Cell transformation cartrdige, cell transformation device, cell transformation system including same and cell transformation method using same

Abstract

A transformation cartridge according to the present invention comprises: a main body portion in which a cell flow path, a material flow path, and a product flow path are formed that meet each other; and an electric field forming portion including an electrode coupled to the main body portion to generate an electric field in the product flow path, wherein the cell flow path and the material flow path are formed in a shape that converges into the product flow path, and the product flow path includes a mixing flow path extending from a location 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

Technology 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] As gene delivery technologies for transformation, viral vector-based transfer methods using viruses as carriers and nonviral delivery techniques using synthetic phospholipids or synthetic cationic polymers are widely known.

[0003] Viruses can be viewed as highly efficient DNA delivery systems because, due to their inherent life mechanism, they parasitize and live by inserting their own DNA into the nucleus of another cell. In other words, DNA delivery systems utilizing viruses involve inserting the gene to be delivered into the inside of a virus that is incapable of replication. The advantage of this technology is that it is highly efficient for transformation. However, this technology has several disadvantages. Fundamentally, although inactivated viruses are used, there are issues such as the potential emergence of RCV (Recombination Competent Virus), the difficulty of repeated use due to immune reactions, the limitation of deliverable substances due to inherent characteristics, and the difficulty of injecting a specific amount of substance into the cell.

[0004] To avoid using viral vectors, a method (electroporation) can be used in which an electric shock is applied to open the phospholipid bilayer while the substance is located around the cell. However, due to the properties of the substance to be delivered, general electroporation may require the cells to be placed in a special buffer (e.g., RNase-free buffer) rather than the culture medium; for this purpose, a pretreatment such as cell washing is required to remove the culture medium from the cells.

[0005] Using such conventional electroporation methods presents problems, including a significant decrease in cell viability and substantial cell loss during the pretreatment process. Additionally, the specialized buffers used are very expensive, making the method uneconomical. Furthermore, since tasks such as pretreatment are difficult to perform in a closed environment, automation is challenging, and maintaining a hygienic working environment is difficult. The problem to be solved

[0006] The present invention has been devised to solve such 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 to occur by electroporation with only the introduction of a cell solution and a material solution. means of solving the problem

[0007] A transformation cartridge according to an embodiment of the present invention comprises: a main body portion in which a cell flow path, a material flow path, and a product flow path are formed that meet each other; and an electric field forming portion including an electrode coupled to the main body portion to generate an electric field in the product flow path, wherein the cell flow path and the material flow path are formed in a shape that converges into the product flow path, and the product flow path includes a mixing flow path extending from a location 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 comprises: a main body portion in which a cell channel, a material channel, and a product channel are formed that meet each other; and an electric field forming portion including an electrode coupled to the main body portion and disposed at the inlet of the material channel and the outlet of the product channel, wherein the cell channel and the material channel are formed in a shape that converges into the product channel, and the product channel includes a mixing channel extending from a location connected to the cell channel and the material channel, and the value obtained by multiplying the thickness of the mixing channel by 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 is smaller than the diameter of the cell flowing in the cell channel.

[0009] A transformation device according to an embodiment of the present invention comprises: a pump unit configured to pump a cell solution and a material solution, respectively, to a cell flow path and a material flow path of a transformation cartridge; a power application unit configured to be connected to a terminal of the transformation cartridge to apply power to an electrode 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 of 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 comprises: a main body portion in which a cell channel, a material channel, and a product channel are formed that meet each other; an electric field forming portion including an electrode coupled to the main body portion and disposed at the inlet of the material channel and the outlet of the product channel; a pump portion arranged to pump a cell solution and a material solution to the cell channel and the material channel, respectively; and a processor electrically connected to the pump portion, wherein the cell channel and the material channel are formed in a shape that converges into the product channel, and the product channel includes a mixing channel extending from a point connected to the cell channel and the material channel, and the processor controls the pump portion such that the thickness of the solution flowing into the mixing channel through the cell channel is smaller than the diameter of the cell contained in the cell solution.

[0011] A transformation method according to an embodiment of the present invention comprises the steps of: preparing a cartridge having a cell channel, a material channel, and a mixing channel formed therein that meet each other; injecting a cell solution containing a cell into the cell channel; and injecting a material solution containing a material for transforming the cell into the material channel, wherein the ratio of the flow rate of the injected cell solution to the flow rate of the material solution is a value such that the cell and the material meet in the mixing channel and transformation occurs. Effects of the invention

[0012] Accordingly, transformation can occur by electroporation with only the introduction of cell solution and material solution.

[0013] Transformation can occur even if the substance solution and the cell solution are not mixed.

[0014] Pretreatment processes such as cell washing for cells or transformed substances can be omitted, which increases cell survival rates during the process and significantly reduces cell and material loss.

[0015] Since no separate special buffer is required, an economical transformation process configuration is possible.

[0016] Since the transformation process can be configured simply by making it possible to inject the solution into a closed and controlled environment without requiring pretreatment, a hygienic working environment can be maintained, which is advantageous from various regulations.

[0017] Since the entire process required for transformation consists of simply injecting a culture medium containing cells and a substance solution and applying an electric field, the process can be easily automated. Brief explanation of the drawing

[0018] FIG. 1 is a perspective view of a transformation cartridge according to a first embodiment of the present invention. FIG. 2 is an exploded perspective view of a transformation cartridge according to a first embodiment of the present invention. FIG. 3 is a plan view of a transformation cartridge according to a first embodiment of the present invention. FIG. 4 is a conceptual diagram of the main body of a transformation cartridge according to the first embodiment of the present invention. FIG. 5 is a conceptual diagram of the main body of a transformation cartridge according to a second embodiment of the present invention. FIG. 6 is a conceptual diagram of the main body of a transformation cartridge according to a third embodiment of the present invention. FIG. 7 is a perspective view of a transformation cartridge according to a fourth embodiment of the present invention. FIG. 8 is an exploded perspective view of a transformation cartridge according to the fourth embodiment of the present invention. FIG. 9 is a drawing showing a transformation system including a transformation cartridge and a transformation device according to a first embodiment of the present invention. FIG. 10 is a drawing showing a transformation system including a transformation cartridge and a transformation device according to a fourth embodiment of the present invention. FIG. 11 is a diagram illustrating the sedimentation of cells occurring in a transformation cartridge according to the first embodiment of the present invention. FIG. 12 is a diagram illustrating the sedimentation of cells occurring in a transgenic cartridge according to a third embodiment of the present invention. Fig. 13 is Neon TM This shows the experimental results confirming the mRNA transformation efficiency using a Transfection System (ThermoFisher) and the mRNA transformation efficiency using the transformation cartridge of the present invention. Figure 14 shows the experimental results confirming the effect of the flow rate ratio between the two solutions within the mixing channel on the delivery efficiency of substances such as mRNA during the process of performing transformation by separately supplying a cell solution and a substance solution using the transformation cartridge of the present invention. Figure 15 shows the experimental results of confirming, repeated three times, whether a substance can be delivered with high efficiency to primary cells collected from the human body when using the transgenic cartridge of the present invention. Figure 16 shows the experimental results confirming the production efficiency (transduction efficiency) (A) and the degree of killing cancer cells (B) of CAR-NK cells produced using the transgenic cartridge of the present invention. Specific details for implementing the invention

[0019] Hereinafter, some embodiments of the present invention will be described in detail with reference to exemplary drawings. It should be noted that in assigning reference numerals to the components of each drawing, the same components are given the same reference numeral whenever possible, even if they are shown in different drawings. Furthermore, in describing the embodiments of the present invention, if it is determined that a detailed description of related known components or functions would hinder understanding of the embodiments of the present invention, such detailed description is omitted.

[0020] In addition, terms such as first, second, A, B, (a), (b), etc., may be used when describing the components of the embodiments of the present invention. These terms are intended only to distinguish the components from other components, and the essence, order, or sequence of the components is not limited by the terms. Where it is stated that a component is "connected," "combined," or "connected" to another component, it should be understood that the component may be directly connected or connected to the other component, but that another component may also be "connected," "combined," or "connected" between each component.

[0021] First embodiment

[0022] 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 a transformation cartridge (1) according to a first embodiment of the present invention. FIG. 3 is a plan view of a transformation cartridge (1) according to a first embodiment of the present invention.

[0023] A transformation cartridge (1) according to the first embodiment of the present invention includes a main body (10) and an electric field forming part (20). In the specification of the present invention, the up, down, left, right, front, and back directions are mutually orthogonal directions used for convenience of explanation and are relative directions that can change depending on the arrangement state of the transformation cartridge (1).

[0024] The electric field forming part (20) is a component that forms an electric field in the main body part (10). The electric field forming part (20) is coupled to the main body part (10) and includes electrodes disposed at the inlet (130) of the material flow path (13) and the 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.

[0025] The electric field forming part (20) may include a plate-shaped electric field forming body (21). The electric field forming body (21) may be composed of a multilayer epoxy resin, but the material is not limited thereto. Electrodes may be formed on the electric field forming body (21), and each electrode may be formed in an annular shape to surround each hole formed in the electric field forming part (20) so as to correspond to each flow path of the main body part (10).

[0026] The electrodes (22, 23, 24) may be formed from a conductor such as metal. The electrodes may be formed by coating copper onto the electric field forming body (21). The electrodes of the electrode 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) that are electrically connected to each electrode (22, 23, 24) and can be connected to a power source. The terminals (26, 27) may be formed from the same material as the electrodes (22, 23, 24). The transgenic cartridge (1) according to the first embodiment of the present invention may include a material terminal (26) that is electrically connected to the material electrode (23) and a result terminal (27) that is electrically connected to the result electrode (24). Each terminal (26, 27) can be placed on the outside of the electric field forming body (21) to facilitate connection to a power source.

[0027] The electric field forming part may be configured to include electrodes that are coupled to the main body and arranged to surround each flow path on the inner side of the main body. These electrodes may also be arranged in the mixing flow path.

[0028] The electric field forming unit (20) can form an electric field using a simple DC power source, but can also form an electric field using a pulse-shaped power source. A processor (201) of a transformation device (2 in FIG. 9) to be described later can control the electric field forming unit (20) in a PWM control manner using a power application unit (203) electrically connected thereto. The frequency of the pulse may be 1 kHz or higher and 100 kHz or lower, and the duty ratio may be 10% or higher and 90% or lower. The electric field forming unit (20) can form the electric field while changing the magnitude of the electric field. The magnitude of the electric field can change by decreasing up to 50% of the maximum value of the magnitude of the electric field formed by the electric field forming unit (20).

[0029] The transformation cartridge (1) according to the first embodiment of the present invention may include a sealing sheet (30). The sealing sheet (30) is positioned between the main body (10) and the lower mount (63) to be described later, so as to prevent impact from being directly transmitted from the lower mount (63) to the main body (10) and to stably support the main body (10). The sealing sheet (30) may be formed from a material including silicone, 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 condition of each flow path (12, 13, 14) can be checked from the lower side upward.

[0030] The transformation cartridge (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 elastic annular O-rings (42, 43, 44) and is positioned between the electric field forming portion (20) and the guide portion (50) to be described later, thereby maintaining watertightness between the electric field forming portion (20) and the guide portion (50) and holding the needle inserted into the guide portion (50) to ensure stable injection of the solution. The O-ring portion (40) may be formed from a material including silicone, but the material is not limited thereto. The O-ring portion (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 result O-ring (44) positioned between the result electrode (24) and the result guide (54).

[0031] A transformation cartridge (1) according to the first embodiment of the present invention may include a guide section (50). The guide section (50) may serve as an inlet and outlet for a solution. The guide section (50) may include a plate-shaped guide body (51), a cell guide (52), a material guide (53), and a result guide (54) that are coupled to the guide body (51). Each guide (52, 53, 54) may be formed as a pipe extending upward from the guide body (51). Each guide (52, 53, 54) has a shape that penetrates vertically and is positioned at a location corresponding to each electrode (22, 23, 24) and a flow path (12, 13, 14). Thus, when a needle is inserted into the guide (52, 53, 54) and connected to a pump to discharge liquid, the discharged liquid can be delivered through each electrode to each flow path. A needle can be stably inserted into each guide (52, 53, 54) to enable the discharge of liquid at an appropriate location. An O-ring (42, 43, 44) may be placed on the lower side of each guide (52, 53, 54), and an 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.

[0032] A 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 on the upper side of the guide portion (50), and the lower mount (63) may be located on the lower side of the sealing sheet (30). The upper mount (61) and the lower mount (63) may be fastened to each other by means of fasteners such as bolts, thereby fixing the sealing sheet (30), the main body portion (10), the electric field forming portion (20), the O-ring portion (40), and the guide portion (50) located between them in a stacked state. The components between them are pressed by the upper mount (61) and the lower mount (63), so that sealing can occur effectively. A hole through which the guide can pass may be formed in the upper mount (61), and a hole may be formed in the lower mount (63) 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 including aluminum, and such aluminum may be anodized aluminum, but the material is not limited thereto.

[0033] The transformation cartridge (1) according to the first embodiment of the present invention may include a main body mount (62). The main body mount (62) may include a hole in the center into which the main body part (10) is inserted, so that the main body part (10) may be aligned with other components while positioned in an appropriate location. The main body mount (62) may be located between the upper mount (61) and the lower mount (63) and may be further connected through a fastener. The main body mount (62) may be composed of a material including aluminum, and such aluminum may be anodized aluminum, but the material is not limited thereto.

[0034] FIG. 4 is a conceptual diagram of the main body (10) of a transformation cartridge (1) according to the first embodiment of the present invention.

[0035] In the main body (10), cell channels (12), material channels (13), and product channels (14) that meet each other are formed. These channels (12, 13, 14) can be formed by penetrating the main body (11). The main body (11) can be formed of a material including glass, and such glass may be borosilicate glass, but the material is not limited thereto.

[0036] An inlet (120, 130) or outlet (140) of each fluid channel (12, 13, 14) may be arranged on the upper surface of the main body (11). The inlet (130) of the material fluid channel (13), the inlet (120) of the cell fluid channel (12), and the outlet (140) of the product fluid channel (14) may be arranged in the order of the inlet (130) of the material fluid channel (13), the inlet (120) of the cell fluid channel (12), and the outlet (140) of the product fluid channel (14) as they move forward in the reference direction. A cell solution (SC) may be introduced into the inlet (120) of the cell fluid channel (12), a material solution (SM) may be introduced into the inlet (130) of the material fluid channel (13), and a product solution may be discharged through the outlet (140) of the product fluid channel (14).

[0037] The cell flow channel (12) is a channel into which a cell solution (SC) is introduced and flows. The cell solution (SC) may consist of a culture medium containing cells (C0) or an electrolyte. The cells (C0) contained in the cell solution (SC) may be blood cells among somatic cells, and among them may be immune cells. The immune cells may be used without limitation as long as they are cells capable of inducing immunity to produce a desired therapeutic effect, and for example, may be 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, but are not limited thereto. The cell flow channel (12) may include a cell inflow channel (121) extending from the inlet (120) of the cell flow channel (12). The cell inflow channel (121) may extend downward from the inlet (120) of the cell flow channel (12).

[0038] The material channel (13) is a channel into 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 intended to be introduced into the interior of the cell (C0), such as the cytoplasm or nucleus, to perform a function within the cell (C0), and is not limited to any form, such as protein, peptide, or nucleic acid, as long as it is a substance capable of performing a function within the cell (C0). In particular, the transforming material (M) may be a nucleic acid, specifically mRNA. The material channel (13) may include a material inflow channel (131) extending from the inlet (130) of the material channel (13). The material inflow channel (131) may extend downward from the inlet (130) of the material channel (13). The material channel (13) may include a material transfer channel (132) extending from the bottom of the material inflow channel (131) toward the resulting channel (14). The material transfer channel (132) can be extended forward in a horizontal direction. Thus, the material channel (13) can be bent at the location where the material inflow channel (131) and the material transfer channel (132) meet, and the angle of bending can be 90 degrees.

[0039] The cell channel (12) and the material channel (13) can meet in a state where the cell channel (12) is located above the material channel (13). The lower end of the cell inflow channel (121) can be located above the front end of the material transfer channel (132). Thus, at the point where the material channel (13) and the cell channel (12) meet, the cell solution (SC) can be introduced into the result channel (14) in a state where it is located above the material solution (SM).

[0040] The resulting fluid channel (14) is a channel through which the resulting solution containing the transformed cell (C1) flows. The resulting solution can be discharged through the outlet (140) of the resulting fluid channel (14). The cell fluid channel (12) and the material fluid channel (13) are formed in a converging manner into the resulting fluid channel (14). Thus, the cell solution (SC) and the material solution (SM) are combined to form the resulting solution.

[0041] The resulting flow path (14) includes a mixing flow path (141) extending from a point connected to the cell flow path (12) and the material flow path (13). The mixing flow path (141) may extend horizontally forward from said point. The resulting flow path (14) may include a resulting discharge flow path (142) connecting the end of the mixing flow path (141) and the outlet (140) of the resulting flow path (14). The resulting discharge flow path (142) may extend upward from the front end of the mixing flow path (141).

[0042] Since the cell channel (12) meets the material transfer channel (132) and the mixing channel (141) in a state where they are not parallel to each other, the flow of the cell solution (SC) is bent at the point of meeting. The cell solution (SC) can be bent at a 90-degree angle. Since the direction of flow of the cell solution (SC) changes at the point where the channels meet, the cell (C0) can sink downward due to the centrifugal acceleration resulting therefrom and easily meet with the material (M).

[0043] The electric field forming unit (20) can form an electric field along the solution flowing in each channel (12, 13, 14) using each electrode (22, 23, 24). The electric field forming unit (20) can form an electric field in the material channel (13) and the product channel (14) using the material electrode (23) and the product electrode (24). The electric field forming unit (20) can form an electric field in the cell channel (12) using the cell electrode (22). The electric field formed in the direction starting from the cell channel (12) or the material channel (13) and going out through the product channel (14) is indicated by a dotted line in the drawing. By forming an electric field, the flow direction within the channels (12, 13, 14) of each solution (SC, SM) can be formed, and transformation by electroporation can occur in the mixing channel (141) to be described later.

[0044] In the mixing channel (141), the transforming substance (M) of the substance solution (SM) can enter the interior of the cell (C0) of the cell solution (SC). Since the phospholipid bilayer of the cell (C0) is locally opened by the electric field applied to the mixing channel (141), the transforming substance (M) can enter the cell (C0).

[0045] 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, 14) refers to the degree to which the boundary of each channel (12, 13, 14) is spread out in a direction perpendicular to the direction in which the solution (SC, SM) flows within the channel (12, 13, 14) in a cross-section cut by a plane perpendicular to the left-right direction as shown in FIG. 4. If each channel (12, 13, 14) is formed as a pipe shape extending from a cylinder, the thickness of the channel (12, 13, 14) may be the inner diameter of the channel (12, 13, 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㎛ or more and 400㎛ or less.

[0046] 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) that converges into the resulting channel (14), so that the thickness (T11) of the cell solution (SC) within 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) within the cell channel (12). Similarly, the thickness (T21) of the material solution (SM) within 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) within the material channel (13). In this case, if the thickness (T11) of the cell solution (SC) within the mixing channel (141) is smaller than the diameter of the cell (C0), a part of the cell (C0) may be exposed to the material solution (SM) and placed in a state where it can come into contact with the transformation material (M). As an electric field is formed in the mixing channel (141), the transforming substance (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.

[0047] In order to enable such flow, the value obtained by dividing the thickness of the cell channel (12) by the sum of the thickness of the cell channel (12) and the thickness of the material channel (13), and then multiplying the value by the thickness of the mixing channel (141), may be smaller than the diameter of the cell (C0) flowing in the cell channel (12).

[0048] The thickness (T11) of the cell solution (SC) flowing within the mixing channel (141) can be determined by controlling the flow rate of each solution injected. The processor (201) can control the pump unit (202) such that the thickness (T11) of the cell solution (SC), which is the solution flowing into the mixing channel (141) through the cell channel (12), within the mixing channel (141) is smaller than the diameter of the cell contained in the cell solution (SC). Under the same conditions, if the pressure or flow rate of the pump unit (202) pumping the cell solution (SC) increases, the thickness (T11) of the cell solution (SC) within the mixing channel (141) may increase.

[0049] In order for the cell (C0) to be easily exposed to the material solution (SM) in the mixing channel (141), the cell channel (12) described above may meet with the material channel (13) in a state where it is located above the material channel (13). This is because, due to this channel arrangement, the cell solution (SC) will be located above the material solution (SM) within the mixing channel (141), so the cell (C0) carried by the cell solution (SC) can move toward the material solution (SM) below under the influence of gravity and be more easily exposed to the material solution (SM).

[0050] Due to the structure of the main body (10) described above, transformation can occur simply by injecting the cell or transformation material (M) into the main body (10) and applying an electric field, without performing separate pretreatment on the cell or transformation material (M), and a transformed cell (C1) can be obtained. Therefore, pretreatment processes such as cell washing can be omitted, and the loss of cells and materials during the process can be significantly reduced. In addition, since a separate special buffer does not need to be used, an economical process configuration is possible. Furthermore, since no pretreatment is required, the process can be configured simply by making it possible to inject the solution into a sealed and controlled environment, thereby maintaining a hygienic working environment and being advantageous from various regulations. Since the entire process required for transformation is simply injecting a culture medium containing cells and a material solution and applying an electric field, it is easy to automate the process.

[0051] 2nd embodiment

[0052] FIG. 5 is a conceptual diagram of the main body (10b) of a transformation cartridge according to a second embodiment of the present invention.

[0053] Since the transformation cartridge according to the second embodiment of the present invention is identical to the transformation cartridge (1) according to the first embodiment except that it further has a second material flow path (1302b), the parts that differ are further explained, and the description of the transformation cartridge (1) according to the first embodiment may be applied as is to the remaining components. In the electric field forming part, guide, O-ring part, etc. of the transformation cartridge according to the second embodiment, a part for the second material flow path (1302b) is formed.

[0054] 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 material (M1) flows, and a second material flow path (1302b) through which a second material solution (SM2) containing a second transforming material (M2) flows. The first transforming material (M1) and the second transforming material (M2) may be different from each other. The first material flow path (1301b) may be identical to the material flow path (13) according to the first embodiment. Therefore, the first material flow path (1301b) is considered to be identical to the material flow path (13) of the first embodiment, and the second material flow path (1302b) will be described further.

[0055] A first substance channel (1301b) may meet the cell channel (12b) at one side of the cell channel (12b), and a second substance channel (1302b) may meet the cell channel (12b) at the other side of the cell channel (12b). The first substance channel (1301b) may meet the cell channel (12b) at the lower side of the cell channel (12b), and the second substance channel (1302b) may meet the cell channel (12b) at the upper side of the cell channel (12b). Accordingly, in the resulting channel (14b), the first substance solution (SM1) may flow to the upper side of the cell solution (SC) and the second substance solution (SM2) may flow to the lower side by means of the action described in the first embodiment, and the cell may be exposed to the first substance solution (SM1) and the second substance solution (SM2). Cells exposed to each substance solution (SM) are opened by an electric field, allowing the first transforming substance (M1) and the second transforming substance (M2) to enter the cell and transformation to occur.

[0056] Accordingly, according to the second embodiment, a plurality of different types of transformation substances (M1, M2) can be injected into a cell simultaneously using a single transformation cartridge to form a transformed cell (C2).

[0057] Third embodiment

[0058] FIG. 6 is a conceptual diagram of the main body (10c) of a transformation cartridge according to the third embodiment of the present invention.

[0059] 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, 14c) formed in the main body (10c). Therefore, the parts that differ are further explained, and the description of the transformation cartridge (1) according to the first embodiment can be applied as is to the remaining components.

[0060] 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 channel (13c) according to the third embodiment may have the shape of a downwardly convex arc. The material flow channel (13c) and the resulting flow channel (14c) may have the shape of a continuous downwardly convex arc. That is, the material flow channel (13c) and the resulting flow channel (14c) may be connected by drawing a continuous downwardly convex arc. Although the cell flow channel (12c) is also depicted as having a curved profile such as having the shape of an arc, the cell flow channel (12c) may have a shape that extends downward. If the cell flow channel (12c) has the shape of an arc, the radius of curvature of the cell flow channel (12c) may be smaller than the radius of curvature of the material flow channel (13c).

[0061] Since the material channel (13c) has an arc shape, centrifugal acceleration is applied to the transforming material introduced into the material channel (13c), making it easier for the transforming material to come into contact with the cell.

[0062] An 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) of the second embodiment.

[0063] 4th embodiment

[0064] FIG. 7 is a perspective view of a transformation cartridge (1d) according to a fourth embodiment of the present invention. FIG. 8 is an exploded perspective view of a transformation cartridge (1d) according to a fourth embodiment of the present invention.

[0065] The transformation cartridge (1d) according to the fourth embodiment of the present invention has the same components as the main body (10), O-ring (40), and sealing sheet (30) of the transformation cartridge (1) according to the first embodiment, and since there are some differences in shape regarding the remaining components, the parts with differences are further explained, and the description of the transformation cartridge (1) according to the first embodiment can be applied as is to the corresponding components.

[0066] The guide section (50d) of the transformation cartridge (1d) according to the fourth embodiment can receive and mix a plurality of solutions and deliver them to the material flow path of the main body (10d). To this end, the guide section (50d) may include a plurality of material guides (53d). Different material solutions are injected into each material guide (53d), and while the different material solutions are delivered to the inlet of the material flow path through the guide section (50d), they are integrated into a single flow path and delivered, so they can be mixed with each other during the flow process.

[0067] The guide section (50d) can discharge the resulting solution into multiple guides. Accordingly, the resulting guide (54d) may include a main resulting guide (541d) and an auxiliary resulting guide (542d). The main resulting guide (541d) may have a pipe shape that extends upward and then bends to the left. The auxiliary resulting guide (542d) may have a pipe shape that extends upward. The resulting solution discharged from the resulting flow path may be split at the guide section (50d) into the main resulting guide (541d) and the auxiliary resulting guide (542d) and discharged respectively.

[0068] The guide section (50d) may include a power supply guide (55d). Multiple power supply guides (55d) may be formed. The power supply guide (55d) allows each terminal (25d, 26d, 27d) of the electric field forming section (20d) located on the lower side of the guide body (51d) to be exposed to the outside of the guide section (50d), and allows a connection port for connecting to each terminal (25d, 26d, 27d) to stably contact and be fixed to each terminal (25d, 26d, 27d). The power supply guide (55d) may also be formed in a pipe shape extending upward from the guide body (51d).

[0069] The electric field forming part (2d0) may include a material terminal (26d) electrically connected to a material electrode (23d), a product terminal (27d) electrically connected to a product electrode (24d), and a cell terminal (25d) electrically connected to a cell electrode (22d). Each terminal (25d, 26d, 27d) may be positioned on the electric field forming body (21d) at a location corresponding to a power supply guide (55d). The electric field forming part (20d) may be coupled to the guide part (50d) through a fastener.

[0070] The main body mount (62d) includes a hole in the center into which the main body part (10d) is inserted, so that the main body part (10d) can be aligned with other components while positioned in an appropriate location. However, a step is formed in the hole of the main body mount (62d) so that the main body part (10d) and the sealing sheet (30d) do not deviate downward while being inserted. The main body mount (62d) can be connected to the electric field forming part (20d) through a fastener.

[0071] FIG. 9 is a drawing 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 drawing showing a transformation system (100d) including a transformation cartridge (1d) and a transformation device (2d) according to a fourth embodiment of the present invention.

[0072] Referring to the drawings, the transformation system (100, 100d) according to the first and fourth embodiments of the present invention includes a transformation cartridge (1, 1d) and a transformation device (2, 2d). The transformation system (100, 100d) may include a syringe (3). Since there is a difference between the first and fourth embodiments only in the number of syringes (3), the number of pumps, the number of guides, etc., the method for transforming cells according to the present invention will be described with reference to FIG. 9 as a representative reference.

[0073] Transformation can occur by injecting each solution into the transformation cartridge (1) by the transformation device (2). A transformation cartridge (1) is prepared in which a cell flow path (12), a material flow path (13), and a product flow path (14) are formed. That is, the transformation method may include the step of preparing the transformation cartridge (1). A syringe (3) containing the cell solution and the material solution, respectively, can be inserted into the guide part (50) of the transformation cartridge (1). The transformation cartridge (1) with the syringe (3) inserted can be introduced into the interior of the transformation device (2).

[0074] 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 may include a power application unit (203).

[0075] The pump unit (202) is configured to pump the cell solution (SC) and the material solution (SM) to the cell flow path (12) and the material flow path (13), respectively. Accordingly, the pump unit (202) may include a plurality of pumps for pumping each solution. The pumps of the pump unit can transfer the solution from a storage tank containing each solution to each flow path of the main body (10), which will be described later, through each pipe.

[0076] 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. Thus, when the transformation cartridge (1) with the syringe (3) inserted into the interior of the transformation device (2) is entered, the fluid can be pumped by the pump approaching and contacting the piston of the syringe (3) and pushing the syringe (3) as operation begins. However, the pump is not limited to a syringe pump, and the pump may be configured such that the piping is directly connected to each flow path of the cartridge and pumps the fluid.

[0077] The pump unit (201) can bring a pressurizing member to contact the piston to push the piston when the transformation cartridge (1) enters the interior of the transformation device (2). At this time, the pump unit (201) can measure the distance the pressurizing member has moved and allow the processor (201) to calculate the volume of the solution stored in the syringe (3).

[0078] 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 that includes an element capable of performing logical operations to execute control commands, and may include a CPU (Central Processing Unit), etc. 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 receive acquired information in the form of signals by connecting to various sensors or acquisition units. Since the processor (201) can be electrically connected to each component, it may be connected by a wire or may have a communication module capable of wireless communication to communicate with each other.

[0079] The transformation device (2) further includes a storage medium so that control commands performed by the processor can be stored in the storage medium and utilized. The storage medium may be a device such as an HDD (Hard Disk Drive), SSD (Solid State Drive), server, volatile medium, non-volatile medium, etc., but its type is not limited thereto. In addition to this, data required by the processor (201) to perform tasks may also be stored in the storage medium.

[0080] The transformation device (2) can receive necessary information through the input unit (204). The input unit (204) can receive information by means such as buttons, switches, and touchscreens, and can also receive information by scanning codes such as barcodes, QR codes, and RFID scanners. Information regarding the transformation cartridge (1) entering the transformation device (2) can be received through the input unit (204). The information received through the input unit (204) is transmitted to the processor (201).

[0081] The processor (201) can determine the flow rate of each solution delivered by the pump unit to the transformation cartridge (1) using input information and pre-set stored information. The processor (201) can determine the flow rate of each solution to a value that causes the cell (C0) and the substance (M) to meet in the mixing channel (141) and transformation to occur. When the processor (201) knows which substance solution (SM) and which cell solution (SC) will be used, it can retrieve the optimal flow rate ratio corresponding to the substance solution (SM) and cell solution (SC) from the stored data. The processor (201) can also retrieve the optimal exposure time for transformation treatment 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 retrieved flow rate ratio and exposure time with the input information of the transformation cartridge (1) (cartridge channel length, channel shape, etc.), and can cause the pump unit (202) to operate according to the determined flow rate to push the syringe (3). Here, the flow rate ratio determines what proportion of the total thickness of the mixing channel (141) the material solution (SM) and the cell solution (SC) will occupy within the mixing channel (141). The optimal exposure time means a sufficient amount of time for the cell (C0) and the material (M) to pass through the mixing channel (141) and for transformation to occur.

[0082] The above flow rate ratio may be a value that causes the cell (C0) and the substance (M) to meet in the mixing channel (141) and transformation to occur. The flow rate ratio may be a value that causes the thickness (T11) of the solution flowing into the mixing channel (141) through the cell channel (11) within the mixing channel (141) to be three times or less the diameter of the cell (C0), and preferably a value that causes it to be smaller than the diameter of the cell (C0).

[0083] The transformation method may include the step of operating a pump unit (202) according to a determined flow rate ratio and flow rate value to inject a cell solution (SC) into a cell flow channel (12) and inject a material solution (SM) into a material flow channel (13).

[0084] The power application unit (203) is electrically connected to the processor (201) and can be connected to terminals (26, 27) to apply power to electrodes (22, 23, 24) included in the electric field forming unit (20) of the transformation cartridge (1). When the transformation cartridge (1) enters the interior of the transformation device (2), an electrical connection can be made by the connecting arm of the power application unit (203) approaching and contacting the terminals (26, 27). When injecting a cell solution (SC) into the cell channel (12) and injecting a material solution (SM) into the material channel (13) using the pump unit (202), the power application unit (203) can apply power to the electric field forming unit (20) so that an electric field is formed. A solution is supplied and an electric field is formed so that transformation occurs in the mixing channel (141), and a solution containing transformed cells (C1) can be discharged through the outlet (140) of the resulting channel (14).

[0085] FIG. 11 is a diagram illustrating the sedimentation of cells (C0) occurring in a transformation cartridge (1) according to the first embodiment of the present invention.

[0086] Using FIG. 11, the flow rate of the solution that the transformation device (2) can provide when the transformation cartridge (1) according to the first embodiment of the present invention is provided to the transformation device (2) is explained. When the cell channel (12) and the material transfer channel (132) meet and lead to the mixing channel (141), the flow of the cell solution (SC) flowing in the cell channel (12) can be bent at a right angle, and the flow of the cell solution (SC) can be approximated as performing a circular motion at a corner. At this time, S is the distance at which the cell (C0) sinks downward in the mixing channel (141) due to the centrifugal force of the aforementioned circular motion. SIt can be expressed as follows using an equation related to centrifugation. S S It can be 1㎛ or more and 20㎛ or less.

[0087]

[0088] Here, V S is the velocity of the downward-facing cell (C0) as depicted, and t S ε is the time required to pass through a 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 that the cell (C0) has in the mixing channel (141).

[0089] The transformation device (2) is the above-described S S Cell solution (SC) and material solution (SM) can be supplied to the transformation cartridge (1) at a flow rate ratio such that the thickness of the cell solution (SC) within the mixing channel (141) becomes smaller. With each solution supplied at this flow rate ratio, the cell (C0) can meet the material (M) in the mixing channel (141) and transformation can occur.

[0090] FIG. 12 is a diagram illustrating the sedimentation of cells occurring in a transgenic cartridge according to a third embodiment of the present invention.

[0091] Using FIG. 12, the flow rate of the solution that the transformation device (2) can provide when the transformation cartridge according to the third embodiment of the present invention is provided to the transformation device (2) is explained. Since the mixing channel, which is a part of the resulting channel (14c), may have the shape of an arc as illustrated, the flow of the cell solution can be approximated as circular motion. At this time, S is the distance the cell sinks downward in the mixing channel due to the centrifugal force of the circular motion described above. S can be expressed as follows. S SIt can be 2㎛ or more and 30㎛ or less.

[0092]

[0093] Here, L E is the length of the mixing channel, and R is the radius of curvature of the mixing channel.

[0094] The transformation device (2) is the above-described S S Cell solution and substance solution can be supplied to the transformation cartridge at a flow rate ratio that causes the thickness of the cell solution within the mixing channel to be reduced. With each solution supplied at this flow rate ratio, the cell can encounter the substance in the mixing channel, allowing transformation to occur.

[0095] Experimental Example 1

[0096] First, the transformation efficiency of mRNA using the transformation cartridge of the present invention was confirmed.

[0097] To this end, using the transformation cartridge of the present invention described above, 4x10 7 1 mL of a cell solution containing NK-92 cells (ATCC CAT# CRL-2407) at a concentration of 1 / mL 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, respectively. Then, while applying an electric field of 1200 V between the inlet of the substance channel and the discharge channel, the cell solution and the substance solution passed through a mixing channel (depth 30 μm, width 3 mm, length 24 mm) with the electric field applied at a flow rate ratio of 1:2, thereby obtaining a transformant in which the eGFP mRNA was introduced into the NK-92 cells, and this was used as the experimental group.

[0098] Meanwhile, Neon TM Using a Transfection System (ThermoFisher), a solution of material containing the same eGFP mRNA used in the preparation process of the transformants of the above experimental group, Neon TMInstead of using R-buffer, a buffer solution dedicated to the Transfection System, a cell solution containing NK-92 cells was mixed with the same cell culture medium used in the preparation process of the transformants of the experimental group above, and the transformants were obtained according to the manufacturer's instructions for the remaining details, and this was used as a positive control. Additionally, a cell solution containing NK-92 cells without mRNA introduction was used as a negative control.

[0099] Flow cytometry was performed on each of the experimental group, positive and negative control groups obtained as described above. Cells with a stronger fluorescence emission than the top 1% signal value of the negative control group were classified as successful expression cells, and the expression efficiency (eTX) was analyzed. Cell viability was measured using the Texas Red fluorescence staining technique.

[0100] As a result, the negative control group and the experimental group produced using the transgenic cartridge of the present invention showed a high cell survival rate of approximately 95%, and using the cells on the culture medium as is without pretreatment, Neon TM The positive control group produced by the Transfection System was also confirmed to exhibit a high cell viability of approximately 90%. However, as shown in Fig. 13, the expression efficiency (eTX) in the positive control group was 0.78%, indicating that there were almost no transformants expressing GFP fluorescence (left graph of Fig. 13), whereas in the experimental group produced using the transfection cartridge of the present invention, the expression efficiency (eTX) was 98.3%, confirming that virtually all cells were transformants expressing GFP fluorescence (right graph of Fig. 13).

[0101] Because mRNA is easily destroyed in cell culture media containing various components necessary for cell growth, Neon TMIn conventional mixed delivery technologies such as Transfection Systems, the cells to be transformed must be washed and then a dedicated buffer (Neon TM In the case of a transfection system, it must be transferred to R-buffer and mixed with mRNA in that state. Nevertheless, in the positive control group as described above, Neon TM It is determined that because NK-92 cells were used as they were in the cell culture medium without using R-buffer, a buffer solution dedicated to the Transfection System, the eGFP mRNA was completely destroyed in the mixture of eGFP mRNA and NK-92 cells, resulting in the failure to produce the transformant in which eGFP mRNA was introduced into the NK-92 cells. From the above results, Neon TM It can be seen that with existing mixed delivery technologies, such as transfection systems, it is virtually impossible to directly deliver mRNA to cells in a culture medium without pretreatment, such as washing the cells or using dedicated buffer solutions.

[0102] In contrast, when using the transformation cartridge of the present invention described above, although NK-92 cells are used as they are contained in the cell culture medium, the cell solution and the solution containing eGFP mRNA are supplied through separate channels. Consequently, eGFP mRNA can be delivered to NK-92 cells without being destroyed by components within the cell culture medium, and as a result, eGFP mRNA is introduced into almost all NK-92 cells, allowing for the production of transformants with high efficiency. From the above results, it can be seen that mRNA can be delivered directly to cells in the culture medium by using the transformation cartridge of the present invention. Therefore, when using the transformation cartridge of the present invention, no pretreatment such as a cell washing process or the use of a dedicated buffer solution is required. Consequently, cell loss or damage during the cell washing process is not induced, and not only is it unnecessary to use expensive consumables such as dedicated buffer solutions, but the transformation process itself can also be significantly simplified, as has been clearly confirmed through these embodiments.

[0103] In particular, compared to conventional mixed delivery technology that proceeds with transformation by transferring cells to a dedicated buffer solution after a cell washing process that causes cell loss or damage, it can be seen that transformation using the transformation cartridge of the present invention is significantly advantageous not only in terms of expression efficiency and cell viability, which are two important factors in intracellular material delivery, but also in terms of process automation rate, process occlusion rate, and production yield, which are the three most important factors in the cell therapy manufacturing process.

[0104] Experimental Example 2

[0105] Next, during the process of performing transformation by separately supplying a cell solution and a material solution using the transformation cartridge of the present invention, the effect of the flow rate ratio between the two solutions within the mixing channel on the delivery efficiency of a substance such as mRNA was confirmed.

[0106] Fluid flow within a mixed channel is laminar and exhibits a parabolic velocity profile with respect to depth; the velocity is maximum at the center of the channel and becomes zero at the top and bottom surfaces. Consequently, changes in the velocity profiles of the cell solution and the substance solution occur depending on their respective flow rate ratios, which can affect the efficiency of substance transfer. Furthermore, from a geometric perspective, due to the fixed height of the mixed channel, the height of the flow decreases as the flow rate decreases. Therefore, in the case of cell flow, the fluid thickness of the cell flow can be controlled by adjusting the flow rate, allowing for regulation according to cell size. The fundamental trend is that as the thickness of the cell fluid flow decreases, it interacts more effectively with the substance flow within the cells, resulting in higher efficiency.

[0107] To measure this, the delivery and expression efficiency of eGFP mRNA was measured by varying the flow ratio (material flow rate / cell flow rate) to 0.5, 1, and 2. Specifically, using the transgenic cartridge of the present invention described above, 4x10 71 mL of a cell solution containing NK-92 cells (ATCC CAT# CRL-2407) at a concentration of 1 / mL 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, respectively. Then, while applying an electric field of 1200 V between the inlet of the substance channel and the product outlet, the cell solution and the substance solution passed through a mixing channel (depth 30 μm, width 3 mm, length 24 mm) with the electric field applied at flow rate ratios of 2, 1, and 0.5 (substance flow rate / cell flow rate), thereby obtaining a transformant in which the eGFP mRNA was introduced into the NK-92 cells, which were designated as transformant #1, #2, and #3, respectively.

[0108] For each of the transformants #1, #2, and #3 obtained as described above, flow cytometry was performed in the same manner as in Example 1 to analyze the delivery and expression efficiency. As a result, as shown in Figure 14, higher delivery efficiency was observed as the cell flow rate decreased. For NK-92 cells with an average size of approximately 15 µm, it was analyzed that the thickness of the cell flow was formed at 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% was observed, indicating that almost all cells were expressed. When the cell solution flow had a thickness similar to the cell diameter, a high expression rate of about 75% was maintained, while when it was thicker than the cell diameter, the expression efficiency dropped to the 40% level.

[0109] From the results described above, it can be seen that the mass transfer process within the mixing channel of the transformation cartridge of the present invention enables mass transfer even if effective mixing between the two fluids does not occur, by allowing cells within the cell solution to move effectively toward the substance solution and come into contact. In other words, in the case of fluid flow in the laminar region, since there is no fluid mixing effect caused by inertial forces occurring in the turbulent region, two or more fluid flows introduced separately cannot be effectively mixed within a short period of time. However, even under laminar flow conditions where the cell solution and the substance solution cannot be directly mixed, as in the transformation cartridge of the present invention, effective intracellular mass transfer and transformation can be achieved by setting the cells within the cell solution to move directly toward the substance solution and come into immediate contact with the substance solution. Furthermore, through this experimental example, it can be confirmed that almost all cells can be transformed at a cell fluid flow thickness less than the cell diameter, and that transformation occurs for some cells under cell solution flow conditions thicker than the cell thickness, and that the proportion of transformed cells decreases as the thickness increases.

[0110] Experimental Example 3

[0111] Furthermore, it was confirmed whether the substance can be delivered with high efficiency to primary cells collected from the human body when using the transgenic cartridge of the present invention.

[0112] Neon TMIn the case of existing mixed delivery technologies such as Transfection Systems, cell loss and damage are inevitably induced during the process of washing cells to remove the cell culture medium. While cell viability is known to be relatively good for cell lines established for laboratory culture and use due to their ability to proliferate indefinitely, primary cells collected from human blood or tissue generally face a problem where the yield is significantly reduced during the process of producing transformants to be used as cell therapies. This is because, compared to the aforementioned cell lines, proliferation through culture is generally more difficult, and more cell damage is induced during the washing process. However, as confirmed in Example 1 above, when performing transformation using the transformation cartridge of the present invention, substances can be delivered directly to cells on the cell culture medium without pretreatment such as cell washing or the use of a dedicated buffer solution. Therefore, it is expected that substances can be delivered with high efficiency even to primary cells collected from the human body.

[0113] To verify this, using the transformation cartridge of the present invention described above, 5x10 in a cell culture medium 71 mL of a cell solution containing human-collected hPBMC (human Peripheral Blood Monoclonal Cells) (Lonza, CAT# CC-2702) at a concentration of 1 / mL 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, respectively. Then, while applying an electric field of 1800 V between the inlet of the substance channel and the discharge channel, the cell solution and the substance solution passed through a mixing channel (depth 30 μm, width 3 mm, length 24 mm) with the electric field applied at a flow rate ratio of 2:1, thereby obtaining a transformant in which the eGFP mRNA was introduced into the hPBMC, and this was used as the experimental group.

[0114] For the transformants obtained as described above, the expression efficiency (eTX) of eGFP and cell viability were measured in the same manner as in Example 1. As a result, as shown in Fig. 15, compared to the negative control group without material delivery, the transformants of the experimental group, which are hPBMCs into which eGFP mRNA was introduced using the transforming cartridge of the present invention, not only exhibited an equivalent level of cell viability, but also showed high cell viability while simultaneously maintaining GFP expression for more than one week, and the expression efficiency reached a level of up to 98% or higher.

[0115] From the results described above, it can be seen that when performing transformation using the transformation cartridge of the present invention, substances can be delivered directly to cells in a cell culture medium without pretreatment such as cell washing or the use of dedicated buffer solutions. Consequently, effective intracellular substance delivery can be achieved without causing cell damage at a level leading to apoptosis, and due to these advantages, substances can be delivered with high efficiency even to primary cells harvested from the human body. Furthermore, since the transformed human-harvested cells produced in this way can exhibit maximum expression efficiency after about one week, it can be seen that when applied as a cell therapy, the therapeutic effect can last for more than one week. This is Neon TM As an excellent effect that could not be achieved with existing mixed delivery technologies such as transfection systems, this experimental example clearly demonstrates that the transfection technology using the transfection cartridge of the present invention can be very useful in processes utilizing human harvested cells, where the number of usable cells is inevitably extremely limited.

[0116] Experimental Example 4

[0117] Based on the excellent transformation efficiency for various cells, such as cell lines and human cells, confirmed in the above examples, CAR-NK cells were produced by introducing mRNA of a CAR (chimeric antigen receptor) into NK-92 cells (ATCC CAT# CRL-2407) using the transformation cartridge of the present invention.

[0118] NK cells are one of the most difficult cells to transform using the various transformation technologies currently applied in cell manipulation, posing significant challenges to the production of CAR-NK. First, NK cells account for less than 5% of all blood cells, meaning the absolute quantity obtainable from human harvesting is small; furthermore, the transformation efficiency induced by viral infection is known to be less than 1%. Therefore, Neon TM It is known that when producing transformants from human-harvested NK cells using existing mixed delivery technologies such as Transfection Systems, the production yield is significantly poor due to cell loss and cell damage occurring during the cell washing process. However, as confirmed in the above examples, it has been confirmed that when using the transforming cartridge of the present invention, cell loss is minimized and substances can be delivered with high viability and efficiency even in human-derived cells. Therefore, it is expected that CAR-NK therapeutic agents can also be produced with high yield by confirming the expression of CARs with proven anticancer therapeutic effects.

[0119] To verify this, using the transformation cartridge of the present invention described above, 1.0 x 10 7 1 mL of a cell solution containing NK-92 cells (ATCC CAT# CRL-2407) at a concentration of 1 / mL was injected into the cell channel, and a substance solution containing CD19 CAR mRNA (RiboPro, CAT# RB-073-3) at a concentration of 500 μg / mL was injected into the substance channel, respectively. Then, while applying an electric field of 1200 V between the inlet of the substance channel and the product discharge channel, the cell solution and the substance solution passed through a mixing channel (depth 30 μm, width 3 mm, length 24 mm) with the electric field applied at a flow rate ratio of 2:1, thereby obtaining a transformant in which the CD19 CAR mRNA was introduced into the NK-92 cells, which was designated as NKL-CD19-500.

[0120] For the transformants obtained as described above, the expression efficiency (eTX) of the CD19 CAR was measured using an anti-FMC63-PE antibody, and the cytotoxicity of Nalm6 cancer cells was measured by mixing and culturing the transformants with the CD19 overexpressing cancer cell line Nalm6 (ATCC CAT# CRL-3273) at a ratio of 2:1 (CAR-NK cells : Nalm6 cells).

[0121] As a result, as shown in Fig. 16, it was confirmed that CD19 CAR was expressed in more than 90% of the cells (Fig. 16 (A)), and more than 80% of Nalm6 cancer cells were killed within one day (Fig. 16 (B)).

[0122] From the results above, when performing transformation using the transformation cartridge of the present invention, since the substance can be directly delivered to cells on the cell culture medium without pretreatment such as a cell washing process or the use of a dedicated buffer solution, Neon TM It can be seen that the efficient production of transformants using human-harvested NK cells, which was virtually impossible with existing mixed delivery technologies such as transfection systems, is possible. This is Neon TM Through this experimental example, we have confirmed the great potential for the development and production of CAR-NK anticancer immunotherapy drugs, which are expected to bring groundbreaking innovation to the treatment of solid tumors, as excellent effects that could not be achieved with existing mixed delivery technologies such as Transfection Systems. With high production performance that has not been reported in existing NK cell transformation technologies, it is clear that the transformation technology using the transformation cartridge of the present invention can be very useful in the field of human-harvested NK cell-based anticancer immunotherapy drugs.

[0123] In the foregoing, although all components constituting the embodiments of the present invention have been described as being combined or operating as a single unit, the present invention is not necessarily limited to such embodiments. That is, within the scope of the purpose of the present invention, all such components may be selectively combined and operated in one or more ways. Furthermore, terms such as "include," "constitute," or "have" described above, unless specifically stated otherwise, mean that the relevant component may be inherent; thus, they should be interpreted as allowing for the inclusion of additional components rather than excluding other components. All terms, including technical or scientific terms, have the same meaning as generally understood by those skilled in the art to which the present invention pertains, unless otherwise defined. Terms commonly used, such as those defined in advance, should be interpreted in accordance with their meaning in the context of the relevant technology and, unless explicitly defined in the present invention, should not be interpreted in an ideal or overly formal sense.

[0124] The foregoing description is merely an illustrative explanation of the technical concept of the present invention, and those skilled in the art to which the present invention pertains will be able to make various modifications and variations within the scope of the essential characteristics of the present invention. Accordingly, the embodiments disclosed in the present invention are intended to explain, not limit, the technical concept of the present invention, and the scope of the technical concept of the present invention is not limited by these embodiments. The scope of protection of the present invention shall be interpreted by the claims below, and all technical concepts within an equivalent scope shall be interpreted as being included within the scope of rights of the present invention. Explanation of the symbols

[0125] 1, 1d : Transformation cartridge 2, 2d : Transformation device 3 : Syringe 10, 10b, 10c, 10d : Main body 11, 11b, 11c: Main body 12, 12b, 12c: Cellular Euro 13, 13b, 13c: Material flow path 14, 14b, 14c : Result Euro 20, 20d : Electric field forming part 21, 21d : Electric field forming body 22, 22d: Cell electrode 23, 23d: Material electrode 24, 24d: Result electrode 25d: Cell terminal 26, 26d : Material terminal 27, 27d : Result terminal 30, 30d: Sealing sheet 40, 40d: O-ring part 42 : Cell O-ring 43 : Material O-ring 44 : Result O-ring 50, 50d: Guide section 51, 51d: Guide body 52, 52d: Cell guide 53, 53d: Material Guide 54, 54d: Result Guide 55d: Power On Guide 61: Upper mount 62, 62d: Main body mount 63 : Lower mount 100, 100d : Transformation system 120: Entrance to the cell euro 121 : Cell inflow channel 130: Entrance to the Material Euro 131 : Material Inflow Channel 132 : Mass transfer channel 140 : Result Euro Exit 141 : Mixed oil 142 : Result discharge channel 201 : Processor 202 : Pump section 203 : Power Supply Section 204 : Input section 541d : Main Output Guide 542d: Auxiliary Output Guide 1301b: First substance flow path 1302b: Secondary substance flow path C0: Cells before transformation C1, C2: Transformed cells M: Transforming substance M1: First transforming substance M2: Second transformation material SC: Cell solution SM: Substance solution SM1: First substance solution SM2: Second substance solution

Claims

Claim 1 A transformation cartridge comprising: a main body portion in which cell channels, material channels, and product channels are formed that meet each other; and an electric field forming portion including an electrode coupled to the main body portion to generate an electric field in the product channels, wherein the cell channels and material channels are formed in a shape that converges into the product channels, and the product channels include a mixing channel extending from a point connected to the cell channels and material channels, and the thickness of the mixing channel is smaller than the thickness of the cell channels. Claim 2 A transformation cartridge in which the cell flow path and the material flow path meet in a state where the cell flow path is located above the material flow path, in the first aspect. Claim 3 A transformation cartridge according to claim 1, wherein the cell flow path comprises a cell inflow path extending downward from the inlet. Claim 4 In claim 1, the transformation cartridge extending in a horizontal direction from a point connected to the cell channel and the material channel. Claim 5 In claim 1, the material flow path is a transformation cartridge having the shape of a downwardly convex arc. Claim 6 In paragraph 5, the material flow path and the resulting flow path have the shape of a continuous arc that is convex downward, in a transformation cartridge. Claim 7 A transformation cartridge according to claim 1, wherein the material flow path comprises a material inlet path extending downward from the inlet and a material transfer path extending horizontally from the bottom of the material inlet path toward the result path. Claim 8 A transformation cartridge according to claim 1, wherein the inlet of the substance flow path, the inlet of the cell flow path, and the outlet of the product flow path are arranged along a reference direction in the order of the inlet of the substance flow path, the inlet of the cell flow path, and the outlet of the product flow path. Claim 9 A transformation cartridge according to claim 1, wherein the material flow path comprises: a first material flow path meeting the cell flow path at one side of the cell flow path; and a second material flow path meeting the cell flow path at the other side of the cell flow path. Claim 10 A transformation cartridge according to claim 9, wherein the first substance channel meets the cell channel at the lower side of the cell channel, and the second substance channel meets the cell channel at the upper side of the cell channel. Claim 11 In claim 1, the electric field forming part is a transformation cartridge configured to form an electric field using a pulse-shaped power source. Claim 12 A transformation cartridge according to claim 1, wherein the result flow path further comprises a result discharge flow path connecting the end of the mixing flow path and the outlet of the result flow path. Claim 13 A transformation cartridge according to claim 1, wherein the thickness of the mixing channel is 6㎛ or more and 400㎛ or less. Claim 14 A transformation device comprising: a pump unit arranged to pump a cell solution and a material solution, respectively, into a cell flow path and a material flow path of a transformation cartridge; a power application unit arranged to be connected to a terminal of the transformation cartridge to apply power to an electrode 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 of the transformation cartridge, a preset flow rate ratio, and a preset exposure time. Claim 15 A transformation system comprising: a main body portion in which cell channels, material channels, and product channels are formed that meet each other; an electric field forming portion including an electrode coupled to the main body portion and disposed at the inlet of the material channel and the outlet of the product channel; a pump portion arranged to pump a cell solution and a material solution to the cell channels and the material channels, respectively; and a processor electrically connected to the pump portion, wherein the cell channels and the material channels are formed in a shape that converges into the product channels, and the product channels include a mixing channel extending from a point connected to the cell channels and the material channels, and the processor controls the pump portion such that the thickness of the solution flowing into the mixing channel through the cell channels is smaller than the diameter of the cell contained in the cell solution. Claim 16 A transformation method comprising the steps of: preparing a cartridge having a cell channel, a material channel, and a mixing channel formed therein that meet each other; injecting a cell solution containing a cell into the cell channel; and injecting a material solution containing a material for transforming the cell into the material channel, wherein the ratio of the flow rate of the injected cell solution to the flow rate of the material solution is a value such that the cell and the material meet in the mixing channel and transformation occurs. Claim 17 A transformation method according to claim 16, wherein the above flow rate ratio is a value such that the thickness of the solution flowing into the mixing channel through the cell channel is less than three times the diameter of the cell. Claim 18 A transformation method according to claim 17, wherein the above flow rate ratio is a value such that the thickness of the solution flowing into the mixing channel through the cell channel is smaller than the diameter of the cell. Claim 19 A transformation method according to claim 17, wherein the above flow rate ratio is a value such that the thickness of the solution flowing into the mixing channel through the cell channel is smaller than the distance the cell sinks downward in the mixing channel.

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