forms an electric field in the body part (10). The electric field forming part (20) includes electrodes connected to the body part (10) and arranged at an inlet (130) of the material flow path (13) and an outlet (140) of the result flow path (14) to generate an electric field in the result flow path (14). The electric field forming part (20) may be a printed circuit board, but its type is not limited thereto.

[0043] The electric field forming part (20) may include a plate-shaped electric field forming body (21). The plate-shaped electric field forming body (21) may be composed of multiple layers of epoxy resin, but its material is not limited thereto. Electrodes may be formed in the electric field forming body (21), and each electrode may be formed in a ring shape to surround each hole formed in the electric field forming part (20) so as to correspond to each flow path of the body part (10).

[0044] The electrodes (22, 23, 24) may be formed of a conductor such as a metal. The electrodes may be formed by coating copper on the electric field forming body (21). The electrodes of the electric field forming part (20) may include a cell electrode (22), a material electrode (23), and a result electrode (24). The electric field forming part (20) may include terminals (26, 27) which may be electrically connected to each electrode (22, 23, 24) and may be connected to a power source. The terminals (26, 27) may be formed of the same material as the electrodes (22, 23, 24). The cell transformation box (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, 27) may be arranged outside the electric field forming body (21) so as to be easily connected to a power source.

[0045] The electric field forming part may be configured to include electrodes arranged to surround each flow path in the body part and coupled to the body part. These electrodes may also be placed in the mixing flow path.

[0046] The electric field forming unit (20) can form the electric field using a simple DC power supply, but can also form the electric field using a pulse power supply. Fig. 9 2) The processor (201) (described later) can control the electric field forming unit (20) by PWM control using the power applying unit (203) electrically connected thereto. The frequency of the pulse can be greater than 1 kHz and less than 100 kHz, and the duty cycle can be greater than 10% and less than 90%. The electric field forming unit (20) can be formed while changing the size of the electric field. The size of the electric field can be changed by reducing it to 50% of the maximum value of the size of the electric field formed by the electric field forming unit (20).

[0047] The cell transformation box (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 the lower support (63) (described later), thereby preventing the impact from being directly transmitted from the lower support (63) to the main body (10) and stably supporting the main body (10). The sealing sheet (30) may be formed of a material including silicon, but the material is not limited thereto. A hole is formed on the sealing sheet (30) at a position corresponding to the lower side of the inlet of each flow path (12, 13, 14), so that the state of each flow path (12, 13, 14) can be checked from the lower side to the upper side.

[0048] The cell transformation box (1) according to the first embodiment of the present invention may include an O-ring portion (40). The O-ring portion (40) is composed of an elastic annular O-ring (42, 43, 44) and is located between the electric field forming portion (20) and the guide portion (50) (described later), so that the water tightness between the electric field forming portion (20) and the guide portion (50) can be maintained, and the needle inserted into the guide portion (50) can be maintained so that the solution can be stably injected. The O-ring portion (40) can be formed of a material containing silicon, but the material is not limited thereto. The O-ring portion (40) may include a cell O-ring (42) located between the cell electrode (22) and the cell guide (52); a substance O-ring (43) located between the substance electrode (23) and the substance guide (53); and a result O-ring (44) located between the result electrode (24) and the result guide (54).

[0049] The cell transformation box (1) according to the first embodiment of the present invention may include a guide part (50). The guide part (50) may be used as an inlet and an outlet of a solution. The guide part (50) may include a plate-shaped guide body (51), a cell guide (52) coupled to the guide body (51), a substance guide (53), and a resultant guide (54). Each guide (52, 53, 54) may be formed into a tubular shape extending upward from the guide body (51). Each guide (52, 53, 54) has a vertically penetrating shape and is arranged at a position corresponding to each electrode (22, 23, 24) and a flow path (12, 13, 14); therefore, when a needle is inserted into the guide (52, 53, 54) and liquid is discharged in a manner that it is connected to a pump or the like, the discharged liquid can be delivered to each flow path through each electrode. The needle can be stably inserted into each guide (52, 53, 54) to enable the liquid to be discharged at an appropriate position. O-rings (42, 43, 44) may be provided on the lower side of each guide member (52, 53, 54); the electric field forming part (20) may be located on the lower side of the guide body (51). The guide part (50) may be formed of a material including polycarbonate, but the material is not limited thereto.

[0050] The cell transformation box (1) according to the first embodiment of the present invention may include an upper bracket (61) and a lower bracket (63). The upper bracket (61) may be located above the guide portion (50), and the lower bracket (63) may be located below the sealing sheet (30). The upper bracket (61) and the lower bracket (63) may be fastened to each other by fasteners such as bolts, thereby fixing the sealing sheet (30), the body portion (10), the electric field forming portion (20), the O-ring portion (40), and the guide portion (50) located therebetween in a stacked state. The components therebetween may be squeezed by the upper bracket (61) and the lower bracket (63), thereby effectively sealing. A hole through which the guide can pass is formed on the upper bracket (61), and a hole is formed on the lower bracket (63) at a position corresponding to the hole of the sealing sheet (30). The upper bracket (61) and the lower bracket (63) may be composed of a material containing aluminum, and the aluminum may be anodized aluminum, but is not limited thereto.

[0051] The cell transformation box (1) according to the first embodiment of the present invention may include a body support (62). The body support (62) may include a hole located at the center thereof, into which the body portion (10) is inserted, so that the body portion (10) can be aligned with other components while being placed in an appropriate position. The body support (62) may be located between the upper support (61) and the lower support (63) and may be further connected by a fastener. The body support (62) may be composed of a material containing aluminum, and the aluminum may be anodized aluminum, but its material is not limited thereto.

[0052] Figure 4 1 is a conceptual diagram of the main body (10) of the cell transformation cassette (1) according to the first embodiment of the present invention.

[0053] A cell flow path (12), a material flow path (13), and a result flow path (14) that merge with each other are formed in the body part (10). These flow paths (12, 13, 14) may be formed through the body part (11). The body part (11) may be formed of a material including glass, and the glass may be borosilicate glass, but the material is not limited thereto.

[0054] The inlet (120, 130) or outlet (140) of each flow path (12, 13, 14) may be arranged on the upper surface of the main body (11). The inlet (130) of the material flow path (13), the inlet (120) of the cell flow path (12), and the outlet (140) of the result flow path (14) may be arranged forward in the order of the inlet (130) of the material flow path (13), the inlet (120) of the cell flow path (12), and the outlet (140) of the result flow path (14), which is a standard direction. The cell solution (SC) may be introduced into the inlet (120) of the cell flow path (12), the material solution (SM) may be introduced into the inlet (130) of the material flow path (13), and the result solution may be discharged to the outlet (140) of the result flow path (14).

[0055] The cell flow path (12) is a flow path for introducing a cell solution (SC). The cell solution (SC) may be composed of a culture medium containing cells (C0) or electrolytes. The cells (C0) contained in the cell solution (SC) may be blood cells among somatic cells, and may be immune cells therein. Immune cells may be used without restriction as long as they are cells capable of inducing immunity to induce the desired therapeutic effect, for example, any one of the group consisting of natural killer cells (NK cells), T cells, natural killer T cells (NKT cells), cytokine-induced killer cells (CIK cells), macrophages, and dendritic cells, but not limited thereto. The cell flow path (12) may include a cell introduction flow path (121) extending from the inlet (120) of the cell flow path (12). The cell introduction flow path (121) may extend downward from the inlet (120) of the cell flow path (12).

[0056] The material flow path (13) is a flow path through which a material solution (SM) is introduced and flows. The material solution (SM) may be water or a buffer solution containing a transformation factor (M). The transformation factor (M) may be introduced into the interior of a cell (C0), for example, into the cytoplasm or the nucleus, and function in the cell (C0), and is not limited to any form such as protein, peptide, nucleic acid, etc., as long as it is a substance that can function in the cell (C0). In particular, the transformation factor (M) may be a nucleic acid, and may be mRNA therein. The material flow path (13) may include a material introduction flow path (131) extending from an inlet (130) of the material flow path (13). The material introduction flow path (131) may extend downward from the inlet (130) of the material flow path (13). The material flow path (13) may include a material transfer flow path (132) extending from the lower end of the material introduction flow path (131) to the result flow path (14). The material transfer flow path (132) may extend forward in a horizontal direction. Therefore, the material flow path (13) may be bent at a position where the material introduction flow path (131) and the material transfer flow path (132) merge with each other, and the bending angle may be 90 degrees.

[0057] The cell flow path (12) and the material flow path (13) can be merged in a state where the cell flow path (12) is located on the upper side of the material flow path (13). The lower end of the cell introduction flow path (121) can be located on the upper side of the front end of the material transfer flow path (132). Therefore, the cell solution (SC) can be introduced into the result flow path (14) at the position where the material flow path (13) and the cell flow path (12) merge with each other in a state where the cell solution (SC) is located on the upper side of the material solution (SM).

[0058] The result flow path (14) is a flow path through which the result solution containing the transformed cells (C1) flows. The result solution can be discharged through the outlet (140) of the result flow path (14). The cell flow path (12) and the substance flow path (13) are formed to converge to the result flow path (14). Therefore, the cell solution (SC) and the substance solution (SM) are combined to form the result solution.

[0059] The result flow path (14) includes a mixing flow path (141) extending from a position connected to the cell flow path (12) and the material flow path (13). The mixing flow path (141) can extend horizontally forward from the position. The result flow path (14) can include a result discharge flow path (142) connecting one end of the mixing flow path (141) and an outlet (140) of the result flow path (14). The result discharge flow path (142) can extend upward from the front end of the mixing flow path (141).

[0060] Since the cell flow path (12) merges with the substance transfer flow path (132) and the mixing flow path (141) in a non-parallel state, the flow of the cell solution (SC) bends at the confluence point. The cell solution (SC) can bend 90 degrees. Since the flow direction of the cell solution (SC) changes at the point where each flow path merges, the cell (C0) sinks due to centrifugal acceleration and easily merges with the substance (M).

[0061] The electric field forming section (20) can form an electric field along the solution flowing in each flow path (12, 13, 14) using each electrode (22, 23, 24). The electric field forming section (20) can form an electric field in the material flow path (13) and the result flow path (14) using the material electrode (23) and the result electrode (24). The electric field forming section (20) can form an electric field in the cell flow path (12) and the result flow path (14) using the cell electrode (22) and the result electrode (24). The electric field formed in the direction starting from the cell flow path (12) or the material flow path (13) and going out through the result flow path (14) is indicated by a dotted line in the figure. By forming an electric field, a flow direction can be formed in the flow paths (12, 13, 14) of each solution (SC, SM), and electroporation transformation can occur in the mixing flow path (141) (described later).

[0062] In the mixing flow path (141), the conversion factor (M) of the substance solution (SM) can enter the cells (C0) of the cell solution (SC). Since the phospholipid bilayer of the cells (C0) is partially opened by applying an electric field to the mixing flow path (141), the conversion factor (M) can enter the cells (C0).

[0063] The thickness (T11+T21) of the mixing channel (141) may be smaller than the thickness (T10) of the cell channel (12). In particular, the thickness of the channels (12, 13, 14) refers to the extent to which the boundaries of the channels (12, 13, 14) extend in a direction orthogonal to the direction in which the solutions (SC, SM) in the channels (12, 13, 14) flow. Figure 4 In the cross section of each flow path (12, 13, 14) cut in a plane orthogonal to the left and right directions shown. When each flow path (12, 13, 14) is formed into a tubular shape obtained by extending a cylinder, the thickness of the flow path (12, 13, 14) can be the inner diameter of the flow path (12, 13, 14). The thickness (T11+T21) of the mixing flow path (141) can be less than the thickness (T20) of the material flow path (13). The thickness of the mixing flow path (141) can be greater than 6 μm and less than 400 μm.

[0064] Since the thickness (T11+T21) of the mixing flow path (141) is smaller than the thickness (T10) of the cell flow path (12) or the thickness (T20) of the material flow path (13) that converges to the result flow path (14), the thickness (T11) of the cell solution (SC) in the mixing flow path (141) can be smaller than the thickness of the cell flow path (12) occupied by the cell solution (SC). Similarly, the thickness (T21) of the material solution (SM) in the mixing flow path (141) can be smaller than the thickness (T20) occupied by the material solution (SM) in the material flow path (13). In particular, the thickness (T11) of the cell solution (SC) in the mixing flow path (141) is smaller than the diameter of the cell (C0), so that a part of the cell (C0) is exposed to the material solution (SM), so that it is in contact with the transformation factor (M). When an electric field is formed in the mixing flow path (141), the transformation factor (M) can enter the cell (C0) to form a transformed cell (C1). Since a portion of the cell (C0) is exposed to the substance solution (SM), transformation can occur even if the substance solution (SM) and the cell solution (SC) are not mixed.

[0065] In order to enable flow, the value obtained by multiplying (the thickness of the mixing flow path (141)) by (the value obtained by dividing 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)) can be smaller than the diameter of the cell (C0) flowing in the cell flow path (12).

[0066] The thickness (T11) of the cell solution (SC) flowing in the mixing flow path (141) can be determined by controlling the flow rate of each solution injected. The processor (201) can control the pump unit (202) so that the thickness (T11) of the cell solution (SC), that is, the solution flowing into the mixing flow path (141) through the cell flow path (12) in the mixing flow path (141) is smaller than the diameter of the cells contained in the cell solution (SC). When other conditions are the same, when the pressure of the cell solution (SC) supplied or the flow rate of the pump unit (202) increases, the thickness (T11) of the cell solution (SC) in the mixing flow path (141) can increase.

[0067] In order to make it easy for the cells (C0) to contact the substance solution (SM) in the mixing flow path (141), the cell flow path (12) and the cell flow path (13) can be merged with each other in a state where they are located above the substance flow path (13). This is because, through such a flow path configuration, the cell solution (SC) is located above the substance solution (SM) in the mixing flow path (141), so the cells (C0) carried by the cell solution (SC) move toward the substance solution (SM) on the lower side under the influence of gravity, thereby being fully exposed to the substance solution (SM).

[0068] Due to the structure of the main body (10), transformation and obtained transformed cells (C1) can be simply carried out by injecting into the main body (10) and applying an electric field, without the need for separate pretreatment of the cells or the transformation factor (M). Therefore, pretreatment processes such as cell washing can be omitted, and the loss of cells and substances in the process can be significantly reduced. In addition, since there is no need to use a separate special buffer, an economical method can be constituted. In addition, since no pretreatment is required, the method can be constituted only by injecting the solution into a closed and controlled environment, so according to various regulations, it has the advantage that a hygienic working environment can be maintained. Simply injecting a culture medium and a substance solution containing cells and applying an electric field are steps necessary for transformation; therefore, it is easy to automate the process.

[0069] Second embodiment

[0070] Figure 5 1 is a conceptual diagram of a main body (10b) of a cell transformation cassette according to a second embodiment of the present invention.

[0071] Since the cell transformation cassette according to the second embodiment of the present invention is the same as the cell transformation cassette (1) according to the first embodiment except that it has the second substance flow path (1302b), only the different parts will be further described, and the description of the cell transformation cassette (1) according to the first embodiment can be applied to the remaining parts as is. In the electric field forming portion, the guide, the O-ring portion, etc. of the cell transformation cassette according to the second embodiment, a portion for the second substance flow path (1302b) is formed.

[0072] 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) including a first conversion factor (M1) flows, and a second material flow path (1302b) through which a second material solution (SM2) including a second conversion factor (M2) flows. The first conversion factor (M1) and the second conversion factor (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 further explanation of the second material flow path (1302b) will be provided below.

[0073] The first material flow path (1301b) may merge with the cell flow path (12b) at one side of the cell flow path (12b), and the second material flow path (1302b) may merge with the cell flow path (12b) at the other side of the cell flow path (12b). The first material flow path (1301b) may merge with the cell flow path (12b) at the lower side of the cell flow path (12b), and the second material flow path (1302b) may merge with the cell flow path (12b) at the upper side of the cell flow path (12b). Therefore, by the same action as described in the first embodiment in the result flow path (14), the first material solution (SM1) may flow to the upper side of the cell solution (SC), and the second material solution (SM2) may flow to the lower side of the cell solution (SC), and the cells may be exposed to the first material solution (SM1) and the second material solution (SM2). The cells exposed to the respective substance solutions (SM) can be opened by the electric field, so that the first conversion factor (M1) and the second conversion factor (M2) can enter the cells and thus undergo conversion.

[0074] Therefore, according to the second embodiment, transformed cells (C2) can be formed by simultaneously injecting a plurality of different types of transformation factors (M1, M2) into cells using one cell transformation cassette.

[0075] Third embodiment

[0076] Figure 6 1 is a conceptual diagram of a main body (10c) of a cell transformation cassette according to a third embodiment of the present invention.

[0077] Since the cell transformation cassette according to the third embodiment of the present invention is the same as the cell transformation cassette (1) according to the first embodiment except for the shape of the flow path (12c, 13c, 14c) formed in the main body (10c), only the different parts are further described, and the description of the cell transformation cassette (1) according to the first embodiment can be applied to the remaining parts as is.

[0078] 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 flow path (13c) according to the third embodiment may have an arc that bulges downward. The material flow path (13c) and the result flow path (14c) may have a continuous arc that bulges downward. That is, the material flow path (13c) and the result flow path (14c) may be connected by forming a continuous arc that bulges downward. Although the cell flow path (12c) is also shown as having a curved profile, such as an arc, the cell flow path (12c) may have a shape that extends downward. When the cell flow path (12c) has an arc, the radius of curvature of the cell flow path (12c) may be smaller than the radius of curvature of the material flow path (13c).

[0079] Since the raw material flow path (13c) has an arc shape, centrifugal acceleration is applied to the transforming factor injected into the raw material flow path (13c), and the transforming factor is easily integrated with the cells.

[0080] The arcuate 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) of the second embodiment.

[0081] Fourth embodiment

[0082] Figure 7 is a perspective view of a cell transformation cassette (1d) according to a fourth embodiment of the present invention. Figure 8 1 is an exploded perspective view of a cell transformation cassette (1d) according to a fourth embodiment of the present invention.

[0083] The cell transformation cassette (1d) according to the fourth embodiment of the present invention has the same components as the body portion (10), the O-ring portion (40), and the sealing sheet (30) of the cell transformation cassette (1) according to the first embodiment. Since there are some structural differences in the remaining components, only the different parts will be further described, and the description of the cell transformation cassette (1) according to the first embodiment can be applied to the remaining components as is.

[0084] The guide portion (50d) of the cell transformation box (1d) according to the fourth embodiment of the present invention can receive and mix a plurality of solutions and deliver them to the material flow path of the body portion (10d). To this end, the guide portion (50d) may include a plurality of material guides (53d). Since each material guide (53d) of the cell transformation box (1d) according to the fourth embodiment is injected with material solutions different from each other, and the material solutions different from each other are integrated into one flow path and are transported while being transported to the inlet of the material flow path through the guide portion (50d), these material solutions different from each other can be mixed with each other during the flow process.

[0085] The guide portion (50d) may discharge the result solution through a plurality of guides. Therefore, the result guide (54d) may include a main result guide (541d) and an auxiliary result guide (542d). The main result guide (541d) may have a tubular shape that bends to the left after extending upward. The auxiliary result guide (542d) may have a tubular shape that extends upward. The result solution discharged from the result flow path may be separated from the guide portion (50d) into the main result guide (541d) and the auxiliary result guide (542d) and discharged.

[0086] The guide portion (50d) may include a power supply application guide (55d). The power supply application guide (55d) may be formed in plural. The guide portion (50d) may allow each terminal (25d, 26d, 27d) of the electric field forming portion (20d) located at the lower side of the guide body (51d) to be exposed to the outside of the guide portion (50d), and allow the connector connected to each terminal (25d, 26d, 27d) to stably contact and fix each terminal (25d, 26d, 27d). The power supply application guide (55d) may also be formed in a tubular shape extending upward from the guide body (51d).

[0087] The electric field forming part (20d) may include a substance terminal (26d) electrically connected to the substance electrode (23d), a result terminal (27d) electrically connected to the result electrode (24d); and a cell terminal (25d) electrically connected to the cell electrode (22d). Each of the terminals (25d, 26d, 27d) may be arranged at a position on the electric field forming body (21d) corresponding to the power supply applying guide (55d). The electric field forming part (20d) may be connected by a guide part (50d) and a fastener.

[0088] The body bracket (62d) may include a hole in the center into which the body portion (10d) is inserted so that the body portion (10d) can be aligned with other components in an appropriate position. However, a step is formed in the hole of the body mounting portion 62d so that the body portion (10d) and the sealing sheet (30d) do not separate downward when the body portion (10d) and the sealing sheet (30d) are inserted. The body bracket (62d) can be coupled to the electric field forming portion (20d) by a fastener.

[0089] Fig. 9 1 is a diagram showing a cell transformation system (100) including a cell transformation box (1) and a cell transformation device (2) according to a first embodiment of the present invention. Fig.10 1 is a view showing a cell transformation system (100d) including a cell transformation box (1d) and a cell transformation device (2d) according to a fourth embodiment of the present invention.

[0090] Referring to the drawings, the cell transformation system (100, 100d) according to the first embodiment and the fourth embodiment of the present invention comprises a cell transformation box (1, 1d) and a cell transformation device (2, 2d). The cell transformation system (100, 100d) may comprise a syringe (3). Since the difference between the first embodiment and the second embodiment is only the number of syringes (3), the number of pumps, the number of catheters, etc., reference will be made to the drawings. Fig. 9 The cell transformation method of the present invention is described as a representative.

[0091] Transformation can occur by injecting each solution into a cell transformation box (1) through a cell transformation device (2). A cell transformation box is prepared, wherein a cell flow path (12), a substance flow path (13), and a result flow path (14) are formed. That is, the cell transformation method may include preparing a cell transformation box (1), and a syringe (3) including each cell solution and substance solution can be inserted into a guide portion (50) of such a cell transformation box (1). The cell transformation box (1) with the syringe (3) inserted can be introduced into the interior of the cell transformation device (2).

[0092] The cell transformation device (2) according to the first embodiment of the present invention includes a pump unit (202) and a processor (201). The cell transformation device 2 may include an input unit (204) and may include a power application unit (203).

[0093] The pump unit (202) is configured to supply a cell solution (SC) and a substance solution (SM) to the cell flow path (12) and the substance flow path (13) under pressure, respectively. Therefore, the pump unit (202) may include a plurality of pumps for supplying each solution under pressure. The pumps of the pump unit may transport the solutions from the storage tanks containing each solution to each flow path of the main body (10) (described later) through respective pipelines.

[0094] Each pump may be a syringe pump capable of contacting the syringe (3) and pushing the piston of the syringe (3) at a desired speed. Therefore, once the cell transformation box (1) with the syringe (3) inserted enters the interior of the cell transformation device (2), as the operation starts, the fluid can be transported by pressurization in a manner such that the pump approaches and contacts the piston of the syringe (3) and pushes the syringe (3). However, the pump is not limited to the syringe pump, and the pump may be constructed in such a manner that the pipeline is directly connected to each flow path of the box and the fluid is transported by pressurization.

[0095] When the cell transformation box (1) enters the interior of the cell transformation device (2), the pump unit (202) can allow the pressure element for pushing the piston to contact the piston. In particular, the pump unit (202) can measure the distance moved by the pressure element, so that the processor (201) can calculate the volume of the solution stored in the syringe (3).

[0096] The processor (201) can be electrically connected to the pump unit (202) to control at least one of the pressure or flow rate of each solution pressurized and supplied by the pump unit (202). The processor (201) is a component that includes a logical operation element capable of executing a control command, and may include a central processing unit (CPU) or the like. The processor (201) is connected to each component of the cell transformation device (2) according to an embodiment of the present invention, so that a signal according to the control command can be sent to each component, and information obtained by connecting to each sensor or acquisition unit in the form of a signal is received. Since the processor (201) can be electrically connected to each component, it can be connected with a wire or can communicate with each other by having a communication module capable of wireless communication.

[0097] The cell transformation device (2) may further include a storage medium, so that the control command executed by the processor can 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, a non-volatile medium, etc., but the type is not limited thereto. In addition, data required for the processor (201) to perform an operation may be further stored in the storage medium.

[0098] The cell transformation device (2) can receive necessary information through the input unit (204). The input unit (204) can receive information including devices such as buttons, switches and touch screens, and can also input information by scanning codes including bar codes, QR codes and RFID scanners. Information about the cell transformation box (1) entering the cell transformation device (2) can be input through the input unit (204). The information input to the input unit (204) is sent to the processor (201).

[0099] The processor (201) can determine the flow rate of each solution delivered to the cell transformation box (1) by the pump unit using input information and information preset and stored therein. The processor (201) can determine the flow rate of each solution to a value that allows the cells (C0) and the substance (M) to merge to cause transformation in the mixing flow path (141). Once the processor (201) knows which substance solution (SM) and which cell solution (SC) will be used, it can extract the optimal flow ratio corresponding to the substance solution (SM) and the cell solution (SC) from the stored data. The processor (201) can also extract the optimal exposure time for the transformation process from the stored data. The processor (201) can determine the flow rate of the cell solution (SC) and the flow rate of the substance solution (SM) by combining the extracted flow rate and exposure time with the input information of the cell transformation box (1) (the flow path length of the box, the shape of the flow path shape, etc.), and can operate the pump unit (202) according to the determined flow rate, thereby pushing the syringe (3). In particular, the flow ratio determines to what extent the substance solution (SM) and the cell solution (SC) in the mixing channel (141) will occupy the total thickness of the mixing channel (141). The optimal exposure time refers to the time sufficient for the cells (C0) and the substance (M) to undergo transformation when passing through the mixing channel (141).

[0100] The flow ratio may be a value that allows conversion to occur when the cell (C0) and the substance (M) merge in the mixing flow path (141). The flow ratio may be a value that allows the solution to flow into the mixing flow path through the cell flow path while having a thickness (T11) in the mixing flow path (141) equal to or less than three times the diameter of the cell (C0), and preferably, may be a value that allows the thickness (T11) to be less than the diameter of the cell (C0).

[0101] The cell transformation method may include operating a pump unit (202) according to the determined flow ratio and flow value to inject a cell solution (SC) into a cell flow path (12) and inject a substance solution (SM) into a substance flow path (13).

[0102] The power applying section (203) is electrically connected to the processor (201) and can be connected to the terminals (26, 27) to apply power to the electrodes (22, 23, 24) included in the electric field forming section (20) of the cell transformation box (1). Once the cell transformation box (1) enters the interior of the cell transformation device (2), the connecting arm of the power applying section (203) approaches and contacts the terminals (26, 27), thereby allowing electrical connection. When the cell solution (SC) is injected into the cell flow path (12) and the material solution (SM) is injected into the material flow path (13) using the pump section (202), the power applying section (203) can apply power to the electric field forming section (20) so that an electric field can be formed. When the solution is supplied and the electric field is formed, transformation occurs in the mixing flow path (141), and the solution containing the transformed cells (C1) can be discharged through the outlet (140) of the result flow path (14).

[0103] Fig.11 It is a diagram showing cell sedimentation occurring in the cell transformation cassette (1) according to the first embodiment of the present invention.

[0104] use Fig.11 , describes the flow rate of the solution that the cell transformation device (2) can provide when the cell transformation box (1) according to the first embodiment of the present invention is provided to the cell transformation device (2). When the cell flow path (12) and the substance transfer flow path (132) merge and lead to the mixing flow path (141), the flow of the cell solution (SC) flowing in the cell flow path (12) can be bent at right angles, and the flow of the cell solution (SC) can be approximated as having a circular motion at the corner. In particular, S S , i.e., the distance that the cells (C0) settle downward due to the centrifugal force of the circular motion in the mixing flow path (141), can be expressed by the equation related to centrifugation as follows. S It can be 1 μm or more and 20 μm or less.

[0105] [Equation 1]

[0106]

[0107] In the above equation 1, V S is the downward velocity of the cell (C0) as shown in the figure, t S is the time required to pass the corner when the corner is approximated as a quarter circle, r is the radius of the approximated quarter circle, d is the diameter of the cell (C0), μ is the viscosity of the cell solution (SC), Δρ is the density difference between the cell (C0) and the cell solution (SC), ω is the angular velocity of the cell (C0), and V C is the horizontal velocity of the cell (C0) in the mixing flow path (141).

[0108] The cell transformation device (2) can be configured so that the thickness of the cell solution (SC) in the mixing flow path (141) is less than the above-mentioned S S The cell solution (SC) and the substance solution (SM) are provided to the cell transformation box (1) at a flow ratio of . The solutions are provided at such a flow ratio that the cells (C0) can merge with the substance (M) in the mixing flow path (141), thereby allowing transformation to occur.

[0109] Fig.12 It is a diagram showing cell sedimentation occurring in the cell transformation cassette according to the third embodiment of the present invention.

[0110] The flow rate of the solution that can be provided by the cell transformation device (2) is described when the cell transformation box according to the third embodiment of the present invention is provided to the cell transformation device (2). Since the mixing flow path as a part of the result flow path (14c) can have an arc shape as shown in the figure, the flow of the cell solution can be approximated as having a circular motion. In particular, S S It is the distance that cells settle downward in the mixing flow path due to the centrifugal force of the circular motion, which can be expressed as follows. S It can be 2 μm or more and 30 μm or less.

[0111] [Equation 2]

[0112]

[0113] In the above equation 2, L E is the length of the mixing flow path, and R is the radius of curvature of the mixing flow path.

[0114] The cell transformation device (2) can be configured so that the thickness of the cell solution in the mixing flow path is less than the above S S The cell solution and the substance solution are provided to the cell transformation box at a flow rate ratio of . Each solution is provided at such a flow rate that the cells can be transformed by confluence with the substance in the mixing flow path.

[0115] Experimental Example 1

[0116] First, the transformation efficiency of mRNA using the cell transformation cassette of the present invention was confirmed.

[0117] For this purpose, using the cell transformation kit of the present invention, 1 mL of a cell culture medium containing 4×10 7A cell solution of NK-92 cells (ATCC CAT#CRL-2407) at a concentration of 1 cell / mL was added to the cell flow channel, and a substance solution containing 200μg / mL of eGFP mRNA (RiboPro, CAT#RB-079) was added to the substance flow channel. Then, an electric field of 1,200V was applied between the inlet of the substance flow channel and the resultant discharge flow channel, and the cell solution and the substance solution were passed through the mixing flow channel (depth 30μm, width 3mm, length 24mm) with the electric field applied at a flow rate ratio of 1:2, and the transformation factor of eGFP mRNA introduced into NK-92 cells was obtained, which was used as the experimental group.

[0118] Meanwhile, using Neon TM Transfection system (ThermoFisher), a material solution containing the same eGFP mRNA as that used to prepare the transforming factors for the experimental group was mixed with a cell solution containing NK-92 cells in the same cell culture medium as that used to prepare the transforming factors for the experimental group, instead of R buffer (i.e., Neon TM For the rest, the transforming factor was obtained according to the manufacturer's instructions and used as a positive control. In addition, as a negative control, a cell solution containing NK-92 cells into which no mRNA was introduced was used.

[0119] For each of the experimental groups and positive and negative control groups obtained as above, flow cytometry was performed; expression efficiency (eTX) was analyzed by classifying cells with stronger fluorescence as successfully expressed cells based on the signal value of the top 1% of the negative control group, and cell viability was measured by Texas Red fluorescent staining technology.

[0120] As a result, it was confirmed that the negative control group and the experimental group prepared using the cell transformation cassette of the present invention showed high cell viability of about 95%, while the cells on the culture medium were used as they were without pretreatment with Neon TM The positive control prepared by the transfection system also showed high cell viability of about 90%. Fig.13 As shown in FIG. 1 , it was confirmed that the expression efficiency (eTX) in the positive control group was 0.78%, and there was almost no transforming factor expressing GFP fluorescence ( Fig.13 Left figure), while in the experimental group prepared using the cell transformation cassette of the present invention, the expression efficiency (eTX) was 98.3%, and almost all cells were transformation factors expressing GFP fluorescence ( Fig.13 right).

[0121] Since mRNA is easily destroyed in cell culture medium containing various components necessary for cell growth, conventional mixed delivery technologies such as Neon TMIn the transfection system, the cells to be transformed should be washed and transferred to a dedicated buffer (Neon TM However, it seems that in the positive control described above, since NK-92 cells were used in a form contained in a cell culture medium without using R buffer (i.e., Neon TM In the mixture of eGFP mRNA and NK-92 cells, all eGFP mRNA was destroyed, so the transforming factor itself in which eGFP mRNA was introduced into NK-92 cells could not be prepared. TM In the case of transfection systems, it is practically impossible to deliver mRNA directly to cells in culture without pretreatment such as washing the cells or using dedicated buffers.

[0122] In contrast, when the cell transformation cassette of the present invention is used, NK-92 cells are used as contained in the cell culture medium as they are; however, since the cell solution and the substance solution containing eGFP mRNA are supplied through separate flow paths, eGFP mRNA can be delivered to NK-92 cells without being destroyed by the components in the cell culture medium, and as a result, eGFP mRNA can be introduced into almost all NK-92 cells, so transformation factors can be prepared efficiently. As can be seen from the above results, the use of the cell transformation cassette of the present invention can deliver mRNA directly to cells on the culture medium. Therefore, it is clearly confirmed by the examples of the present invention that when the cell transformation cassette of the present invention is used, no pretreatment (e.g., washing cells or using a dedicated buffer) is required; therefore, cell loss or cell damage is not caused during cell washing, and expensive consumables such as a dedicated buffer do not need to be used, and the transformation process itself can also be significantly simplified.

[0123] In particular, it can be seen that, compared to existing hybrid delivery technologies in which transformation is performed after transfer to a dedicated buffer, transformation using the cell transformation cassette of the present invention, after undergoing a cell washing process that causes cell loss or cell damage, has significant advantages not only in terms of expression efficiency and cell viability (i.e., two important factors in intracellular mass transfer), but also in terms of process automation rate, process sealing rate, and manufacturing yield (i.e., the three most important factors in the cell therapy manufacturing process).

[0124] Experimental Example 2

[0125] Next, in the process of transformation by separately supplying a cell solution and a substance solution using the cell transformation cassette of the present invention, the effect of the flow ratio of the two solutions in the mixing flow path on the delivery efficiency of a substance such as mRNA was confirmed.

[0126] The fluid flow in the mixing channel is laminar flow with a parabolic velocity curve in the form of a quadratic function depending on the depth, where the velocity becomes maximum at the center of the flow path, and the velocity is 0 at the top and bottom surfaces. Therefore, depending on the flow ratio of the cell solution and the substance solution, there may be changes in the velocity distribution of each flow, thereby affecting the transfer efficiency of the substance. In addition, from a geometric point of view, since the height of the mixing flow path is fixed, the lower the flow rate, the smaller the height of the flow path. Therefore, in the case of cell flow, the thickness of the fluid in the cell flow can be adjusted by controlling the flow rate, so that the thickness can be adjusted according to the size of the cell. The basic trend is that the smaller the thickness of the fluid in the cell flow, the more effective its interaction with the substance flow in the cell is, resulting in higher efficiency.

[0127] To measure this, the delivery and expression efficiency of eGFP mRNA was measured by changing the flow rate (flow rate of substance / flow rate of cells) ratio to 0.5, 1, and 2. Specifically, using the cell transformation kit of the present invention, 1 mL of a cell culture medium containing 4×10 7 A cell solution of NK-92 cells (ATCC CAT#CRL-2407) at a concentration of 1 cell / mL was injected into the cell flow path, and a material solution containing eGFP mRNA (RiboPro, CAT#RB-079) at a concentration of 200 μg / mL was injected into the material flow path. An electric field of 1,200 V was applied between the inlet of the material flow path and the resultant material discharge flow path, and the cell solution and the material solution were passed through a mixing flow path (depth 30 μm, width 3 mm, length 24 mm) to which the electric field was applied at flow ratios of 2, 1, and 0.5 (material flow rate / cell flow rate); as a result, transformation factors for introducing eGFP mRNA into NK-92 cells were obtained and were named transformation factors #1, #2, and #3, respectively.

[0128] For each of the transformation factors #1, #2, and #3 obtained as described above, flow cytometry was performed in the same manner as in Experimental Example 1 to analyze the delivery and expression efficiency. Fig.14 As shown, the smaller the flow rate of the cell flow, the higher the transfer efficiency. When the average size of NK-92 cells is about 15μm, the thickness of the cell flow at each mixing ratio is analyzed to be about 10μm, 15μm, and 20μm. In the case of a thickness of 10μm less than the average cell diameter, almost all cells show an expression rate of more than 90%, which has become the expression level. It can be seen that when the cell solution flow has a thickness similar to the cell diameter, a high expression rate of about 75% is maintained, and when the cell solution flow has a thickness higher than the cell diameter, the expression efficiency is reduced to a level of 40%.

[0129] From the above results, it can be confirmed that the substance transfer process in the mixed flow path of the cell transformation box of the present invention can induce substance transfer by allowing the cells in the cell solution to effectively move toward the substance solution and contact, even if the mixing of the two fluids cannot effectively occur. That is, for the fluid flow in the laminar flow region, due to the inertial force generated in the turbulent flow region, there is no fluid mixing effect. Therefore, even under laminar flow conditions where the cell solution and the substance solution cannot be directly mixed as in the cell transformation box of the present invention, two or more fluid flows introduced separately cannot be effectively mixed in a short time. By arranging the cells in the cell solution so that they can move directly toward the substance solution and immediately contact the substance solution, effective intracellular substance transfer and conversion can be performed. In addition, it can be confirmed through this experimental example that when the thickness of the cell solution flow is less than the cell diameter, almost all cells can be converted, and it can also be seen that under the cell solution flow condition where the thickness is greater than the cell thickness, some cells are converted, and the proportion of converted cells decreases with the increase of thickness.

[0130] Experimental Example 3

[0131] Furthermore, it was confirmed that when the cell transformation cassette of the present invention is used, substances can be delivered at high efficiency even to cells collected from the human body (primary cells).

[0132] In conventional hybrid delivery technologies such as Neon TM In the case of the transfection system, cell loss and damage are inevitably induced in the process of washing cells to remove the cell culture medium. In the case of cells established for use as cell lines by laboratory culture, it is known that cell viability is relatively good due to their unlimited proliferation potential; however, in the case of primary cells collected from human blood or tissues, there is a problem that not only is it generally more difficult to proliferate by culture than the above-mentioned cell lines, but more cell damage is induced in the cell washing process, so the yield is significantly reduced in the process of producing the conversion factor used as a cell therapeutic agent. However, as confirmed in Example 1 above, in the case of transformation using the cell transformation cassette of the present invention, since the substance can be directly delivered to the cells on the cell culture medium without pretreatment such as cell washing or use of a dedicated buffer, it is expected that the substance can be efficiently delivered even to primary cells collected from the human body.

[0133] To confirm this, 1 mL of a cell culture medium containing 5×10 7A cell solution of human peripheral blood monoclonal cells (hPBMC) (Lonza, CAT#CC-2702) at a concentration of 1.5 cells / mL was added to the cell flow path, and a substance solution containing 200μg / mL eGFP mRNA (RiboPro, CAT#RB-079) was added to the substance flow path. Then, an electric field of 1,800V was applied between the inlet of the substance flow path and the resultant discharge flow path, and the cell solution and the substance solution were passed through the mixing flow path (depth 30μm, width 3mm, length 24mm) with an electric field applied at a flow ratio of 2:1, and the conversion factor for introducing eGFP mRNA into hPBMC was obtained, which was used as the experimental group.

[0134] For the transforming factors obtained as described above, the eGFP expression efficiency (eTX) and cell viability were measured in the same manner as in Example 1. Fig.15 As shown, it was confirmed that the hPBMC transformation factors introduced with eGFP mRNA using the cell transformation cassette of the present invention not only showed the same level of cell viability as the negative control group without substance transfer, but also maintained GFP expression for more than one week while showing such high cell viability and the expression efficiency reached a level as high as 98% or more.

[0135] As can be seen from the above results, in the case of transformation using the cell transformation cassette of the present invention, the substance can be delivered directly to the cells on the cell culture medium without pretreatment such as cell washing or the use of a dedicated buffer; therefore, intracellular substance transfer can be effectively performed without causing cell damage to the level that leads to apoptosis, and due to these advantages, the substance can be efficiently delivered to cells (primary cells) even collected from the human body. In addition, in the case of the transformation factor of human harvested cells prepared in this way, the maximum expression efficiency can be shown at a time point of about one week, and it can be seen that when used as a cell therapy, the efficacy can last for more than one week. This is an advantage of existing hybrid delivery technologies such as Neon TM This experimental example clearly shows that the transformation technology using the cell transformation cassette of the present invention is very useful in processes using human cells where the number of available cells is extremely limited.

[0136] Experimental Example 4

[0137] Based on the excellent transformation efficiency of various cells such as cell lines and cells collected from humans confirmed in the above examples, CAR-NK cells were prepared by introducing mRNA of the chimeric antigen receptor (CAR) into NK-92 cells (ATCC CAT #CRL-2407) using the cell transformation kit of the present invention.

[0138] Since NK cells are one of the most difficult cells to transform using various transformation technologies currently applicable to cell manipulation; therefore, there are significant difficulties in preparing CAR-NK. Since the proportion of NK cells in blood cells is less than 5%, the absolute amount of NK cells that can be obtained from the human body is small; in addition, it is known that the transformation efficiency of NK cells infected by viruses is less than 1%. Therefore, it is known that when using existing hybrid delivery technologies such as Neon TM When the transfection system produces transforming factors from human NK cells, the yield is significantly poor due to cell loss and cell damage occurring during cell washing. However, as confirmed in the above examples, when the cell transformation cassette of the present invention is used, it is confirmed that cell loss can be minimized, and substances can be delivered with high activity and efficiency even in cells of human origin, so it is expected that CAR-NK therapeutic agents can also be produced with high yield by confirming the expression of CAR, and the anti-cancer therapeutic effect of CAR has been confirmed.

[0139] To confirm this, the cell transformation kit of the present invention was used to add 1 mL of a cell culture medium containing 1.0×10 7 A cell solution of NK-92 cells (ATCC CAT#CRL-2407) with a concentration of 1 cell / mL was added to the cell flow channel, and a material solution containing 500μg / mL CD19 CAR mRNA (RiboPro, CAT#RB-073-3) was added to the material flow channel. Then, an electric field of 1,200V was applied between the inlet of the material flow channel and the resultant discharge flow channel, and the cell solution and the material solution were simultaneously passed through the mixed flow channel (depth 30μm, width 3mm, length 24mm) with an electric field applied at a flow ratio of 2:1, and a conversion factor for introducing CD19 CAR mRNA into NK-92 cells was obtained, which was named NKL-CD19-500.

[0140] For the conversion factor obtained as described above, the expression efficiency (eTX) of CD19 CAR was measured using anti-FMC63-PE antibody, and the conversion factor and the CD19 overexpressing cancer cell line Nalm6 (ATCC CAT#CRL-3273) were mixed at a ratio of 2:1 (CAR-NK cells: Nalm6 cells) and cultured to measure the cytotoxicity of Nalm6 cancer cells.

[0141] Results, such as Fig.16 As shown, it was confirmed that CD19 CAR was expressed in more than 90% of cells ( Fig.16 (A)) and confirmed that more than 80% of Nalm6 cancer cells were killed within one day ( Fig.16 (B)).

[0142] From the above results, it can be seen that since the substance can be directly delivered to the cells on the cell culture medium without pretreatment (e.g., cell washing or use of a dedicated buffer) when transformation is performed using the cell transformation cassette of the present invention, it is possible to efficiently produce transformation factors using human NK cells, but this is difficult for existing mixed delivery technologies (e.g., Neon TM transfection system) is virtually impossible. This is the case with existing hybrid delivery technologies (e.g., Neon TM The present invention provides an excellent effect that cannot be achieved by conventional transfection systems, and this experimental example confirms the great potential for the development and production of CAR-NK anti-cancer immune cell therapeutic agents that are expected to produce breakthroughs in the treatment of solid cancers. It can also be clearly seen that the transformation technology using the cell transformation box of the present invention can be very useful in the field of anti-cancer immune cell therapy based on human collected NK cells with high production performance, which has not been reported in existing NK cell transformation technologies.

[0143] Above, only because all the components constituting the embodiments of the present invention are described as being combined or combined into one to operate, the present invention is not necessarily limited to these embodiments. That is, as long as it is within the scope of the purpose of the present invention, all the components can be operated by selectively combining one or more. In addition, the above-mentioned terms such as "including", "constituting" and "having" mean that the corresponding components may be inherent, unless otherwise specified, and therefore, should not be interpreted as excluding other components, but may further include other components. Unless otherwise defined, all terms, including technical or scientific terms, have the same meanings as those generally understood by ordinary technicians in the field to which the present invention belongs. Common terms, such as those defined in dictionaries, should be interpreted as being consistent with the contextual meaning of the relevant technology, and should be interpreted in an ideal or overly formal sense, unless clearly defined in the present invention.

[0144] Since the above description is only an example of the technical idea of ​​the present invention, a person of ordinary skill in the art to which the present invention belongs will be able to make various modifications and changes without departing from the basic features of the present invention. Therefore, the embodiments disclosed in the present invention are not intended to limit the technical idea of ​​the present invention, but to illustrate that the scope of the technical idea of ​​the present invention is not limited by these embodiments. The scope of protection of the present invention should be interpreted by the attached claims, and all technical ideas within the scope of equivalents should be interpreted as included within the scope of the present invention.

[0145] Reference numerals

[0146] 1.1d: Cell transformation cassette

[0147] 2.2d: Cell transformation device

[0148] 3: Syringe

[0149] 10, 10b, 10c, 10d: Main body

[0150] 11, 11b, 11c: Main body

[0151] 12, 12b, 12c: Cell flow path

[0152] 13, 13b, 13c: Material flow path

[0153] 14, 14b, 14c: Results logistics path

[0154] 20, 20d: Electric field forming part

[0155] 21, 21d: Electric field forming body

[0156] 22, 22d: Cell electrode

[0157] 23, 23d: Material electrode

[0158] 24, 24d: Result electrode

[0159] 25d: Cell terminals

[0160] 26, 26d: Material terminal

[0161] 27, 27d: Result terminal

[0162] 30, 30d: Sealing sheet

[0163] 40, 40d: O-ring part

[0164] 42: Cell O-ring

[0165] 43: Material O-ring

[0166] 44: Result O-ring

[0167] 50, 50d: guide part

[0168] 51, 51d: Guide body

[0169] 52, 52d: Cell guide

[0170] 53, 53d: Material guide

[0171] 54, 54d: Result guide

[0172] 55d: Power supply guide

[0173] 61: Upper bracket

[0174] 62, 62d: Main body bracket

[0175] 63: Lower bracket

[0176] 100, 100d: Cell transformation system

[0177] 120: Entrance of the cell flow path

[0178] 121: Cell introduction flow path

[0179] 130: Entrance of material flow path

[0180] 131: Material introduction flow path

[0181] 132: Material transfer flow path

[0182] 140: Result logistics route exit

[0183] 141: Mixing flow path

[0184] 142: Result discharge flow path

[0185] 201: Processor

[0186] 202: Pump Department

[0187] 203: Power application unit

[0188] 204: Input

[0189] 541d: Main result guide

[0190] 542d: Auxiliary result guide

[0191] 1301b: First material flow path

[0192] 1302b: Second material flow path

[0193] C0: cells before transformation

[0194] C1, C2: Transformed cells

[0195] M: Conversion factor

[0196] M1: First conversion factor

[0197] M2: Second conversion factor

[0198] SC: cell solution

[0199] SM: Material Solution

[0200] SM1: First substance solution

[0201] SM2: Second substance solution

Claims

1. A cell transformation cassette, comprising: a main body portion in which a cell flow path, a substance flow path, and a result flow path that merge with each other are formed; and an electric field forming portion, comprising an electrode connected to the body portion to generate an electric field in the resultant flow path, wherein the cell flow path and the substance flow path are formed in a shape converging to the result flow path, wherein the result flow path includes a mixing flow path extending from a position connected to the cell flow path and the substance flow path, and Wherein, the thickness of the mixing flow path is smaller than the thickness of the cell flow path. 2 . The cell transformation cassette according to claim 1 , wherein the cell flow path and the substance flow path merge with each other in a state where the cell flow path is located above the substance flow path.

3. The cell transformation cassette according to claim 1, wherein the cell flow path comprises a cell introduction flow path extending downward from an inlet thereof. 4 . The cell transformation cassette according to claim 1 , wherein the mixing flow path extends in a horizontal direction from a position connected to the cell flow path and the substance flow path. The cell transformation cassette according to claim 1 , wherein the material flow path has an arc shape convex downward. 6 . The cell transformation cassette according to claim 5 , wherein the material flow path and the resultant flow path have a downwardly convex continuous arc shape.

7. The cell transformation cassette according to claim 1, wherein the material flow path comprises a material introduction flow path extending downward from an inlet thereof; and a material transfer flow path extending horizontally from a lower end of the material introduction flow path to a result flow path.

8. The cell transformation cassette of claim 1, wherein the inlet of the material flow path, the inlet of the cell flow path, and the outlet of the result flow path are arranged in this order along a reference direction.

9. The cell transformation cassette according to claim 1, wherein the material flow path comprises: a first material flow path, merging with the cell flow path at one side of the cell flow path; and The second material flow path merges with the cell flow path on the other side of the cell flow path. 10 . The cell transformation cassette according to claim 9 , wherein the first substance flow path merges with the cell flow path at a lower side of the cell flow path, and the second substance flow path merges with the cell flow path at an upper side of the cell flow path.

11. The cell transformation cassette according to claim 1, wherein the electric field forming section is provided to form the electric field using a pulse type power source.

12. The cell transformation cassette according to claim 1, wherein the resultant flow path further comprises a resultant discharge flow path connecting one end of the mixing flow path and an outlet of the resultant flow path. 13 . The cell transformation cassette according to claim 1 , wherein the mixing channel has a thickness of 6 μm or more and 400 μm or less.

14. A cell transformation device, comprising: A pump unit configured to supply a cell solution and a substance solution to a cell flow path and a substance flow path of the cell transformation cartridge under pressure, respectively; a power applying unit, configured to be connected to a terminal of the cell transformation box so as to apply power to an electrode of the cell transformation box; and a processor electrically connected to the power applying section and the pump section, Wherein, the processor controls the pump unit based on the information of the cell transformation box, a preset flow ratio, and a preset exposure time.

15. A cell transformation system, comprising: a main body portion in which a cell flow path, a substance flow path, and a result flow path that merge with each other are formed; an electric field forming part, comprising electrodes connected to the body part and disposed at an inlet of the substance flow path and an outlet of the result flow path; A pump unit configured to supply a cell solution and a substance solution to the cell flow path and the substance flow path under pressure, respectively; and a processor electrically connected to the pump portion, wherein the cell flow path and the substance flow path are formed in a shape converging to the result flow path, wherein the result flow path includes a mixing flow path extending from a position connected to the cell flow path and the substance flow path, and The processor controls the pump unit so that the thickness of the solution flowing into the mixing flow path through the cell flow path is smaller than the diameter of the cells contained in the cell solution.

16. A method for cell transformation, comprising: preparing a cassette in which a cell flow path, a substance flow path, and a mixing flow path that merge with each other are formed; injecting a cell solution including cells into the cell flow path; and injecting a solution containing substances for cell transformation into the substance flow path, Here, the ratio of the flow rate of the cell solution to the flow rate of the injected substance solution is a value that allows conversion to occur when the cells and the substance meet in the mixing flow path. 17 . The cell transformation method according to claim 16 , wherein the ratio is a value that allows the solution to flow into the mixing flow path through the cell flow path while having a thickness in the mixing flow path that is less than three times the diameter of the cell. 18 . The cell transformation method according to claim 17 , wherein the ratio is a value that allows the solution to flow into the mixing flow path through the cell flow path while having a thickness in the mixing flow path that is smaller than a diameter of the cell.

19. The cell transformation method according to claim 17, wherein the ratio is a value that allows the solution to flow into the mixing flow path through the cell flow path while having a thickness in the mixing flow path that is smaller than a distance that the cells settle downward in the mixing flow path.

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