Method for preparing cells for expressing exogenous genes through electrotransfection

Abstract

The invention provides a method for preparing cells for expressing exogenous genes through electrotransfection. The method comprises the following steps: 1) treating the cells by using a pretreatment solution before electrotransfection; and / or 2) introducing a nucleic acid construct containing the coding exogenous gene through the electrically diverted cells by using the electrically diverted liquid; wherein the pretreatment liquid contains divalent ions, and / or the electrotransduction liquid contains monovalent cations. According to the method, the electrotransfection efficiency and the cell survival rate are remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and more specifically, the present invention relates to a method for preparing cells expressing foreign genes by electrotransformation. Background Art

[0002] Cell transfection is one of the commonly used methods in the field of molecular biology to transfer foreign genes into cells. It can be divided into three categories: chemical method, physical method and biologically mediated method. Electroporation (or electroporation), as a representative physical transfection method, first appeared in 1982. It relies on pulsed current to make holes in the cell membrane to introduce nucleic acids into the cell, thereby integrating foreign genes into the host gene and stably expressing them. As a universal and easy-to-use cell transfection method, electroporation has advantages that other methods cannot match, such as simple operation, good repeatability, low cost, low safety risk, and a wide range of applications. Under certain conditions, it can achieve both electroporation efficiency and cell viability, especially for primary cells and cells that are generally considered difficult to transfect. It has a good transfection efficiency.

[0003] The electroporation buffer system is one of the decisive factors affecting electroporation efficiency and cell viability. When using existing electroporation buffers for electroporation, there are still problems such as high cell mortality rate and low positive rate of exogenous gene expression after electroporation.

[0004] Therefore, there is an urgent need in the art to improve electroporation buffers and provide matching electroporation methods to increase electroporation efficiency and cell viability. Summary of the invention

[0005] The object of the present invention is to provide a method for preparing cells expressing exogenous genes by electroporation, comprising the following steps: 1) treating the cells with a pretreatment solution before electroporation; and 2) introducing a nucleic acid construct encoding the exogenous gene into the cells by electroporation using an electroporation solution; wherein the pretreatment solution contains divalent ions, and / or the electroporation solution contains monovalent cations. The method significantly improves electroporation efficiency and cell survival rate.

[0006] In a first aspect of the present invention, a method for preparing cells expressing exogenous genes by electroporation is provided, comprising the following steps: treating the cells with divalent cations before electroporation, and / or electroporating the cells under conditions containing monovalent cations.

[0007] In one or more embodiments, the method comprises the steps of: 1) treating the cells with a pretreatment solution before electroporation; and 2) introducing a nucleic acid construct encoding the exogenous gene into the cells by electroporation; or

[0008] The method comprises the steps of: 1) introducing a nucleic acid construct encoding the exogenous gene into cells by electroporation in an electroporation solution; or

[0009] The method comprises the steps of: 1) treating the cells with a pretreatment solution before electrotransformation; and 2) introducing a nucleic acid construct encoding the exogenous gene into the cells in the electrotransformation solution by electrotransformation;

[0010] Wherein, the pretreatment solution contains divalent cations, and the electrotransfer solution contains monovalent cations.

[0011] In one or more embodiments, the concentration of cells in the treated system is 1×10 5 Up to 1×10 10 / mL, preferably 1×10 5 Up to 1×10 7 / mL, more preferably (5±0.1)×10 6 Pieces / mL.

[0012] In one or more embodiments, the method further comprises the steps of: (4) expanding the culture of the cells after electroporation, and / or (5) culturing the cells in the presence of an anti-CD3 antibody (eg, OKT-3).

[0013] In one or more embodiments, the divalent cation is selected from Ca 2+ Mg 2+ , Mn 2+ Any one, any two or all of; preferably, the concentration of the divalent cation in the pretreatment solution is 0.1-90, 0.2-80, 0.3-70, 0.4-60, 0.5-50, 1-50, 1-25, 1-20, 0.5-20, 0.5-15, 0.5-10, 1-10 or 2-10 mM.

[0014] In one or more embodiments, the monovalent cation is selected from Na + , K + , Li + Any one, any two or all of; preferably, the concentration of the monovalent cation in the electrotransfer solution is 0.2-500, 0.5-500, 0.5-400, 0.5-300, 0.5-200, 0.5-150, 0.5-100, 1-500, 1-250, 1-200, 1-100, 1-50, 2-50 or 5-50 mM.

[0015] In one or more embodiments, the method comprises the steps of: 1) incubating the cells in a pretreatment solution for at least 0.5 hours, such as at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 16, at least 18, at least 20, at least 24, at least 30, or at least 36 hours, such as 2-30 hours, and 2) introducing a nucleic acid construct encoding the exogenous gene into the cells by electrophoresis. In one or more embodiments, the pretreatment solution contains Ca 2+ Mg 2+ , Mn 2+ In one or more embodiments, the pretreatment solution contains any one or more of CaCl2, Ca(NO3)2, MnCl2, MgCl2, and MgSO4.

[0016] In one or more embodiments, the method comprises the steps of: 1) incubating the cells in an electroporation solution for at least 0.1 hour, such as at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 hours, such as 0.1-5 hours, wherein the electroporation solution contains a nucleic acid construct encoding the exogenous gene and a monovalent cation, and 2) introducing the nucleic acid construct into the cells by electroporation in the electroporation solution. In one or more embodiments, the electroporation solution contains Na + , K + , Li + In one or more embodiments, the electroporation solution contains any one or more of NaCl, KCl, LiCl, sodium pyruvate (NaC3H3O3) and sodium citrate (Na3C6H8O7).

[0017] In one or more embodiments, the method comprises the steps of: 1) incubating the cells in a pretreatment solution for at least 0.5 hour, such as at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 16, at least 18, at least 20, at least 24, at least 30, or at least 36 hours, such as 2-30 hours, 2) incubating the cells in an electroporation solution for at least 0.1 hour, such as at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 hours, such as 0.1-5 hours, wherein the electroporation solution contains a nucleic acid construct encoding the exogenous gene and a monovalent cation, and 3) introducing the nucleic acid construct into the cells by electroporation in the electroporation solution.

[0018] In one or more embodiments, the pretreatment solution contains Ca 2+ , and the electrotransfer solution contains Na+ Preferably, the pretreatment solution contains CaCl2, and the electro-conversion solution contains NaCl. Preferably, the pretreatment solution contains CaCl2, and the electro-conversion solution contains NaC3H3O3.

[0019] In one or more embodiments, the pretreatment solution contains Ca 2+ , and the electroporation solution contains K + Preferably, the pretreatment solution contains Ca(NO3)2, and the electro-conversion solution contains KCl.

[0020] In one or more embodiments, the pretreatment solution contains Mn 2+ , and the electrotransfer solution contains Na + Preferably, the pretreatment solution contains MnCl2, and the electro-conversion solution contains NaCl.

[0021] In one or more embodiments, the pretreatment solution contains Ca 2+ , and the electrotransfer solution contains Li + Preferably, the pretreatment solution contains CaCl2, and the electro-conversion solution contains LiCl.

[0022] In one or more embodiments, the pretreatment solution contains Mg 2+ , and the electrotransfer solution contains Na + Preferably, the pretreatment solution contains MgCl2, and the electro-conversion solution contains NaCl.

[0023] In one or more embodiments, the pretreatment solution contains Ca 2+ , and the electrotransfer solution contains Na + and K + Preferably, the pretreatment solution contains CaCl2, and the electro-conversion solution contains NaCl and KCl.

[0024] In one or more embodiments, the pretreatment solution contains Ca 2+ and Mg 2+ , and the electrotransfer solution contains Na + Preferably, the pretreatment solution contains CaCl2 and MgSO4, and the electro-conversion solution contains NaCl.

[0025] In one or more embodiments, the pretreatment solution contains Ca 2+ and Mg 2+ , and the electrotransfer solution contains Na + and K + Preferably, the pretreatment solution contains CaCl2 and MgSO4, and the electro-conversion solution contains NaCl and KCl.

[0026] In one or more embodiments, the pretreatment solution further contains a cell culture medium. In one or more embodiments, the cells are animal cells, and the cell culture medium includes AIM-V, X-VIVO, DMEM, RPMI1640. In one or more embodiments, the cell culture medium further contains serum.

[0027] In one or more embodiments, the pretreatment solution further comprises cytokines.

[0028] In one or more embodiments, the cytokines include one or more selected from the following: interleukins, tumor necrosis factor (TNF), interferon (IFN), colony stimulating factor (CSF) and tumor growth factor (TGF). The interleukins include but are not limited to IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-18 and IL-21; the tumor necrosis factor includes but is not limited to TNF-α and TNF-β; the interferon includes but is not limited to IFN-α, IFN-β and IFN-γ; the colony stimulating factor includes but is not limited to M-CSF, G-CSF and GM-CSF; the tumor growth factor includes but is not limited to TGF-α, TGF-β1, TGF-β2 and TGF-β3.

[0029] In one or more embodiments, the concentration of IL-2 in the pretreatment solution is 200-6000 IU / mL, more preferably 200-950, 200-850, 200-750, 200-650, 200-550 or 200-500 IU / mL.

[0030] In one or more embodiments, the electrotransfer solution further comprises an electrotransfer buffer.

[0031] In one or more embodiments, the cell is an immune cell. Preferably, the immune cell is selected from any one or more of T cells, DN T cells, NK cells, NKT cells, CAR-T cells, TCR-T cells, TIL, CTL, LAK cells and CIK cells; more preferably, T cells or TIL cells.

[0032] In one or more embodiments, the treatment in step 1) is incubation; preferably, incubation is performed in an incubator.

[0033] In one or more embodiments, the treatment duration in step 1) is at least 0.5 hour, for example at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 16, at least 18, at least 20, at least 24, at least 30, or at least 36 hours, for example 2-30 hours.

[0034] In one or more embodiments, the temperature of the treatment in step 1) is 25°C-40°C; preferably 30°C-40°C; more preferably 30°C-37°C.

[0035] In one or more embodiments, the nucleic acid construct in step 2) is DNA or RNA; more preferably, it is any one or more of a plasmid, an adenoviral vector, a lentiviral vector, an adeno-associated viral vector and a non-viral vector; preferably, the non-viral vector is a non-viral vector based on a transposon system.

[0036] The present invention also provides a cell expressing a foreign gene prepared by the method described in any embodiment herein.

[0037] In one or more embodiments, the cell is an immune cell. Preferably, the immune cell is selected from any one or more of T cells, DN T cells, NK cells, NKT cells, CAR-T cells, TCR-T cells, TIL, CTL, LAK cells and CIK cells; most preferably, it is a T cell or a TIL cell.

[0038] The present invention also provides the use of cells expressing exogenous genes prepared by the method described in any embodiment of the present invention in the preparation of drugs for treating diseases.

[0039] In one or more embodiments, the disease is a disease suitable for cell therapy.

[0040] In one or more embodiments, the disease comprises a tumor. Preferably, the tumor comprises: prostate cancer, breast cancer, lung cancer, colorectal cancer, gastric cancer, liver cancer, pancreatic cancer, bladder cancer, skin cancer, kidney cancer, melanoma, cervical cancer, ovarian cancer, head and neck tumors.

[0041] The present invention also provides an electrotransfer solution combination, which includes one or both of a pretreatment solution for treating cells to be electrotransferred before electrotransfer and an electrotransfer solution used for electrotransfer during electrotransfer; wherein the pretreatment solution includes divalent cations, and / or the electrotransfer solution includes monovalent cations.

[0042] In one or more embodiments, the pretreatment solution and the electroporation solution are as described in any of the embodiments herein.

[0043] The present invention also provides an electroporation kit for improving transduction efficiency, survival rate of electroporated cells, number of live cells after electroporation, ratio of electroporation-positive cells, number of electroporation-positive cells and survival of electroporated cells, wherein the kit comprises one or both of a pretreatment solution for treating cells to be electroporated before electroporation and an electroporation solution for electroporation; wherein the pretreatment solution comprises divalent cations, and the electroporation solution comprises monovalent cations.

[0044] In one or more embodiments, the kit further comprises any one or more of a cell culture medium, a cytokine, and an electroporation buffer.

[0045] The present invention also provides a pretreatment solution and / or electrotransfer solution as described in any embodiment of the present invention, an electrotransfer solution combination as described in any embodiment of the present invention, or an electrotransfer kit, for use in improving the transduction efficiency of electrotransfer, the survival rate of electrotransfected cells, the number of live cells after electrotransfer, the proportion of electrotransfer-positive cells, the number of electrotransfer-positive cells and / or the survival of electrotransfected cells.

[0046] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 , Comparison of the number of live cells, cell survival rate, positive cell ratio, and positive cell number of TILs electroporated with EGFP expression vector in Group 1 and Group 2 in Example 3.

[0048] Figure 2 , Comparison of the number of live cells, cell survival rate, positive cell ratio, and positive cell number of TILs electroporated with EGFP expression vector in Group 1 and Group 2 in Example 4.

[0049] Figure 3 , Comparison of the number of live cells, cell survival rate, positive cell ratio, and positive cell number of TILs electroporated with EGFP expression vector in Group 1 and Group 2 in Example 5.

[0050] Figure 4 , Comparison of the number of live cells, cell survival rate, positive cell ratio, and positive cell number of TILs electroporated with the EGFP expression vector in Group 1 and Group 2 in Example 6.

[0051] Figure 5 , Comparison of the number of live cells, cell survival rate, positive cell ratio, and positive cell number of TILs electroporated with EGFP expression vector in Group 1 and Group 2 in Example 7.

[0052] Figure 6 , Comparison of the number of live cells, cell survival rate, positive cell ratio, and positive cell number of TILs electroporated with the EGFP expression vector in Group 1 and Group 2 in Example 8.

[0053] Figure 7 , Comparison of the number of live cells, cell survival rate, positive cell ratio, and positive cell number of TILs electroporated with the EGFP expression vector in Group 1 and Group 2 in Example 9.

[0054] Figure 8, Comparison of the number of live cells, cell survival rate, positive cell ratio, and positive cell number of TILs electroporated with the EGFP expression vector in Group 1 and Group 2 in Example 10.

[0055] Fig. 9 , Comparison of the number of live cells, cell survival rate, positive cell ratio, and positive cell number of TILs electroporated with the EGFP expression vector in Group 1 and Group 2 in Example 11.

[0056] Fig.10 , Comparison of the number of live cells, cell survival rate, positive cell ratio, and positive cell number of TILs electroporated with EGFP expression vector in Group 1 and Group 2 in Example 15.

[0057] Fig.11 , Comparison of the number of live cells, cell survival rate, positive cell ratio and positive cell number of TILs electroporated with EGFP expression vector in Group 1 and Group 2 in Comparative Example 1.

[0058] Fig.12 , Comparative Example 2, comparison of the number of live cells, cell survival rate, positive cell ratio, and positive cell number of TILs electroporated with EGFP expression vector in Group 1 and Group 2.

[0059] Fig.13 , Comparative Example 3, comparison of the number of live cells, cell survival rate, positive cell ratio, and positive cell number of TILs electroporated with EGFP expression vector in Group 1 and Group 2.

[0060] Fig.14 , Comparative Example 4, comparison of the number of live cells, cell survival rate, positive cell ratio, and positive cell number of TILs electroporated with EGFP expression vector in Group 1 and Group 2. DETAILED DESCRIPTION

[0061] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a preferred technical solution.

[0062] The present invention uses a pretreatment solution to treat the electrotransfected cells before introducing exogenous genes into cells (especially immune cells) using the electrotransfer method, and / or uses an electrotransfer solution to treat the electrotransfected cells when introducing exogenous genes into the cells, which can significantly improve the survival rate and the number of live cells after electrotransfer, and at the same time increase the proportion and number of cells that positively express the exogenous genes.

[0063] the term

[0064] As used herein, "pretreatment solution" refers to a buffer used before electrotransfer. As used herein, "electrotransfer solution" refers to a buffer used during electrotransfer. The "before electrotransfer" refers to the pretreatment of cells to be electrotransferred before the introduction of exogenous nucleic acids into cells, for example, before the step of electrotransfer in an electrotransfer device. "Before electrotransfer" includes the time or step immediately before electrotransfer, and also includes a longer time or multiple steps before electrotransfer. The "during electrotransfer" refers to the time when exogenous nucleic acids are introduced into cells by electrotransfer, for example, when electrotransfer is performed in an electrotransfer device.

[0065] As used herein, the "cell" refers to a cell that can be transfected using an electroporation operation, including an animal cell, a plant cell, or a bacterial cell. The cell includes an immune cell, i.e., a cell that participates in an immune response or is associated with an immune response, including lymphocytes, dendritic cells, monocytes / macrophages, granulocytes, mast cells, etc. In one or more embodiments, the cell includes any one or more of T cells, DN T cells, NK cells, NKT cells, CAR-T cells, TCR-T cells, TIL, CTL, LAK cells, and CIK cells, preferably T cells or TIL cells. In one or more embodiments, the T cell is an αβT cell or a γδT cell, such as a CD3+CD8+T cell or a CD3+CD4+T cell. In one or more embodiments, the TIL cell is a CD3+TIL cell, a CD28+TIL cell, or a CD137+TIL cell.

[0066] As used herein, the term "expression" includes overexpression.

[0067] As used herein, the term "above" or "below" refers to including the number itself.

[0068] As used herein, the "exogenous gene" refers to a non-endogenous gene, that is, a gene originating from outside the organism of interest or study.

[0069] Electroporation fluid combination and its application

[0070] After in-depth research, the inventors provide an electrotransfer fluid combination or an electrotransfer fluid composition, which includes a pretreatment fluid for treating cells to be electrotransferred before electrotransfer and an electrotransfer fluid for electrotransfer during electrotransfer; wherein the pretreatment fluid includes divalent cations, and / or the electrotransfer fluid includes monovalent cations.

[0071] In one or more embodiments, the divalent cation is selected from Ca 2+ Mg 2+ , Mn 2+ Any, two or all of the following.

[0072] In one or more embodiments, the total concentration of divalent cations in the pretreatment solution is 0.1-100 mM, such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 mM, or a range between any two of the above values. Preferably, the total concentration of divalent cations in the pretreatment solution is 0.1-90, 0.2-80, 0.3-70, 0.4-60, 0.5-50, 1-50, 1-25, 1-20, 0.5-20, 0.5-15, 0.5-10, 1-10 or 2-10 mM.

[0073] In one or more embodiments, the monovalent cation is selected from Na + , K + , Li + Any, two or all of the following.

[0074] In one or more embodiments, the total concentration of monovalent cations in the electroporation solution is 0.1-500 mM, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 300, 400, 500 mM, or a range between any two of the above values. Preferably, the total concentration of divalent cations in the pretreatment solution is 0.2-500, 0.5-500, 0.5-400, 0.5-300, 0.5-200, 0.5-150, 0.5-100, 1-500, 1-250, 1-200, 1-100, 1-50, 2-50 or 5-50 mM.

[0075] The pretreatment solution and the electro-conversion solution also contain anions. The type of anions is not limited, including one or more organic and / or inorganic anions. The valence state of the anions is not limited, and monovalent, divalent or more valent anions are all suitable for the present invention. Exemplary anions include but are not limited to Cl - Br - 、NO3 - 、H2PO4 - PO4 3- 、SO3 2- 、SO4 2- 、C6H8O7 3- 、C3H3O3 -In one or more embodiments, the pretreatment solution contains any one or more of CaCl2, Ca(NO3)2, MnCl2, MgCl2, MgSO4. In one or more embodiments, the electro-conversion solution contains any one or more of NaCl, KCl, LiCl, Na3C6H8O7, and NaC3H3O3.

[0076] The pretreatment solution also includes cytokines, including one or more selected from the following: interleukins, tumor necrosis factor (TNF), interferon (IFN), colony stimulating factor (CSF) and tumor growth factor (TGF). The interleukins include but are not limited to IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-18 and IL-21; the tumor necrosis factor includes but is not limited to TNF-α and TNF-β; the interferon includes but is not limited to IFN-α, IFN-β and IFN-γ; the colony stimulating factor includes but is not limited to M-CSF, G-CSF and GM-CSF; the tumor growth factor includes but is not limited to TGF-α, TGF-β1, TGF-β2 and TGF-β3.

[0077] The pretreatment solution may also contain a cell culture medium or a buffer capable of maintaining the cell state. In one or more embodiments, the cell is an animal cell, and the cell culture medium includes AIM-V, X-VIVO, DMEM, RPMI1640, preferably X-VIVO 15. In one or more embodiments, the cell culture medium also contains serum. The buffer includes an isotonic buffer for cells.

[0078] In one or more embodiments, the concentration of cytokines (e.g., IL-2) in the pretreatment solution is 200-6000 IU / mL, such as 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000 IU / mL, or a range between any two of the above values. Preferably, the concentration of IL-2 in the pretreatment solution is 200-950, 200-850, 200-750, 200-650, 200-550, 200-500 IU / mL.

[0079] In one or more embodiments, the pretreatment solution consists of cytokines, divalent cations and cell culture medium. In one or more embodiments, the pretreatment solution consists of divalent cations and cell culture medium.

[0080] The electrotransfer solution also includes an electrotransfer buffer. The electrotransfer buffer suitable for use herein can be any buffer known in the art that is suitable for electrotransfer therein, such as water or 10% glycerol. Depending on the different cells to be transformed, a person skilled in the art can select a suitable electrotransfer buffer. The electrotransfer buffer can also be commercially available, such as Nucleofector solution and optional electrotransfer supplement solution (e.g., LONZA) of LONZA. In one or more embodiments, the volume ratio of the Nucleofector solution and the electrotransfer supplement solution in the electrotransfer solution is between 1:100-100:1, 1:90-90:1, 1:80-80:1, 1:50-50:1, 1:20-20:1, 1:15-15:1, 1:10-10:1, 1:5-5:1, 1:2-5:1, 1:1-5:1, 2:1-5:1, 3:1-5:1 or 4:1-5:1 (e.g., 82:18).

[0081] In one or more embodiments, the electrotransfer solution does not contain divalent cations. In one or more embodiments, the electrotransfer solution consists of electrotransfer buffer and monovalent cations. In one or more embodiments, the electrotransfer solution consists of Nucleofector solution and electrotransfer supplement solution.

[0082] In one or more embodiments, the pretreatment fluid is free of monovalent cations.

[0083] In one or more embodiments, the electroporation solution is free of divalent cations.

[0084] The present invention also provides the use of the electroporation solution combination described in any embodiment of the present invention in improving the electroporation transduction efficiency, electroporation cell survival rate, number of live cells after electroporation, electroporation positive cell ratio, number of electroporation positive cells and / or cell survival. "Transduction efficiency" can be detected directly or indirectly by conventional techniques, for example, reflected by the positive rate.

[0085] Method for preparing cells expressing foreign genes by electroporation, cells expressing foreign genes and their use

[0086] The present invention also provides a method for preparing cells expressing foreign genes by electroporation, comprising the following steps: 1) treating the cells with a pretreatment solution before electroporation; and / or 2) using the electroporation solution to introduce a nucleic acid construct encoding the foreign gene into the cells by electroporation; wherein the pretreatment solution contains divalent cations and the electroporation solution contains monovalent cations. The pretreatment solution, electroporation solution, divalent cations, and monovalent cations are as described in any embodiment of the present invention.

[0087] In one or more embodiments, the method comprises the steps of: 1) treating the cells with a pretreatment solution before electrotransfection, and 2) introducing a nucleic acid construct encoding the exogenous gene into the cells by electrotransfection; or, the method comprises the steps of: 1) introducing a nucleic acid construct encoding the exogenous gene into the cells by electrotransfection in an electrotransfection solution; or, the method comprises the steps of: 1) treating the cells with a pretreatment solution before electrotransfection, and 2) introducing a nucleic acid construct encoding the exogenous gene into the cells by electrotransfection in an electrotransfection solution.

[0088] In one or more embodiments, the cell is an immune cell; preferably, the immune cell is selected from any one or more of T cells, DN T cells, NK cells, NKT cells, CAR-T cells, TCR-T cells, TIL, CTL, LAK cells and CIK cells; more preferably, it is a T cell or a TIL cell.

[0089] Herein, "treatment" includes any process of contacting a substance, such as the process of contacting a pretreatment solution with cells. Usually, such treatment is mainly achieved by incubation. The conditions of treatment or incubation, such as temperature, humidity, CO2 concentration, etc., are all suitable for the desired purpose of the treatment (such as maintaining cells, cell growth, cell expansion, cell activation, etc.), and these conditions are well known to those skilled in the art. For example, the conditions for incubating cells can be adjusted by those skilled in the art according to the desired cells. In one or more embodiments, the treatment described in step 1) is incubation, such as placing it in a constant temperature incubator for incubation.

[0090] In the treatment system of step 1), the cell concentration is 1×10 5 Up to 1×10 10 / mL, preferably 1×10 5 Up to 1×10 7 / mL, more preferably (5±0.1)×10 6 In step 2), the cell concentration in the electroporation system is 1×10 5 Up to 1×10 10 / mL, preferably 1×10 5 Up to 1×10 7 / mL, more preferably (5±0.1)×10 6 Pieces / mL.

[0091] The treatment in step 1) comprises: incubating the cells in the pretreatment solution for at least 0.5 hour, for example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 16, at least 18, at least 20, at least 24, at least 30, or at least 36 hours, for example, 2-30 hours, 12 hours-72 hours, 18 hours-48 hours, 18 hours-36 hours or 20 hours-30 hours, preferably 24±1 hour.

[0092] In one or more embodiments, the temperature of the treatment in step 1) is 25°C-40°C, such as 30°C-40°C, preferably 30°C-37°C.

[0093] In one or more embodiments, the nucleic acid construct in step 2) is DNA or RNA, such as a vector, mRNA, or siRNA.

[0094] In one or more embodiments, the nucleic acid construct in step 2) is any one or more of a plasmid, a viral vector (adenoviral vector, lentiviral vector, adeno-associated viral vector) and a non-viral vector (e.g., a non-viral vector based on a transposon system).

[0095] Herein, "electroporation", "electrofection" and "electroporation" are used interchangeably and include any process in which the cell membrane has a certain permeability through the action of a transient electric field, so that charged exogenous substances enter the cell membrane. Those skilled in the art are aware of the process, reagents and conditions of electrotransfer. For commercially available electrotransfer equipment and reagents, refer to the product manual. In one or more embodiments, the step of electrotransferring cells in step 2) includes: preparing an electrotransfer solution according to the electrotransfer method used, adding a nucleic acid construct (e.g., a plasmid, mRNA, siRNA) and treated cells, and selecting an appropriate electrotransfer program in an electrotransfer instrument for electrotransfer. Exemplarily, a LONZA Nucleofector is used. TM 2b electroporator and its matching electroporation fluid, the electroporation program is T-020.

[0096] In one or more embodiments, the method for preparing cells expressing exogenous genes described herein comprises the steps of: 1) incubating the cells in a pretreatment solution for at least 0.5 hours, such as at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 16, at least 18, at least 20, at least 24, at least 30, or at least 36 hours, such as 2-30 hours, 2) introducing a nucleic acid construct encoding the exogenous gene into the cells by electrophoresis. The pretreatment solution contains Ca 2+ Mg 2+ , Mn 2+Preferably, the pretreatment solution contains any one or more of CaCl2, Ca(NO3)2, MnCl2, MgCl2, and MgSO4.

[0097] In one or more embodiments, the method for preparing a cell expressing an exogenous gene described herein comprises the steps of: 1) incubating the cell in an electroporation solution for at least 0.1 hour, such as at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 hours, such as 0.1-5 hours, wherein the electroporation solution contains a nucleic acid construct encoding the exogenous gene and monovalent ions, 2) introducing the nucleic acid construct into the cell by electroporation in the electroporation solution. The electroporation solution contains Na + , K + , Li + In one or more embodiments, the electro-conversion solution contains any one or more of NaCl, KCl, LiCl, Na3C6H8O7 and NaC3H3O3.

[0098] In one or more embodiments, the method for preparing a cell expressing an exogenous gene described herein comprises the steps of: 1) incubating the cell in a pretreatment solution for at least 0.5 hours, such as at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 16, at least 18, at least 20, at least 24, at least 30, or at least 36 hours, such as 2-30 hours, 2) incubating the cell in an electroporation solution for at least 0.1 hours, such as at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 hours, such as 0.1-5 hours, wherein the electroporation solution contains a nucleic acid construct encoding the exogenous gene and monovalent ions, and 3) introducing the nucleic acid construct into the cell by electroporation in the electroporation solution. In one or more embodiments, the pretreatment solution contains Ca 2+ , and the electrotransfer solution contains Na + Preferably, the pretreatment solution contains CaCl2, and the electro-conversion solution contains NaCl. In one or more embodiments, the pretreatment solution contains Ca 2+ , and the electroporation solution contains K + Preferably, the pretreatment solution contains Ca(NO3)2, and the electro-conversion solution contains KCl. In one or more embodiments, the pretreatment solution contains Mn 2+ , and the electrotransfer solution contains Na + Preferably, the pretreatment solution contains MnCl2, and the electro-conversion solution contains NaCl. In one or more embodiments, the pretreatment solution contains Ca 2+, and the electrotransfer solution contains Li + Preferably, the pretreatment solution contains CaCl2, and the electro-conversion solution contains LiCl. In one or more embodiments, the pretreatment solution contains Mg 2+ , and the electrotransfer solution contains Na + Preferably, the pretreatment solution contains MgCl2, and the electro-conversion solution contains NaCl. In one or more embodiments, the pretreatment solution contains Ca 2+ , and the electrotransfer solution contains Na + and K + Preferably, the pretreatment solution contains CaCl2, and the electro-conversion solution contains NaCl and KCl. In one or more embodiments, the pretreatment solution contains Ca 2+ and Mg 2+ , and the electrotransfer solution contains Na + Preferably, the pretreatment solution contains CaCl2 and MgSO4, and the electro-conversion solution contains NaCl. In one or more embodiments, the pretreatment solution contains Ca 2+ and Mg 2+ , and the electrotransfer solution contains Na + and K + Preferably, the pretreatment solution contains CaCl2 and MgSO4, and the electro-conversion solution contains NaCl and KCl. In one or more embodiments, the pretreatment solution contains Ca 2+ , and the electrotransfer solution contains Na + Preferably, the pretreatment solution contains CaCl2, and the electro-conversion solution contains NaC3H3O3.

[0099] The method for preparing cells expressing exogenous genes described herein may also include the step of culturing the electrotransfected cells with a cell culture medium after electrotransformation. In some embodiments, the cell culture medium is AIM-V, X-VIVO, DMEM or RPMI1640, preferably AIM-V or X-VIVO. The step of culturing cells is common knowledge of those skilled in the art. For example, the method for culturing TIL cells can refer to WO2022 / 111571. In certain embodiments, the method further includes the step of expanding and culturing the cells after electrotransformation. In certain embodiments, the method further includes the step of culturing cells in the presence of an anti-CD3 antibody (eg, OKT-3) after electrotransformation. Alternatively, the method further includes the steps of (a) expanding and culturing the cells, and (b) culturing the cells in the presence of an anti-CD3 antibody (eg, OKT-3) after electrotransformation. The present invention also provides cells expressing exogenous genes obtained by the method for preparing cells expressing exogenous genes by electrotransformation according to the present invention. Preferably, the cells are immune effector cells, as described in any embodiment of the present invention.

[0100] The present invention also provides the use of the cell expressing the exogenous gene of the present invention in the preparation of a drug for treating a disease. In one or more embodiments, the disease is a disease that can be treated with cell therapy (especially immune cell therapy). In one or more embodiments, the disease includes a tumor, such as but not limited to: prostate cancer, breast cancer, lung cancer, colorectal cancer, gastric cancer, liver cancer, pancreatic cancer, bladder cancer, skin cancer, kidney cancer, melanoma, cervical cancer, ovarian cancer, head and neck tumors.

[0101] Electroporation Kit

[0102] The present invention also provides an electroporation kit for improving transduction efficiency and cell survival, the kit comprising the pretreatment solution and electroporation solution of the present invention, wherein the pretreatment solution contains divalent cations, and / or the electroporation solution contains monovalent cations. Preferably, the pretreatment solution, electroporation solution, divalent cations, and monovalent cations are as described in any embodiment of the present invention. The kit optionally further comprises a cell culture medium (e.g., AIM-V or X-VIVO), a cytokine (e.g., IL-2), and an electroporation buffer.

[0103] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0104] Unless otherwise indicated, percentages and parts are by weight.

[0105] In the unpaired t test, * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001.

[0106] Example

[0107] The EGFP expression vector pKB20-EGFP used in the example was constructed according to the method described in Example 1 on page 21 of the specification of WO2022078310A1, and its entire contents are incorporated herein by reference.

[0108] Example 1: Isolation and culture of TIL cells from human melanoma tissue

[0109] Freshly excised human liver cancer tissue specimens were collected and immediately processed under sterile conditions. The liver cancer tissue of this embodiment was processed and cultured to obtain TIL according to the culture medium described in Implementation 1 of WO2022111571A1 and the tumor sample processing method and TIL culture method described in Example 2. WO2022111571A1 is incorporated herein by reference in its entirety.

[0110] The details are as follows:

[0111] 1) Prepare physiological saline containing a final concentration of 100 U / mL penicillin, 100 μg / mL streptomycin and 50 μg / mL gentamicin for later use;

[0112] 2) In a sterile environment in a Class II biosafety cabinet, the freshly isolated tumor tissue sample from the tumor patient was placed in a 10 cm dish to which 30 mL of the physiological saline prepared in step 1) had been added for washing, and then transferred to a new 10 cm dish to which 30 mL of the physiological saline prepared in step 1) had been added for washing, and the washing was repeated 3 times in total;

[0113] 3) Use a sterile surgical blade to remove fat tissue and necrotic tissue, and cut the tumor tissue into pieces with a diameter of 3×3×3mm 3 2 G-REX100 culture tanks (purchased from Wilsonwolf) were taken, 42 randomly selected tumor tissue blocks were placed in each G-REX100 culture tank, and seed cell culture medium was added to the culture tank, the components of the seed culture medium included: 3000IU / mL IL-2, 20ng / mL IL-7, 20ng / mL IL-15, 500U / mL GM-CSF, 1000IU / mL IFN-γ, 3μg / mL anti-CD137mAb, 3μg / mL anti-CD28 mAb, 3μg / mL anti-PD-1mAb, 10ng / mL TNF-α, 5% v / v human AB serum, 1×PS double antibody and X-VIVO 15 basal culture medium supplemented to the final volume (1L); the excess tumor tissue blocks were frozen with CryoStor10 (purchased from Bio LifeSolutions) cryopreservation solution by liquid nitrogen in a program cooling instrument;

[0114] 4) After adding 1 L of the above seed cell culture medium to the G-REX100 culture tank containing the tumor tissue block in 3), the tumor tissue block was cultured at 37° C. and 5% CO2. Half of the volume of the old seed cell culture medium was removed every 4 days, and half of the volume of fresh seed cell culture medium was added. After the TIL seed cells were harvested by centrifugation on the 12th day, the total number of cells and the viability were counted; some seed cells were collected to prepare for subsequent experiments directly, and the remaining seed cells continued to step 5);

[0115] 5) Prepare expansion medium, the components of which include: 500 IU / mL IL-2, 7 ng / mL IL-7, 30 ng / mL IL-15, 5% v / v human AB serum, 1×PS bispecific antibody and X-VIVO 15 supplemented to a final volume (1 L); the expansion medium is used for seed cell reselection and expansion culture in a G-REX500M culture tank.

[0116] Take some of the seed cells harvested in 4) and resuspend them in expansion medium to 5.0×105 / mL, added to the cell culture vessels pre-treated with anti-CD3 mAb, anti-CD28 mAb and anti-CD137 mAb, activated at 37℃5% CO2 for 2 days, then the activated cells were collected by centrifugation and inoculated into G-REX500M culture tanks with pre-heated expansion medium. The volume of expansion medium in each G-REX500M was 5L. The activated seed cells were cultured at 2.5×10 5 / cm 2 The cells were inoculated at a density of 1.0 × 10 and cultured at 37 °C with 5% CO2. After counting the cells every 4 days, half of the old expansion medium was removed and half of the fresh expansion medium was added. The total number of cells in each G-REX500M tank reached 1.0 × 10 10 After that, the cells were divided into bottles at a ratio of 1:2, and each bottle was supplemented with fresh expansion medium to 5 L before continuing to culture. After a total of 12 days of culture in the expansion medium of the G-REX500M culture tank, the cells were harvested to obtain TIL finished cells.

[0117] The phenotypes of the above-mentioned TIL seed cells and TIL finished cells were detected by flow cytometry, and the results showed that the proportion of CD3+ cells exceeded 95%.

[0118] Example 2. Preparation of human activated T cells

[0119] A six-well plate was coated with a coating solution containing 5 μg / ml anti-CD3 antibody and 5 μg / ml anti-CD28 antibody at room temperature for 2-4 hours. After the coating solution was removed, the plate was washed with physiological saline for 1-3 times, and AIM-V medium containing 2% FBS was added for standby use; human peripheral blood PBMC (purchased from ALLCELLS) was resuscitated in a 37°C water bath, and the PBMC was cultured for 2-4 hours by adhesion. The non-adherent suspension cells were the initial T cells. The suspension cells were collected in a 15 ml centrifuge tube, centrifuged at 1200 rpm for 3 minutes, the supernatant was discarded, physiological saline was added, centrifuged at 1200 rpm for 3 minutes, the physiological saline was discarded, and this step was repeated; the washed initial T cells were then transferred to the antibody-coated wells containing the standby culture medium, and cultured at 37°C, 5% CO2 for 3-4 days before subsequent experiments.

[0120] Example 3: CaCl2 and NaCl are used for electroporation to prepare TIL cells expressing EGFP

[0121] The TIL finished cells obtained in Example 1 were used to prepare TIL overexpressing EGFP according to the following steps:

[0122] 1) The TIL finished cells prepared in Example 1 were divided into two groups (Group 1 and Group 2). The cells in Group 1 were resuspended to 5×10 6 / mL, IL-2 was added to a final concentration of 500 IU / mL and CaCl2 to a final concentration of 5 mM, and the cells were placed in a 30°C 5% v / v CO2 incubator for 24 h for pretreatment. After 24 h, the cells were centrifuged at 800 g for 10 min, the supernatant was discarded, the cell pellet was resuspended and washed with PBS, the centrifugation was repeated and the supernatant was discarded. The cell pellet was resuspended with PBS and divided into 3 equal portions, each containing 5.0 × 10 6 The cells in group 2 were not treated as above, but were directly centrifuged at 800 g for 5 min, the supernatant was discarded, the cell pellet was resuspended in PBS, washed, and resuspended in PBS again and divided into 3 portions, each containing 5.0×10 6 Live cells are available for backup;

[0123] 2) Add the expansion medium containing 500 IU / mL IL-2, 7 ng / mL IL-7, 30 ng / mL IL-15, 5% v / v human AB serum, 1× PS dual antibody and X-VIVO 15 supplemented to the final volume described in Example 1 into a 6-well plate, 3 mL per well, and then transfer to a cell culture incubator at 37°C and 5% CO2 for preheating for 1 hour;

[0124] 3) Take each portion of cells from group 1 and group 2 in 1), centrifuge at 800 g for 10 min, resuspend and wash the precipitated cells with PBS, centrifuge again at 800 g for 10 min, and discard the supernatant;

[0125] 4) Prepare the electrotransfer solution ratio for each well according to Table 1:

[0126] Table 1

[0127] <![CDATA[100μL Nucleocuvette TM Strip(μL)]]> <![CDATA[Nucleofector TM Volume of solution]]> 82 Electroporation Supplement Solution 18

[0128] 5) For each cell in group 1, add 3 μL of plasmid pKB20-EGFP at a concentration of 1 μg / μL and 1 μL of 5M NaCl to the prepared electrotransfer solution (to a final concentration of 50 mM NaCl in the electrotransfer solution), and then let stand at room temperature for less than 30 min; for each cell in group 2, add 3 μL of plasmid pKB20-EGFP at a concentration of 1 μg / μL to the prepared electrotransfer solution, and then let stand at room temperature for less than 30 min;

[0129] 6) Use the plasmid-containing electroporation solution prepared in 5) for group 1 and group 2 cell samples to resuspend the cell pellets of group 1 and group 2 obtained in 3) respectively, 100 μL per tube, carefully pipette the cell resuspension into the LONZA 100 μL electroporation cup, and place the electroporation cup into the LONZA Nucleofector TM 2b In the electroporation tank, select T-020 as the electroporation program and start the electroporation program;

[0130] 7) After the electroporation is completed, carefully remove the electroporation cup, aspirate the cell suspension and transfer it to the EP tube, add 200 μL of the preheated expansion medium described in Example 1 to each tube, and then transfer it to the wells of the 6-well plate containing the preheated expansion medium in 2), and culture at 37°C, 5% v / v CO2 for 24 hours; during this period, pre-coat a new 6-well plate with antibody OKT-3, add 1 mL of 5 μg / μL OKT-3 antibody coating solution to each well, incubate at 37°C for 4 hours, discard the antibody coating solution, and wash 3 times with PBS for later use;

[0131] 8) After culturing at 37°C and 5% v / v CO2 for 24 h, the expansion culture medium suspension containing each cell was transferred to the wells of a six-well plate coated with OKT-3 and cultured for another 24 h under the stimulation of the antibody OKT-3;

[0132] 9) Each cell was centrifuged at 800 g for 10 min, the supernatant was discarded, the cell pellet was resuspended with the expansion medium described in Example 1 and then transferred to the wells of a new six-well plate for continued culture. Half the volume of the old medium was removed every two days, and half the volume of fresh expansion medium was added. After continued culture for 10 days, cell counting and flow cytometry were performed on each cell of Group 1 and Group 2, and the average values ​​of the survival rate, number of live cells, and proportion and number of EGFP-positive cells of three samples of each of Group 1 and Group 2 were calculated.

[0133] The results are as follows Figure 1 Compared with the untreated group, the number of live cells, the proportion of EGFP-positive cells and the number of EGFP-positive cells in the CaCl2-treated + NaCl-electroporated group after electroporation were significantly higher.

[0134] Example 4: CaCl2 is used for electroporation to prepare TIL expressing EGFP

[0135] The TIL seed cells obtained in Example 1 were used to prepare TIL overexpressing EGFP according to the following steps:

[0136] 1) The TIL seed cells prepared in Example 2 were divided into two groups (Group 1 and Group 2). The cells in Group 1 were resuspended to 4×10 6 / mL, IL-2 was added to a final concentration of 500 IU / mL and CaCl2 to a final concentration of 5 mM, and the cells were placed in a 30°C 5% v / v CO2 incubator for 16 h for pretreatment. After 16 h, the cells were centrifuged at 800 g for 10 min, the supernatant was discarded, the cell pellet was resuspended and washed with PBS, the centrifugation was repeated and the supernatant was discarded. The cell pellet was resuspended with PBS and divided into 3 equal portions, each containing 5.0 × 10 6The cells in group 2 were not treated as above, but were directly centrifuged at 800 g for 5 min, the supernatant was discarded, the cell pellet was resuspended in PBS, washed, and resuspended in PBS again and divided into 3 portions, each containing 5.0×10 6 Live cells are available for backup;

[0137] 2) Add the expansion medium containing 500 IU / mL IL-2, 7 ng / mL IL-7, 30 ng / mL IL-15, 5% v / v human AB serum, 1× PS dual antibody and X-VIVO 15 supplemented to the final volume described in Example 1 into a 6-well plate, 3 mL per well, and then transfer to a cell culture incubator at 37°C and 5% CO2 for preheating for 1 hour;

[0138] 3) Take each portion of cells from group 1 and group 2 in 1), centrifuge at 800 g for 10 min, resuspend and wash the precipitated cells with PBS, centrifuge again at 800 g for 10 min, and discard the supernatant;

[0139] 4) Prepare the electroporation solution ratio for each well according to Table 1;

[0140] 5) Add 3 μL of plasmid pKB20-EGFP at a concentration of 1 μg / μL to the prepared electroporation solution, and then let it stand at room temperature for less than 30 minutes;

[0141] 6) Use the plasmid-containing electroporation solution prepared in 5) to resuspend the cell pellets of group 1 and group 2 obtained in 3) respectively, 100 μL per tube, carefully pipette the cell resuspension into the LONZA 100 μL electroporation cup, and place the electroporation cup into the LONZA Nucleofector TM 2b In the electroporation tank, select T-020 as the electroporation program and start the electroporation program;

[0142] 7) After the electroporation is completed, carefully remove the electroporation cup, aspirate the cell suspension and transfer it to the EP tube, add 200 μL of the preheated expansion medium described in Example 1 to each tube, and then transfer it to the wells of the 6-well plate containing the preheated expansion medium in 2), and culture at 37°C, 5% v / v CO2 for 24 hours; during this period, pre-coat a new 6-well plate with antibody OKT-3, add 1 mL of 5 μg / μL OKT-3 antibody coating solution to each well, incubate at 37°C for 4 hours, discard the antibody coating solution, and wash 3 times with PBS for later use;

[0143] 8) After culturing at 37°C and 5% v / v CO2 for 24 h, the expansion culture medium suspension containing each cell was transferred to the wells of a six-well plate coated with OKT-3 and cultured for another 24 h under the stimulation of the antibody OKT-3;

[0144] 9) Each cell was centrifuged at 800 g for 10 min, the supernatant was discarded, the cell pellet was resuspended with the expansion medium described in Example 1 and then transferred to the wells of a new six-well plate for continued culture. Half the volume of the old medium was removed every two days, and half the volume of fresh expansion medium was added. After continued culture for 10 days, cell counting and flow cytometry were performed on each cell of Group 1 and Group 2, and the average values ​​of the survival rate, number of live cells, and proportion and number of EGFP-positive cells of three samples of each of Group 1 and Group 2 were calculated.

[0145] The results are as follows Figure 2 As shown. Compared with the untreated group, the total number of living cells, the proportion of EGFP-positive cells and the total number of positive cells in the CaCl2-treated group were significantly higher. The cell survival rates of the untreated group and the CaCl2-treated group were both around 90%, which was slightly higher in the CaCl2-treated group than in the untreated group, and there was a significant difference.

[0146] Example 5: MgSO4 is used for electroporation to prepare T cells expressing EGFP

[0147] The activated T cells obtained in Example 1 were used to prepare T cells overexpressing EGFP according to the following steps:

[0148] 1) The human activated T cells prepared in Example 2 were divided into two groups (Group 1 and Group 2). The cells in Group 1 were resuspended to 1×10 6 / mL, IL-2 was added to a final concentration of 500 IU / mL and MgSO4 to a final concentration of 2.5 mM, and the cells were placed in a 30°C 5% v / v CO2 incubator for 8 h for pretreatment. After 8 h, the cells were centrifuged at 800 g for 10 min, the supernatant was discarded, the cell pellet was resuspended and washed with PBS, the centrifugation was repeated and the supernatant was discarded. The cell pellet was resuspended with PBS and divided into 3 equal portions, each containing 5.0 × 10 6 The living cells of group 2 were resuspended in 1 mL PBS and prepared for the next experiment. The cells of group 2 were not treated as above, but were directly centrifuged at 800 g for 5 min, the supernatant was discarded, the cell pellet was resuspended in PBS, washed, and resuspended in PBS again and divided into 3 portions, each containing 5.0×10 6 Live cells are available for backup;

[0149] 2) Add the expansion medium containing 500 IU / mL IL-2, 7 ng / mL IL-7, 30 ng / mL IL-15, 5% v / v human AB serum, 1× PS dual antibody and X-VIVO 15 supplemented to the final volume described in Example 1 into a 6-well plate, 3 mL per well, and then transfer to a cell culture incubator at 37°C and 5% CO2 for preheating for 1 hour;

[0150] 3) Take each portion of cells from group 1 and group 2 in 1), centrifuge at 800 g for 10 min, resuspend and wash the precipitated cells with PBS, centrifuge again at 800 g for 10 min, and discard the supernatant;

[0151] 4) Prepare the electrotransfer solution ratio for each well according to Table 1:

[0152] 5) Add 3 μL of plasmid pKB20-EGFP at a concentration of 1 μg / μL to the prepared electroporation solution, and then let it stand at room temperature for less than 30 minutes;

[0153] 6) Use the plasmid-containing electroporation solution prepared in 5) to resuspend the cell pellets of group 1 and group 2 obtained in 3) respectively, 100 μL per tube, carefully pipette the cell resuspension into the LONZA 100 μL electroporation cup, and place the electroporation cup into the LONZA Nucleofector TM 2b In the electroporation tank, select T-020 as the electroporation program and start the electroporation program;

[0154] 7) After the electroporation is completed, carefully remove the electroporation cup, aspirate the cell suspension and transfer it to the EP tube, add 200 μL of the preheated expansion medium described in Example 1 to each tube, and then transfer it to the wells of the 6-well plate containing the preheated expansion medium in 2), and culture at 37°C, 5% v / v CO2 for 24 hours; during this period, pre-coat a new 6-well plate with antibody OKT-3, add 1 mL of 5 μg / μL OKT-3 antibody coating solution to each well, incubate at 37°C for 4 hours, discard the antibody coating solution, and wash 3 times with PBS for later use;

[0155] 8) After culturing at 37°C and 5% v / v CO2 for 24 h, the expansion culture medium suspension containing each cell was transferred to the wells of a six-well plate coated with OKT-3 and cultured for another 24 h under the stimulation of the antibody OKT-3;

[0156] 9) Each cell was centrifuged at 800 g for 10 min, the supernatant was discarded, the cell pellet was resuspended with the expansion medium described in Example 1 and then transferred to the wells of a new six-well plate for continued culture. Half the volume of the old medium was removed every two days, and half the volume of fresh expansion medium was added. After continued culture for 10 days, cell counting and flow cytometry were performed on each cell of Group 1 and Group 2, and the average values ​​of the survival rate, number of live cells, and proportion and number of EGFP-positive cells of three samples of each of Group 1 and Group 2 were calculated.

[0157] The results are as follows Figure 3 Compared with the untreated group, the number of live cells, the proportion of EGFP-positive cells and the number of EGFP-positive cells in the MgSO4-treated group were significantly higher.

[0158] Example 6: Ca(NO3)2 and KCl are used for electroporation to prepare TIL cells expressing EGFP

[0159] The seed cells obtained in Example 1 were used to prepare TILs overexpressing EGFP according to the following steps:

[0160] 1) The TIL seed cells prepared in Example 1 were divided into two groups (Group 1 and Group 2). The cells in Group 1 were resuspended to 3×10 6 / mL, IL-2 was added to a final concentration of 500 IU / mL and Ca(NO3)2 to a final concentration of 2 mM, and the cells were placed in a 30°C 5% v / v CO2 incubator for 4 h for pretreatment. After 4 h, the cells were centrifuged at 800 g for 10 min, the supernatant was discarded, the cell pellet was resuspended and washed with PBS, the centrifugation was repeated and the supernatant was discarded. The cell pellet was resuspended with PBS and divided into 3 equal portions, each containing 5.0×10 6 The cells in group 2 were not treated as above, but were directly centrifuged at 800 g for 5 min, the supernatant was discarded, the cell pellet was resuspended in PBS, washed, and resuspended in PBS again and divided into 3 portions, each containing 5.0×10 6 Live cells are available for backup;

[0161] 2) Add the expansion medium containing 500 IU / mL IL-2, 7 ng / mL IL-7, 30 ng / mL IL-15, 5% v / v human AB serum, 1× PS dual antibody and X-VIVO 15 supplemented to the final volume described in Example 1 into a 6-well plate, 3 mL per well, and then transfer to a cell culture incubator at 37°C and 5% CO2 for preheating for 1 hour;

[0162] 3) Take each portion of cells from group 1 and group 2 in 1), centrifuge at 800 g for 10 min, resuspend and wash the precipitated cells with PBS, centrifuge again at 800 g for 10 min, and discard the supernatant;

[0163] 4) Prepare the electrotransfer solution ratio for each well according to Table 1:

[0164] 5) For each cell in group 1, add 3 μL of plasmid pKB20-EGFP at a concentration of 1 μg / μL and 0.5 μL of 5M KCl to the prepared electrotransfer solution (to a final concentration of 25 mM KCl in the electrotransfer solution), and then let stand at room temperature for less than 30 min; for each cell in group 2, add 3 μL of plasmid pKB20-EGFP at a concentration of 1 μg / μL to the prepared electrotransfer solution, and then let stand at room temperature for less than 30 min;

[0165] 6) Use the plasmid-containing electroporation solution prepared in 5) for group 1 and group 2 cell samples to resuspend the cell pellets of group 1 and group 2 obtained in 3) respectively, 100 μL per tube, carefully pipette the cell resuspension into the LONZA 100 μL electroporation cup, and place the electroporation cup into the LONZA Nucleofector TM 2b In the electroporation tank, select T-020 as the electroporation program and start the electroporation program;

[0166] 7) After the electroporation is completed, carefully remove the electroporation cup, aspirate the cell suspension and transfer it to the EP tube, add 200 μL of the preheated expansion medium described in Example 1 to each tube, and then transfer it to the wells of the 6-well plate containing the preheated expansion medium in 2), and culture at 37°C, 5% v / v CO2 for 24 hours; during this period, pre-coat a new 6-well plate with antibody OKT-3, add 1 mL of 5 μg / μL OKT-3 antibody coating solution to each well, incubate at 37°C for 4 hours, discard the antibody coating solution, and wash 3 times with PBS for later use;

[0167] 8) After culturing at 37°C and 5% v / v CO2 for 24 hours, the expansion culture medium suspension containing each cell was transferred to the wells of the OKT-3-coated six-well plate and cultured for another 24 hours under the stimulation of the antibody OKT-3;

[0168] 9) Each cell was centrifuged at 800 g for 10 min, the supernatant was discarded, the cell pellet was resuspended with the expansion medium described in Example 1 and then transferred to the wells of a new six-well plate for continued culture. Half the volume of the old medium was removed every two days, and half the volume of fresh expansion medium was added. After continued culture for 10 days, cell counting and flow cytometry were performed on each cell of Group 1 and Group 2, and the average values ​​of the survival rate, number of live cells, and proportion and number of EGFP-positive cells of three samples of each of Group 1 and Group 2 were calculated.

[0169] The results are as follows Figure 4 Compared with the untreated group, the number of live cells, the proportion of EGFP-positive cells and the number of EGFP-positive cells in the Ca(NO3)2-treated + KCl-electroporated group were significantly higher.

[0170] Example 7. Sodium citrate (Na3C6H8O7) is used for electroporation to prepare T cells expressing EGFP. T cells overexpressing EGFP are prepared using the activated T cells obtained in Example 1 according to the following steps:

[0171] 1) The human activated T cells prepared in Example 2 were divided into two groups (Group 1 and Group 2), centrifuged at 800 g for 10 min, the supernatant was discarded, the cell pellet was resuspended and washed with PBS, and the centrifugation was repeated. The cell pellet of each group was resuspended with PBS and then divided into 3 equal portions, each containing 5.0×10 6 Resuspend the living cells in 1 mL PBS and prepare for the next experiment;

[0172] 2) Add the expansion medium containing 500 IU / mL IL-2, 7 ng / mL IL-7, 30 ng / mL IL-15, 5% v / v human AB serum, 1× PS dual antibody and X-VIVO 15 supplemented to the final volume described in Example 1 into a 6-well plate, 3 mL per well, and then transfer to a cell culture incubator at 37°C and 5% CO2 for preheating for 1 hour;

[0173] 3) Take the precipitated cells in 1), resuspend them in PBS, wash them, centrifuge them again at 800 g for 10 min, and discard the supernatant;

[0174] 4) Prepare the electrotransfer solution ratio for each well according to Table 1:

[0175] 5) For each cell in group 1, add 3 μL of plasmid pKB20-EGFP at a concentration of 1 μg / μL and 1 μL of 1M Na3C6H8O7 to the prepared electrotransfer solution (to a final concentration of 10 mM Na3C6H8O7 in the electrotransfer solution), and then let stand at room temperature for less than 30 min; for each cell in group 2, add 3 μL of plasmid pKB20-EGFP at a concentration of 1 μg / μL to the prepared electrotransfer solution, and then let stand at room temperature for less than 30 min;

[0176] 6) Centrifuge the cells of group 1 and group 2 at 800g for 10 min, discard the supernatant, and resuspend the cell pellets of group 1 and group 2 respectively with the plasmid-containing electroporation solution prepared in 5) for the cell samples of group 1 and group 2, 100 μL per tube, carefully pipette the cell resuspension into a LONZA 100 μL electroporation cup, and place the electroporation cup into the LONZA Nucleofector TM 2b In the electroporation tank, select T-020 as the electroporation program and start the electroporation program;

[0177] 7) After the electroporation is completed, carefully remove the electroporation cup, aspirate the cell suspension and transfer it to the EP tube, add 200 μL of the preheated expansion medium described in Example 1 to each tube, and then transfer it to the wells of the 6-well plate containing the preheated expansion medium in 2), and culture at 37°C, 5% v / v CO2 for 24 hours; during this period, pre-coat a new 6-well plate with antibody OKT-3, add 1 mL of 5 μg / μL OKT-3 antibody coating solution to each well, incubate at 37°C for 4 hours, discard the antibody coating solution, and wash 3 times with PBS for later use;

[0178] 8) After culturing at 37°C and 5% v / v CO2 for 24 hours, the expansion culture medium suspension containing each cell was transferred to the wells of the OKT-3-coated six-well plate and cultured for another 24 hours under the stimulation of the antibody OKT-3;

[0179] 9) Each cell was centrifuged at 800 g for 10 min, the supernatant was discarded, the cell pellet was resuspended with the expansion medium described in Example 1 and then transferred to the wells of a new six-well plate for continued culture. Half the volume of the old medium was removed every two days, and half the volume of fresh expansion medium was added. After continued culture for 10 days, cell counting and flow cytometry were performed on each cell of Group 1 and Group 2, and the average values ​​of the survival rate, number of live cells, and proportion and number of EGFP-positive cells of three samples of each of Group 1 and Group 2 were calculated.

[0180] The results are as follows Figure 5 Compared with the untreated group, the number of live cells, the proportion of EGFP-positive cells and the number of EGFP-positive cells in the sodium citrate electroporation group were significantly higher.

[0181] Example 8: NaCl is used for electroporation to prepare TIL cells expressing EGFP

[0182] The TIL finished cells obtained in Example 1 were used to prepare TIL cells overexpressing EGFP according to the following steps:

[0183] 1) The human activated T cells prepared in Example 2 were divided into two groups (Group 1 and Group 2), centrifuged at 800 g for 10 min, the supernatant was discarded, the cell pellet was resuspended and washed with PBS, and the centrifugation was repeated. The cell pellet of each group was resuspended with PBS and then divided into 3 equal portions, each containing 5.0×10 6 Resuspend the living cells in 1 mL PBS and prepare for the next experiment;

[0184] 2) Add the expansion medium containing 500 IU / mL IL-2, 7 ng / mL IL-7, 30 ng / mL IL-15, 5% v / v human AB serum, 1× PS dual antibody and X-VIVO 15 supplemented to the final volume described in Example 1 into a 6-well plate, 3 mL per well, and then transfer to a cell culture incubator at 37°C and 5% CO2 for preheating for 1 hour;

[0185] 3) Take the precipitated cells in 1), resuspend them in PBS, wash them, centrifuge them again at 800 g for 10 min, and discard the supernatant;

[0186] 4) Prepare the electrotransfer solution ratio for each well according to Table 1:

[0187] 5) For each cell in group 1, add 3 μL of plasmid pKB20-EGFP at a concentration of 1 μg / μL and 1 μL of 5M NaCl to the prepared electrotransfer solution (to a final concentration of 50 mM NaCl in the electrotransfer solution), and then let stand at room temperature for less than 30 min; for each cell in group 2, add 3 μL of plasmid pKB20-EGFP at a concentration of 1 μg / μL to the prepared electrotransfer solution, and then let stand at room temperature for less than 30 min;

[0188] 6) Centrifuge the cells of group 1 and group 2 at 800g for 10 min, discard the supernatant, and resuspend the cell pellets of group 1 and group 2 respectively with the plasmid-containing electroporation solution prepared in 5) for the cell samples of group 1 and group 2, 100 μL per tube, carefully pipette the cell resuspension into a LONZA 100 μL electroporation cup, and place the electroporation cup into the LONZA Nucleofector TM 2b In the electroporation tank, select T-020 as the electroporation program and start the electroporation program;

[0189] 7) After the electroporation is completed, carefully remove the electroporation cup, aspirate the cell suspension and transfer it to the EP tube, add 200 μL of the preheated expansion medium described in Example 1 to each tube, and then transfer it to the wells of the 6-well plate containing the preheated expansion medium in 2), and culture at 37°C, 5% v / v CO2 for 24 hours; during this period, pre-coat a new 6-well plate with antibody OKT-3, add 1 mL of 5 μg / μL OKT-3 antibody coating solution to each well, incubate at 37°C for 4 hours, discard the antibody coating solution, and wash 3 times with PBS for later use;

[0190] 8) After culturing at 37°C and 5% v / v CO2 for 24 hours, the expansion culture medium suspension containing each cell was transferred to the wells of the OKT-3-coated six-well plate and cultured for another 24 hours under the stimulation of the antibody OKT-3;

[0191] 9) Each cell was centrifuged at 800 g for 10 min, the supernatant was discarded, the cell pellet was resuspended with the expansion medium described in Example 1 and then transferred to the wells of a new six-well plate for continued culture. Half the volume of the old medium was removed every two days, and half the volume of fresh expansion medium was added. After continued culture for 10 days, cell counting and flow cytometry were performed on each cell of Group 1 and Group 2, and the average values ​​of the survival rate, number of live cells, and proportion and number of EGFP-positive cells of three samples of each of Group 1 and Group 2 were calculated.

[0192] The results are as follows Figure 6 Compared with the untreated group, the number of live cells, the proportion of EGFP-positive cells and the number of EGFP-positive cells in the NaCl electroporation group were significantly higher.

[0193] Example 9: MnCl2 and NaCl are used for electroporation to prepare TIL cells expressing EGFP

[0194] The seed cells obtained in Example 1 were used to prepare TILs overexpressing EGFP according to the following steps:

[0195] 1) The TIL seed cells prepared in Example 1 were divided into two groups (Group 1 and Group 2). The cells in Group 1 were resuspended to 5×10 6 / mL, IL-2 was added to a final concentration of 500 IU / mL and MnCl2 to a final concentration of 3 mM, and the cells were placed in a 30°C 5% v / v CO2 incubator for 12 h for pretreatment. After 12 h, the cells were centrifuged at 800 g for 10 min, the supernatant was discarded, the cell pellet was resuspended and washed with PBS, the centrifugation was repeated and the supernatant was discarded. The cell pellet was resuspended with PBS and divided into 3 equal portions, each containing 5.0 × 10 6 The cells in group 2 were not treated as above, but were directly centrifuged at 800 g for 10 min, the supernatant was discarded, the cell pellet was resuspended in PBS, washed, and resuspended in PBS again and divided into 3 portions, each containing 5 × 10 6 Resuspend the living cells in 1 mL PBS for later use;

[0196] 2) Add the expansion medium containing 500 IU / mL IL-2, 7 ng / mL IL-7, 30 ng / mL IL-15, 5% v / v human AB serum, 1× PS dual antibody and X-VIVO 15 supplemented to the final volume described in Example 1 into a 6-well plate, 3 mL per well, and then transfer to a cell culture incubator at 37°C and 5% CO2 for preheating for 1 hour;

[0197] 3) Take each portion of cells from group 1 and group 2 in 1), centrifuge at 800 g for 10 min, resuspend and wash the precipitated cells with PBS, centrifuge again at 800 g for 10 min, and discard the supernatant;

[0198] 4) Prepare the electrotransfer solution ratio for each well according to Table 1:

[0199] 5) For each cell in group 1, add 3 μL of plasmid pKB20-EGFP at a concentration of 1 μg / μL and 0.5 μL of 5M NaCl to the prepared electrotransfer solution (to a final concentration of 25 mM NaCl in the electrotransfer solution), and then let stand at room temperature for less than 30 min; for each cell in group 2, add 3 μL of plasmid pKB20-EGFP at a concentration of 1 μg / μL to the prepared electrotransfer solution, and then let stand at room temperature for less than 30 min;

[0200] 6) Use the plasmid-containing electroporation solution prepared in 5) for group 1 cell samples and group 2 cell samples to resuspend the cell pellets of group 1 and group 2 obtained in 3), 100 μL per tube, carefully pipette the cell resuspension into the LONZA 100 μL electroporation cup, and place the electroporation cup into the LONZA Nucleofector TM 2b In the electroporation tank, select T-020 as the electroporation program and start the electroporation program;

[0201] 7) After the electroporation is completed, carefully remove the electroporation cup, aspirate the cell suspension and transfer it to the EP tube, add 200 μL of the preheated expansion medium described in Example 1 to each tube, and then transfer it to the wells of the 6-well plate containing the preheated expansion medium in 2), and culture at 37°C, 5% v / v CO2 for 24 hours; during this period, pre-coat a new 6-well plate with antibody OKT-3, add 1 mL of 5 μg / μL OKT-3 antibody coating solution to each well, incubate at 37°C for 4 hours, discard the antibody coating solution, and wash 3 times with PBS for later use;

[0202] 8) After culturing at 37°C and 5% v / v CO2 for 24 hours, the expansion culture medium suspension containing each cell was transferred to the wells of the OKT-3-coated six-well plate and cultured for another 24 hours under the stimulation of the antibody OKT-3;

[0203] 9) Each cell was centrifuged at 800 g for 10 min, the supernatant was discarded, the cell pellet was resuspended with the expansion medium described in Example 1 and then transferred to the wells of a new six-well plate for continued culture. Half the volume of the old medium was removed every two days, and half the volume of fresh expansion medium was added. After continued culture for 10 days, cell counting and flow cytometry were performed on each cell of Group 1 and Group 2, and the average values ​​of the survival rate, number of live cells, and proportion and number of EGFP-positive cells of three samples of each of Group 1 and Group 2 were calculated.

[0204] The results are as follows Figure 7 Compared with the untreated group, the number of live cells, the proportion of EGFP-positive cells and the number of EGFP-positive cells in the MnCl2-treated + NaCl-electroporated group were significantly higher.

[0205] Example 10: CaCl2 and LiCl are used for electroporation to prepare T cells expressing EGFP

[0206] The activated T cells obtained in Example 1 were used to prepare T cells overexpressing EGFP according to the following steps:

[0207] 1) The activated T cells prepared in Example 1 were divided into two groups (Group 1 and Group 2). The cells in Group 1 were resuspended to 5×10 6 / mL, IL-2 was added to a final concentration of 500 IU / mL and CaCl2 to a final concentration of 5 mM, and the cells were placed in a 30°C 5% v / v CO2 incubator for 16 h for pretreatment. After 16 h, the cells were centrifuged at 800 g for 10 min, the supernatant was discarded, the cell pellet was resuspended and washed with PBS, the centrifugation was repeated and the supernatant was discarded. The cell pellet was resuspended with PBS and divided into 3 equal portions, each containing 5 × 10 6The cells in group 2 were not treated as above, but were directly centrifuged at 800 g for 5 min, the supernatant was discarded, the cell pellet was resuspended in PBS, washed, and resuspended in PBS again and divided into 3 portions, each containing 5×10 6 Live cells are available for backup;

[0208] 2) Add the expansion medium containing 500 IU / mL IL-2, 7 ng / mL IL-7, 30 ng / mL IL-15, 5% v / v human AB serum, 1× PS dual antibody and X-VIVO 15 supplemented to the final volume described in Example 1 into a 6-well plate, 3 mL per well, and then transfer to a cell culture incubator at 37°C and 5% CO2 for preheating for 1 hour;

[0209] 3) Take each portion of cells from group 1 and group 2 in 1), centrifuge at 800 g for 10 min, resuspend and wash the precipitated cells with PBS, centrifuge again at 800 g for 10 min, and discard the supernatant;

[0210] 4) Prepare the electrotransfer solution ratio for each well according to Table 1:

[0211] 5) Add 3 μL of plasmid pKB20-EGFP at a concentration of 1 μg / μL and 1 μL of 3M LiCl to the prepared electroporation solution (to a final concentration of 30 mM LiCl in the electroporation solution), and then let stand at room temperature for less than 30 min;

[0212] 6) Use the plasmid-containing electroporation solution prepared in 5) for group 1 and group 2 cell samples to resuspend the cell pellets of group 1 and group 2 obtained in 3) respectively, 100 μL per tube, carefully pipette the cell resuspension into the LONZA 100 μL electroporation cup, and place the electroporation cup into the LONZA Nucleofector TM 2b In the electroporation tank, select T-020 as the electroporation program and start the electroporation program;

[0213] 7) After the electroporation is completed, carefully remove the electroporation cup, aspirate the cell suspension and transfer it to the EP tube, add 200 μL of the preheated expansion medium described in Example 1 to each tube, and then transfer it to the wells of the 6-well plate containing the preheated expansion medium in 2), and culture at 37°C, 5% v / v CO2 for 24 hours; during this period, pre-coat a new 6-well plate with antibody OKT-3, add 1 mL of 5 μg / μL OKT-3 antibody coating solution to each well, incubate at 37°C for 4 hours, discard the antibody coating solution, and wash 3 times with PBS for later use;

[0214] 8) After culturing at 37°C and 5% v / v CO2 for 24 hours, the expansion culture medium suspension containing each cell was transferred to the wells of the OKT-3-coated six-well plate and cultured for another 24 hours under the stimulation of the antibody OKT-3;

[0215] 9) Each cell was centrifuged at 800 g for 10 min, the supernatant was discarded, the cell pellet was resuspended with the expansion medium described in Example 1 and then transferred to the wells of a new six-well plate for continued culture. Half the volume of the old medium was removed every two days, and half the volume of fresh expansion medium was added. After continued culture for 10 days, cell counting and flow cytometry were performed on each cell of Group 1 and Group 2, and the average values ​​of the survival rate, number of live cells, and proportion and number of EGFP-positive cells of three samples of each of Group 1 and Group 2 were calculated.

[0216] The results are as follows Figure 8 Compared with the untreated group, the number of live cells, the proportion of EGFP-positive cells and the number of EGFP-positive cells in the CaCl2-treated + LiCl-electroporated group were significantly higher.

[0217] Example 11, MgCl2 and NaCl are used for electroporation to prepare TIL cells expressing EGFP. The TIL finished cells obtained in Example 1 are used to prepare TIL overexpressing EGFP according to the following steps:

[0218] 1) The TIL finished cells prepared in Example 1 were divided into two groups (Group 1 and Group 2). The cells in Group 1 were resuspended to 5×10 6 / mL, IL-2 was added to a final concentration of 500 IU / mL and MgCl2 to a final concentration of 5 mM, and the cells were placed in a 37°C 5% v / v CO2 incubator for 8 h for pretreatment. After 8 h, the cells were centrifuged at 800 g for 10 min, the supernatant was discarded, the cell pellet was resuspended and washed with PBS, the centrifugation was repeated and the supernatant was discarded. The cell pellet was resuspended with PBS and divided into 3 equal portions, each containing 5 × 10 6 The cells in group 2 were not treated as above, but were directly centrifuged at 800 g for 10 min, the supernatant was discarded, the cell pellet was resuspended in PBS, washed, and resuspended in PBS again and divided into 3 portions, each containing 5 × 10 6 Live cells are available for backup;

[0219] 2) Add the expansion medium containing 500 IU / mL IL-2, 7 ng / mL IL-7, 30 ng / mL IL-15, 5% v / v human AB serum, 1× PS dual antibody and X-VIVO 15 supplemented to the final volume described in Example 1 into a 6-well plate, 3 mL per well, and then transfer to a cell culture incubator at 37°C and 5% CO2 for preheating for 1 hour;

[0220] 3) Take each portion of cells from group 1 and group 2 in 1), centrifuge at 800 g for 10 min, resuspend and wash the precipitated cells with PBS, centrifuge again at 800 g for 10 min, and discard the supernatant;

[0221] 4) Prepare the electrotransfer solution ratio for each well according to Table 1:

[0222] 5) For each cell in group 1, add 3 μL of plasmid pKB20-EGFP at a concentration of 1 μg / μL and 0.5 μL of 5M NaCl to the prepared electrotransfer solution (to a final concentration of 25 mM NaCl in the electrotransfer solution), and then let stand at room temperature for less than 30 min; for each cell in group 2, add 3 μL of plasmid pKB20-EGFP at a concentration of 1 μg / μL to the prepared electrotransfer solution, and then let stand at room temperature for less than 30 min;

[0223] 6) Use the plasmid-containing electroporation solution prepared in 5) for group 1 and group 2 cell samples to resuspend the cell pellets of group 1 and group 2 obtained in 3) respectively, 100 μL per tube, carefully pipette the cell resuspension into the LONZA 100 μL electroporation cup, and place the electroporation cup into the LONZA Nucleofector TM 2b In the electroporation tank, select T-020 as the electroporation program and start the electroporation program;

[0224] 7) After the electroporation is completed, carefully remove the electroporation cup, aspirate the cell suspension and transfer it to the EP tube, add 200 μL of the preheated expansion medium described in Example 1 to each tube, and then transfer it to the wells of the 6-well plate containing the preheated expansion medium in 2), and culture at 37°C, 5% v / v CO2 for 24 hours; during this period, pre-coat a new 6-well plate with antibody OKT-3, add 1 mL of 5 μg / μL OKT-3 antibody coating solution to each well, incubate at 37°C for 4 hours, discard the antibody coating solution, and wash 3 times with PBS for later use;

[0225] 8) After culturing at 37°C and 5% v / v CO2 for 24 hours, the expansion culture medium suspension containing each cell was transferred to the wells of the OKT-3-coated six-well plate and cultured for another 24 hours under the stimulation of the antibody OKT-3;

[0226] 9) Each cell was centrifuged at 800 g for 10 min, the supernatant was discarded, the cell pellet was resuspended with the expansion medium described in Example 1 and then transferred to the wells of a new six-well plate for continued culture. Half the volume of the old medium was removed every two days, and half the volume of fresh expansion medium was added. After continued culture for 10 days, cell counting and flow cytometry were performed on each cell of Group 1 and Group 2, and the average values ​​of the survival rate, number of live cells, and proportion and number of EGFP-positive cells of three samples of each of Group 1 and Group 2 were calculated.

[0227] The results are as follows Fig. 9 Compared with the untreated group, the number of live cells, the proportion of EGFP-positive cells and the number of EGFP-positive cells in the CaCl2-treated + NaCl-electroporated group were significantly higher.

[0228] Example 12: CaCl2, NaCl and KCl are used for electroporation to prepare TIL cells expressing EGFP. The TIL finished cells obtained in Example 1 are used to prepare TIL overexpressing EGFP according to the following steps:

[0229] 1) The TIL finished cells prepared in Example 1 were divided into two groups (Group 1 and Group 2). The cells in Group 1 were resuspended to 5×10 6 / mL, IL-2 was added to a final concentration of 500 IU / mL and CaCl2 to a final concentration of 3 mM, and the cells were placed in a 37°C 5% v / v CO2 incubator for 8 h for pretreatment. After 8 h, the cells were centrifuged at 800 g for 10 min, the supernatant was discarded, the cell pellet was resuspended and washed with PBS, the centrifugation was repeated and the supernatant was discarded. The cell pellet was resuspended with PBS and divided into 3 equal portions, each containing 5 × 10 6 The cells in group 2 were not treated as above, but were directly centrifuged at 800 g for 10 min, the supernatant was discarded, the cell pellet was resuspended in PBS, washed, and resuspended in PBS again and divided into 3 portions, each containing 5 × 10 6 Live cells are available for backup;

[0230] 2) Add the expansion medium containing 500 IU / mL IL-2, 7 ng / mL IL-7, 30 ng / mL IL-15, 5% v / v human AB serum, 1× PS dual antibody and X-VIVO 15 supplemented to the final volume described in Example 1 into a 6-well plate, 3 mL per well, and then transfer to a cell culture incubator at 37°C and 5% CO2 for preheating for 1 hour;

[0231] 3) Take each portion of cells from group 1 and group 2 in 1), centrifuge at 800 g for 10 min, resuspend and wash the precipitated cells with PBS, centrifuge again at 800 g for 10 min, and discard the supernatant;

[0232] 4) Prepare the electrotransfer solution ratio for each well according to Table 1:

[0233] 5) For each cell in group 1, add 3 μL of plasmid pKB20-EGFP at a concentration of 1 μg / μL, 0.5 μL of 5M NaCl (to a final concentration of 25 mM NaCl added to the electrotransfer solution), and 0.5 μL of 5M KCl (to a final concentration of 25 mM KCl added to the electrotransfer solution) to the prepared electrotransfer solution, and then let stand at room temperature for less than 30 min; for each cell in group 2, add 3 μL of plasmid pKB20-EGFP at a concentration of 1 μg / μL to the prepared electrotransfer solution, and then let stand at room temperature for less than 30 min;

[0234] 6) Use the plasmid-containing electroporation solution prepared in 5) for group 1 and group 2 cell samples to resuspend the cell pellets of group 1 and group 2 obtained in 3) respectively, 100 μL per tube, carefully pipette the cell resuspension into the LONZA 100 μL electroporation cup, and place the electroporation cup into the LONZA Nucleofector TM 2b In the electroporation tank, select T-020 as the electroporation program and start the electroporation program;

[0235] 7) After the electroporation is completed, carefully remove the electroporation cup, aspirate the cell suspension and transfer it to the EP tube, add 200 μL of the preheated expansion medium described in Example 1 to each tube, and then transfer it to the wells of the 6-well plate containing the preheated expansion medium in 2), and culture at 37°C, 5% v / v CO2 for 24 hours; during this period, pre-coat a new 6-well plate with antibody OKT-3, add 1 mL of 5 μg / μL OKT-3 antibody coating solution to each well, incubate at 37°C for 4 hours, discard the antibody coating solution, and wash 3 times with PBS for later use;

[0236] 8) After culturing at 37°C and 5% v / v CO2 for 24 hours, the expansion culture medium suspension containing each cell was transferred to the wells of the OKT-3-coated six-well plate and cultured for another 24 hours under the stimulation of the antibody OKT-3;

[0237] 9) Each cell was centrifuged at 800 g for 10 min, the supernatant was discarded, the cell pellet was resuspended with the expansion medium described in Example 1 and then transferred to the wells of a new six-well plate for continued culture. Half the volume of the old medium was removed every two days, and half the volume of fresh expansion medium was added. After continued culture for 10 days, cell counting and flow cytometry were performed on each cell of Group 1 and Group 2, and the average values ​​of the survival rate, number of live cells, and proportion and number of EGFP-positive cells of three samples of each of Group 1 and Group 2 were calculated.

[0238] The results showed that compared with the untreated group, the CaCl2 treatment + NaCl and KCl electroporation groups had significantly higher numbers of live cells, EGFP-positive cell ratios and EGFP-positive cell numbers.

[0239] Example 13: MgSO4, CaCl2 and NaCl are used for electroporation to prepare TIL cells expressing EGFP

[0240] The TIL finished cells obtained in Example 1 were used to prepare TIL overexpressing EGFP according to the following steps:

[0241] 1) The TIL finished cells prepared in Example 1 were divided into two groups (Group 1 and Group 2). The cells in Group 1 were resuspended to 5×10 6 / mL, IL-2 was added to a final concentration of 500 IU / mL, MgSO4 to a final concentration of 2.5 mM, and CaCl2 to a final concentration of 2.5 mM, and the cells were placed in a 37°C 5% v / v CO2 incubator for 8 h for pretreatment. After 8 h, the cells were centrifuged at 800 g for 10 min, the supernatant was discarded, the cell pellet was resuspended and washed with PBS, the centrifugation was repeated and the supernatant was discarded. The cell pellet was resuspended with PBS and divided into 3 equal portions, each containing 5 × 10 6 The cells in group 2 were not treated as above, but were directly centrifuged at 800 g for 10 min, the supernatant was discarded, the cell pellet was resuspended in PBS, washed, and resuspended in PBS again and divided into 3 portions, each containing 5 × 10 6 Live cells are available for backup;

[0242] 2) Add the expansion medium containing 500 IU / mL IL-2, 7 ng / mL IL-7, 30 ng / mL IL-15, 5% v / v human AB serum, 1× PS dual antibody and X-VIVO 15 supplemented to the final volume described in Example 1 into a 6-well plate, 3 mL per well, and then transfer to a cell culture incubator at 37°C and 5% CO2 for preheating for 1 hour;

[0243] 3) Take each portion of cells from group 1 and group 2 in 1), centrifuge at 800 g for 10 min, resuspend and wash the precipitated cells with PBS, centrifuge again at 800 g for 10 min, and discard the supernatant;

[0244] 4) Prepare the electrotransfer solution ratio for each well according to Table 1:

[0245] 5) For each cell in group 1, add 3 μL of plasmid pKB20-EGFP at a concentration of 1 μg / μL and 1.0 μL of 5M NaCl to the prepared electroporation solution (to a final concentration of 50 mM NaCl in the electroporation solution), and then let stand at room temperature for less than 30 min; for each cell in group 2, add 3 μL of plasmid pKB20-EGFP at a concentration of 1 μg / μL to the prepared electroporation solution, and then let stand at room temperature for less than 30 min;

[0246] 6) Use the plasmid-containing electroporation solution prepared in 5) for group 1 and group 2 cell samples to resuspend the cell pellets of group 1 and group 2 obtained in 3) respectively, 100 μL per tube, carefully pipette the cell resuspension into the LONZA 100 μL electroporation cup, and place the electroporation cup into the LONZA Nucleofector TM 2b In the electroporation tank, select T-020 as the electroporation program and start the electroporation program;

[0247] 7) After the electroporation is completed, carefully remove the electroporation cup, aspirate the cell suspension and transfer it to the EP tube, add 200 μL of the preheated expansion medium described in Example 1 to each tube, and then transfer it to the wells of the 6-well plate containing the preheated expansion medium in 2), and culture at 37°C, 5% v / v CO2 for 24 hours; during this period, pre-coat a new 6-well plate with antibody OKT-3, add 1 mL of 5 μg / μL OKT-3 antibody coating solution to each well, incubate at 37°C for 4 hours, discard the antibody coating solution, and wash 3 times with PBS for later use;

[0248] 8) After culturing at 37°C and 5% v / v CO2 for 24 hours, the expansion culture medium suspension containing each cell was transferred to the wells of the OKT-3-coated six-well plate and cultured for another 24 hours under the stimulation of the antibody OKT-3;

[0249] 9) Each cell was centrifuged at 800 g for 10 min, the supernatant was discarded, the cell pellet was resuspended with the expansion medium described in Example 1 and then transferred to the wells of a new six-well plate for continued culture. Half the volume of the old medium was removed every two days, and half the volume of fresh expansion medium was added. After continued culture for 10 days, cell counting and flow cytometry were performed on each cell of Group 1 and Group 2, and the average values ​​of the survival rate, number of live cells, and proportion and number of EGFP-positive cells of three samples of each of Group 1 and Group 2 were calculated.

[0250] The results showed that compared with the untreated group, the MgSO4 and CaCl2 treatment + NaCl electroporation groups had significantly higher numbers of live cells, EGFP-positive cell ratios, and EGFP-positive cell numbers.

[0251] Example 14: MgSO4, CaCl2, NaCl and KCl are used for electroporation to prepare TIL cells expressing EGFP

[0252] The TIL finished cells obtained in Example 1 were used to prepare TIL overexpressing EGFP according to the following steps:

[0253] 1) The TIL finished cells prepared in Example 1 were divided into two groups (Group 1 and Group 2). The cells in Group 1 were resuspended to 5×10 6 / mL, IL-2 was added to a final concentration of 500 IU / mL, MgSO4 to a final concentration of 2.5 mM, and CaCl2 to a final concentration of 2.5 mM, and the cells were placed in a 37°C 5% v / v CO2 incubator for 8 h for pretreatment. After 8 h, the cells were centrifuged at 800 g for 10 min, the supernatant was discarded, the cell pellet was resuspended and washed with PBS, the centrifugation was repeated and the supernatant was discarded. The cell pellet was resuspended with PBS and divided into 3 equal portions, each containing 5 × 10 6The cells in group 2 were not treated as above, but were directly centrifuged at 800 g for 10 min, the supernatant was discarded, the cell pellet was resuspended in PBS, washed, and resuspended in PBS again and divided into 3 portions, each containing 5 × 10 6 Live cells are available for backup;

[0254] 2) Add the expansion medium containing 500 IU / mL IL-2, 7 ng / mL IL-7, 30 ng / mL IL-15, 5% v / v human AB serum, 1× PS dual antibody and X-VIVO 15 supplemented to the final volume described in Example 1 into a 6-well plate, 3 mL per well, and then transfer to a cell culture incubator at 37°C and 5% CO2 for preheating for 1 hour;

[0255] 3) Take each portion of cells from group 1 and group 2 in 1), centrifuge at 800 g for 10 min, resuspend and wash the precipitated cells with PBS, centrifuge again at 800 g for 10 min, and discard the supernatant;

[0256] 4) Prepare the electrotransfer solution ratio for each well according to Table 1:

[0257] 5) For each cell in group 1, add 3 μL of plasmid pKB20-EGFP at a concentration of 1 μg / μL, 0.5 μL of 5M NaCl (to a final concentration of 25 mM NaCl added to the electrotransfer solution), and 0.5 μL of 5M KCl (to a final concentration of 25 mM KCl added to the electrotransfer solution) to the prepared electrotransfer solution, and then let stand at room temperature for less than 30 min; for each cell in group 2, add 3 μL of plasmid pKB20-EGFP at a concentration of 1 μg / μL to the prepared electrotransfer solution, and then let stand at room temperature for less than 30 min;

[0258] 6) Use the plasmid-containing electroporation solution prepared in 5) for group 1 and group 2 cell samples to resuspend the cell pellets of group 1 and group 2 obtained in 3) respectively, 100 μL per tube, carefully pipette the cell resuspension into the LONZA 100 μL electroporation cup, and place the electroporation cup into the LONZA Nucleofector TM 2b In the electroporation tank, select T-020 as the electroporation program and start the electroporation program;

[0259] 7) After the electroporation is completed, carefully remove the electroporation cup, aspirate the cell suspension and transfer it to the EP tube, add 200 μL of the preheated expansion medium described in Example 1 to each tube, and then transfer it to the wells of the 6-well plate containing the preheated expansion medium in 2), and culture at 37°C, 5% v / v CO2 for 24 hours; during this period, pre-coat a new 6-well plate with antibody OKT-3, add 1 mL of 5 μg / μL OKT-3 antibody coating solution to each well, incubate at 37°C for 4 hours, discard the antibody coating solution, and wash 3 times with PBS for later use;

[0260] 8) After culturing at 37°C and 5% v / v CO2 for 24 hours, the expansion culture medium suspension containing each cell was transferred to the wells of the OKT-3-coated six-well plate and cultured for another 24 hours under the stimulation of the antibody OKT-3;

[0261] 9) Each cell was centrifuged at 800 g for 10 min, the supernatant was discarded, the cell pellet was resuspended with the expansion medium described in Example 1 and then transferred to the wells of a new six-well plate for continued culture. Half the volume of the old medium was removed every two days, and half the volume of fresh expansion medium was added. After continued culture for 10 days, cell counting and flow cytometry were performed on each cell of Group 1 and Group 2, and the average values ​​of the survival rate, number of live cells, and proportion and number of EGFP-positive cells of three samples of each of Group 1 and Group 2 were calculated.

[0262] The results showed that compared with the untreated group, the MgSO4 and CaCl2 treatment + NaCl and KCl electroporation groups had significantly higher numbers of viable cells, EGFP-positive cell ratios and EGFP-positive cell numbers.

[0263] Example 15: CaCl2 and sodium pyruvate (NaC3H3O3) are used for electroporation to prepare TIL cells expressing EGFP

[0264] The TIL finished cells obtained in Example 1 were used to prepare TIL overexpressing EGFP according to the following steps:

[0265] 1) The TIL finished cells prepared in Example 1 were divided into two groups (Group 1 and Group 2). The cells in Group 1 were resuspended to 5×10 6 / mL, IL-2 was added to a final concentration of 500 IU / mL and CaCl2 to a final concentration of 5 mM, and the cells were placed in a 30°C 5% v / v CO2 incubator for 24 h for pretreatment. After 24 h, the cells were centrifuged at 800 g for 10 min, the supernatant was discarded, the cell pellet was resuspended and washed with PBS, the centrifugation was repeated and the supernatant was discarded. The cell pellet was resuspended with PBS and divided into 3 equal portions, each containing 5.0 × 10 6 The cells in group 2 were not treated as above, but were directly centrifuged at 800 g for 5 min, the supernatant was discarded, the cell pellet was resuspended in PBS, washed, and resuspended in PBS again and divided into 3 portions, each containing 5.0×10 6 Live cells are available for backup;

[0266] 2) Add the expansion medium containing 500 IU / mL IL-2, 7 ng / mL IL-7, 30 ng / mL IL-15, 5% v / v human AB serum, 1× PS dual antibody and X-VIVO 15 supplemented to the final volume described in Example 1 into a 6-well plate, 3 mL per well, and then transfer to a cell culture incubator at 37°C and 5% CO2 for preheating for 1 hour;

[0267] 3) Take each portion of cells from group 1 and group 2 in 1), centrifuge at 800 g for 10 min, resuspend and wash the precipitated cells with PBS, centrifuge again at 800 g for 10 min, and discard the supernatant;

[0268] 4) Prepare the electrotransfer solution ratio for each well according to Table 1:

[0269] 5) For each cell in group 1, add 3 μL of plasmid pKB20-EGFP at a concentration of 1 μg / μL and 1 μL of 500 mM NaC3H3O3 to the prepared electrotransfer solution (to a final concentration of 5 mM NaC3H3O3 in the electrotransfer solution), and then let stand at room temperature for less than 30 min; for each cell in group 2, add 3 μL of plasmid pKB20-EGFP at a concentration of 1 μg / μL to the prepared electrotransfer solution, and then let stand at room temperature for less than 30 min;

[0270] 6) Use the plasmid-containing electroporation solution prepared in 5) for group 1 and group 2 cell samples to resuspend the cell pellets of group 1 and group 2 obtained in 3) respectively, 100 μL per tube, carefully pipette the cell resuspension into the LONZA 100 μL electroporation cup, and place the electroporation cup into the LONZA Nucleofector TM 2b In the electroporation tank, select T-020 as the electroporation program and start the electroporation program;

[0271] 7) After the electroporation is completed, carefully remove the electroporation cup, aspirate the cell suspension and transfer it to the EP tube, add 200 μL of the preheated expansion medium described in Example 1 to each tube, and then transfer it to the wells of the 6-well plate containing the preheated expansion medium in 2), and culture at 37°C, 5% v / v CO2 for 24 hours; during this period, pre-coat a new 6-well plate with antibody OKT-3, add 1 mL of 5 μg / μL OKT-3 antibody coating solution to each well, incubate at 37°C for 4 hours, discard the antibody coating solution, and wash 3 times with PBS for later use;

[0272] 8) After culturing at 37°C and 5% v / v CO2 for 24 h, the expansion culture medium suspension containing each cell was transferred to the wells of a six-well plate coated with OKT-3 and cultured for another 24 h under the stimulation of the antibody OKT-3;

[0273] 9) Each cell was centrifuged at 800 g for 10 min, the supernatant was discarded, the cell pellet was resuspended with the expansion medium described in Example 1 and then transferred to the wells of a new six-well plate for continued culture. Half the volume of the old medium was removed every two days, and half the volume of fresh expansion medium was added. After continued culture for 10 days, cell counting and flow cytometry were performed on each cell of Group 1 and Group 2, and the average values ​​of the survival rate, number of live cells, and proportion and number of EGFP-positive cells of three samples of each of Group 1 and Group 2 were calculated.

[0274] The results are as follows Fig.10 Compared with the untreated group, the number of live cells, the proportion of EGFP-positive cells and the number of EGFP-positive cells in the CaCl2-treated + NaC3H3O3-electroporated group after electroporation were significantly higher.

[0275] Comparative Example 1

[0276] The TIL finished cells obtained in Example 1 were used to prepare TIL overexpressing EGFP according to the following steps:

[0277] 1) The TIL finished cells prepared in Example 1 were divided into two groups (Group 1 and Group 2). The cells in Group 1 were resuspended to 5×10 6 / mL, IL-2 was added to a final concentration of 500 IU / mL, CaCl2 to a final concentration of 2.5 mM, and NaCl to a final concentration of 50 mM, and the cells were placed in a 30°C 5% v / v CO2 incubator for 24 h for pretreatment. After 24 h, the cells were centrifuged at 800 g for 10 min, the supernatant was discarded, the cell pellet was resuspended and washed with PBS, the centrifugation was repeated and the supernatant was discarded. The cell pellet was resuspended with PBS and divided into 3 equal portions, each containing 5 × 10 6 The cells in group 2 were not treated as above, but were directly centrifuged at 800 g for 10 min, the supernatant was discarded, the cell pellet was resuspended in PBS, washed, and resuspended in PBS again and divided into 3 portions, each containing 5 × 10 6 Live cells are available for backup;

[0278] 2) Add the expansion medium containing 500 IU / mL IL-2, 7 ng / mL IL-7, 30 ng / mL IL-15, 5% v / v human AB serum, 1× PS dual antibody and X-VIVO 15 supplemented to the final volume described in Example 1 into a 6-well plate, 3 mL per well, and then transfer to a cell culture incubator at 37°C and 5% CO2 for preheating for 1 hour;

[0279] 3) Take each portion of cells from group 1 and group 2 in 1), centrifuge at 800 g for 10 min, resuspend and wash the precipitated cells with PBS, centrifuge again at 800 g for 10 min, and discard the supernatant;

[0280] 4) Prepare the electrotransfer solution ratio for each well according to Table 1:

[0281] 5) Add 3 μL of plasmid pKB20-EGFP at a concentration of 1 μg / μL to the prepared electroporation solution, and then let it stand at room temperature for less than 30 minutes;

[0282] 6) Use the plasmid-containing electroporation solution prepared in 5) to resuspend the cell pellets of group 1 and group 2 obtained in 3) respectively, 100 μL per tube, carefully pipette the cell resuspension into the LONZA 100 μL electroporation cup, and place the electroporation cup into the LONZA Nucleofector TM 2b In the electroporation tank, select T-020 as the electroporation program and start the electroporation program;

[0283] 7) After the electroporation is completed, carefully remove the electroporation cup, aspirate the cell suspension and transfer it to the EP tube, add 200 μL of the preheated expansion medium described in Example 1 to each tube, and then transfer it to the wells of the 6-well plate containing the preheated expansion medium in 2), and culture at 37°C, 5% v / v CO2 for 24 hours; during this period, pre-coat a new 6-well plate with antibody OKT-3, add 1 mL of 5 μg / μL OKT-3 antibody coating solution to each well, incubate at 37°C for 4 hours, discard the antibody coating solution, and wash 3 times with PBS for later use;

[0284] 8) After culturing at 37°C and 5% v / v CO2 for 24 hours, the expansion culture medium suspension containing each cell was transferred to the wells of the OKT-3-coated six-well plate and cultured for another 24 hours under the stimulation of the antibody OKT-3;

[0285] 9) Each cell was centrifuged at 800 g for 10 min, the supernatant was discarded, the cell pellet was resuspended with the expansion medium described in Example 1 and then transferred to the wells of a new six-well plate for continued culture. Half the volume of the old medium was removed every two days, and half the volume of fresh expansion medium was added. After continued culture for 10 days, cell counting and flow cytometry were performed on each cell of Group 1 and Group 2, and the average values ​​of the survival rate, number of live cells, and proportion and number of EGFP-positive cells of three samples of each of Group 1 and Group 2 were calculated.

[0286] The results are as follows Fig.11 Compared with the untreated group, the number of living cells in the CaCl2+NaCl-treated group was significantly less, and there were no significant differences in cell survival rate, EGFP-positive cell ratio, and EGFP-positive cell number.

[0287] Comparative Example 2

[0288] The TIL finished cells obtained in Example 1 were used to prepare TIL overexpressing EGFP according to the following steps:

[0289] 1) The TIL finished cells prepared in Example 1 were divided into two groups (Group 1 and Group 2). The cells in Group 1 were resuspended to 5×10 6 / mL, IL-2 was added to a final concentration of 500 IU / mL and NaCl to a final concentration of 50 mM, and the cells were placed in a 30°C 5% v / v CO2 incubator for 24 h for pretreatment. After 24 h, the cells were centrifuged at 800 g for 10 min, the supernatant was discarded, the cell pellet was resuspended and washed with PBS, the centrifugation was repeated and the supernatant was discarded. The cell pellet was resuspended with PBS and divided into 3 equal portions, each containing 5 × 10 6 The cells in group 2 were not treated as above, but were directly centrifuged at 800 g for 10 min, the supernatant was discarded, the cell pellet was resuspended in PBS, washed, and resuspended in PBS again and divided into 3 portions, each containing 5 × 10 6 Live cells are available for backup;

[0290] 2) Add the expansion medium containing 500 IU / mL IL-2, 7 ng / mL IL-7, 30 ng / mL IL-15, 5% v / v human AB serum, 1× PS dual antibody and X-VIVO 15 supplemented to the final volume described in Example 1 into a 6-well plate, 3 mL per well, and then transfer to a cell culture incubator at 37°C and 5% CO2 for preheating for 1 hour;

[0291] 3) Take each portion of cells from group 1 and group 2 in 1), centrifuge at 800 g for 10 min, resuspend and wash the precipitated cells with PBS, centrifuge again at 800 g for 10 min, and discard the supernatant;

[0292] 4) Prepare the electrotransfer solution ratio for each well according to Table 1:

[0293] 5) Add 3 μL of plasmid pKB20-EGFP at a concentration of 1 μg / μL to the prepared electroporation solution, and then let it stand at room temperature for less than 30 minutes;

[0294] 6) Use the plasmid-containing electroporation solution prepared in 5) to resuspend the cell pellets of group 1 and group 2 obtained in 3) respectively, 100 μL per tube, carefully pipette the cell resuspension into the LONZA 100 μL electroporation cup, and place the electroporation cup into the LONZA Nucleofector TM 2b In the electroporation tank, select T-020 as the electroporation program and start the electroporation program;

[0295] 7) After the electroporation is completed, carefully remove the electroporation cup, aspirate the cell suspension and transfer it to the EP tube, add 200 μL of the preheated expansion medium described in Example 1 to each tube, and then transfer it to the wells of the 6-well plate containing the preheated expansion medium in 2), and culture at 37°C, 5% v / v CO2 for 24 hours; during this period, pre-coat a new 6-well plate with antibody OKT-3, add 1 mL of 5 μg / μL OKT-3 antibody coating solution to each well, incubate at 37°C for 4 hours, discard the antibody coating solution, and wash 3 times with PBS for later use;

[0296] 8) After culturing at 37°C and 5% v / v CO2 for 24 hours, the expansion culture medium suspension containing each cell was transferred to the wells of the OKT-3-coated six-well plate and cultured for another 24 hours under the stimulation of the antibody OKT-3;

[0297] 9) Each cell was centrifuged at 800 g for 10 min, the supernatant was discarded, the cell pellet was resuspended with the expansion medium described in Example 1 and then transferred to the wells of a new six-well plate for continued culture. Half the volume of the old medium was removed every two days, and half the volume of fresh expansion medium was added. After continued culture for 10 days, cell counting and flow cytometry were performed on each cell of Group 1 and Group 2, and the average values ​​of the survival rate, number of live cells, and proportion and number of EGFP-positive cells of three samples of each of Group 1 and Group 2 were calculated.

[0298] The results are as follows Fig.12 Compared with the untreated group, the number of living cells in the NaCl-treated group was significantly less, and there were no significant differences in cell survival rate, EGFP-positive cell ratio, and EGFP-positive cell number.

[0299] Comparative Example 3

[0300] The TIL finished cells obtained in Example 1 were used to prepare TIL overexpressing EGFP according to the following steps:

[0301] 1) The TIL finished cells prepared in Example 1 were taken and divided into 2 groups (Group 1 and Group 2), centrifuged at 800 g for 10 min, the supernatant was discarded, the cell pellet was resuspended and washed with PBS, the centrifugation was repeated and the supernatant was discarded, the cell pellet of each group was resuspended with PBS and then divided into 3 equal portions, each portion containing 5×10 6 Living cells, ready for the next experiment;

[0302] 2) Add the expansion medium containing 500 IU / mL IL-2, 7 ng / mL IL-7, 30 ng / mL IL-15, 5% v / v human AB serum, 1× PS dual antibody and X-VIVO 15 supplemented to the final volume described in Example 1 into a 6-well plate, 3 mL per well, and then transfer to a cell culture incubator at 37°C and 5% CO2 for preheating for 1 hour;

[0303] 3) Take each portion of cells from group 1 and group 2 in 1), centrifuge at 800 g for 10 min, resuspend and wash the precipitated cells with PBS, centrifuge again at 800 g for 10 min, and discard the supernatant;

[0304] 4) Prepare the electrotransfer solution ratio for each well according to Table 1:

[0305] 5) For each cell in group 1, add 3 μL of plasmid pKB20-EGFP at a concentration of 1 μg / μL and 1 μL of 0.5M CaCl2 to the prepared electroporation solution (to a final concentration of 50 mM CaCl2 in the electroporation solution), and then let stand at room temperature for less than 30 min; for each cell in group 2, add 3 μL of plasmid pKB20-EGFP at a concentration of 1 μg / μL to the prepared electroporation solution, and then let stand at room temperature for less than 30 min;

[0306] 6) Use the plasmid-containing electroporation solution prepared in 5) for group 1 and group 2 cell samples to resuspend the cell pellets of group 1 and group 2 obtained in 3) respectively, 100 μL per tube, carefully pipette the cell resuspension into the LONZA 100 μL electroporation cup, and place the electroporation cup into the LONZA Nucleofector TM 2b In the electroporation tank, select T-020 as the electroporation program and start the electroporation program;

[0307] 7) After the electroporation is completed, carefully remove the electroporation cup, aspirate the cell suspension and transfer it to the EP tube, add 200 μL of the preheated expansion medium described in Example 1 to each tube, and then transfer it to the wells of the 6-well plate containing the preheated expansion medium in 2), and culture at 37°C, 5% v / v CO2 for 24 hours; during this period, pre-coat a new 6-well plate with antibody OKT-3, add 1 mL of 5 μg / μL OKT-3 antibody coating solution to each well, incubate at 37°C for 4 hours, discard the antibody coating solution, and wash 3 times with PBS for later use;

[0308] 8) After culturing at 37°C and 5% v / v CO2 for 24 hours, the expansion culture medium suspension containing each cell was transferred to the wells of the OKT-3-coated six-well plate and cultured for another 24 hours under the stimulation of the antibody OKT-3;

[0309] 9) Each cell was centrifuged at 800 g for 10 min, the supernatant was discarded, the cell pellet was resuspended with the expansion medium described in Example 1 and then transferred to the wells of a new six-well plate for continued culture. Half the volume of the old medium was removed every two days, and half the volume of fresh expansion medium was added. After continued culture for 10 days, cell counting and flow cytometry were performed on each cell of Group 1 and Group 2, and the average values ​​of the survival rate, number of live cells, and proportion and number of EGFP-positive cells of three samples of each of Group 1 and Group 2 were calculated.

[0310] The results are as follows Fig.13Compared with the untreated group, the number of live cells, the proportion of EGFP-positive cells, and the number of EGFP-positive cells in the CaCl2 electroporation group were significantly less, and there was no significant difference in cell survival rate.

[0311] Comparative Example 4

[0312] The TIL finished cells obtained in Example 1 were used to prepare TIL overexpressing EGFP according to the following steps:

[0313] 1) The TIL finished cells prepared in Example 1 were taken and divided into 2 groups (Group 1 and Group 2), centrifuged at 800 g for 10 min, the supernatant was discarded, the cell pellet was resuspended and washed with PBS, the centrifugation was repeated and the supernatant was discarded, the cell pellet of each group was resuspended with PBS and then divided into 3 equal portions, each portion containing 5×10 6 Living cells, ready for the next experiment;

[0314] 2) Add the expansion medium containing 500 IU / mL IL-2, 7 ng / mL IL-7, 30 ng / mL IL-15, 5% v / v human AB serum, 1× PS dual antibody and X-VIVO 15 supplemented to the final volume described in Example 1 into a 6-well plate, 3 mL per well, and then transfer to a cell culture incubator at 37°C and 5% CO2 for preheating for 1 hour;

[0315] 3) Take each portion of cells from group 1 and group 2 in 1), centrifuge at 800 g for 10 min, resuspend and wash the precipitated cells with PBS, centrifuge again at 800 g for 10 min, and discard the supernatant;

[0316] 4) Prepare the electrotransfer solution ratio for each well according to Table 1:

[0317] 5) For each cell in group 1, add 3 μL of plasmid pKB20-EGFP at a concentration of 1 μg / μL and 1 μL of 0.25M MgSO4 to the prepared electroporation solution (to a final concentration of MgSO4 of 2.5 mM), and then let stand at room temperature for less than 30 min; for each cell in group 2, add 3 μL of plasmid pKB20-EGFP at a concentration of 1 μg / μL to the prepared electroporation solution, and then let stand at room temperature for less than 30 min;

[0318] 6) Use the plasmid-containing electroporation solution prepared in 5) for group 1 and group 2 cell samples to resuspend the cell pellets of group 1 and group 2 obtained in 3) respectively, 100 μL per tube, carefully pipette the cell resuspension into the LONZA 100 μL electroporation cup, and place the electroporation cup into the LONZA Nucleofector TM 2b In the electroporation tank, select T-020 as the electroporation program and start the electroporation program;

[0319] 7) After the electroporation is completed, carefully remove the electroporation cup, aspirate the cell suspension and transfer it to the EP tube, add 200 μL of the preheated expansion medium described in Example 1 to each tube, and then transfer it to the wells of the 6-well plate containing the preheated expansion medium in 2), and culture at 37°C, 5% v / v CO2 for 24 hours; during this period, pre-coat a new 6-well plate with antibody OKT-3, add 1 mL of 5 μg / μL OKT-3 antibody coating solution to each well, incubate at 37°C for 4 hours, discard the antibody coating solution, and wash 3 times with PBS for later use;

[0320] 8) After culturing at 37°C and 5% v / v CO2 for 24 hours, the expansion culture medium suspension containing each cell was transferred to the wells of the OKT-3-coated six-well plate and cultured for another 24 hours under the stimulation of the antibody OKT-3;

[0321] 9) Each cell was centrifuged at 800 g for 10 min, the supernatant was discarded, the cell pellet was resuspended with the expansion medium described in Example 1 and then transferred to the wells of a new six-well plate for continued culture. Half the volume of the old medium was removed every two days, and half the volume of fresh expansion medium was added. After continued culture for 10 days, cell counting and flow cytometry were performed on each cell of Group 1 and Group 2, and the average values ​​of the survival rate, number of live cells, and proportion and number of EGFP-positive cells of three samples of each of Group 1 and Group 2 were calculated.

[0322] The results are as follows Fig.14 Compared with the untreated group, the number of live cells, the proportion of EGFP-positive cells, and the number of EGFP-positive cells in the MgSO4 electroporation group were significantly less, and there was no significant difference in cell survival rate.

[0323] Comparative Example 5

[0324] The TIL finished cells obtained in Example 1 were used to prepare TIL overexpressing EGFP according to the following steps:

[0325] 1) The TIL finished cells prepared in Example 1 were taken and divided into 2 groups (Group 1 and Group 2), centrifuged at 800 g for 10 min, the supernatant was discarded, the cell pellet was resuspended and washed with PBS, the centrifugation was repeated and the supernatant was discarded, the cell pellet of each group was resuspended with PBS and then divided into 3 equal portions, each portion containing 5×10 6 Living cells, ready for the next experiment;

[0326] 2) Add the expansion medium containing 500 IU / mL IL-2, 7 ng / mL IL-7, 30 ng / mL IL-15, 5% v / v human AB serum, 1× PS dual antibody and X-VIVO 15 supplemented to the final volume described in Example 1 into a 6-well plate, 3 mL per well, and then transfer to a cell culture incubator at 37°C and 5% CO2 for preheating for 1 hour;

[0327] 3) Take each portion of cells from group 1 and group 2 in 1), centrifuge at 800 g for 10 min, resuspend and wash the precipitated cells with PBS, centrifuge again at 800 g for 10 min, and discard the supernatant;

[0328] 4) Prepare the electrotransfer solution ratio for each well according to Table 1:

[0329] 5) For each cell in group 1, add 3 μL of plasmid pKB20-EGFP at a concentration of 1 μg / μL, 0.25 M NaCl (to a final concentration of NaCl added to the electrotransfer solution) and 1 μL of 0.25 M CaCl2 (to a final concentration of CaCl2 added to the electrotransfer solution of 2.5 mM) to the prepared electrotransfer solution, and then let stand at room temperature for less than 30 min; for each cell in group 2, add 3 μL of plasmid pKB20-EGFP at a concentration of 1 μg / μL to the prepared electrotransfer solution, and then let stand at room temperature for less than 30 min;

[0330] 6) Use the plasmid-containing electroporation solution prepared in 5) for group 1 and group 2 cell samples to resuspend the cell pellets of group 1 and group 2 obtained in 3) respectively, 100 μL per tube, carefully pipette the cell resuspension into the LONZA 100 μL electroporation cup, and place the electroporation cup into the LONZA Nucleofector TM 2b In the electroporation tank, select T-020 as the electroporation program and start the electroporation program;

[0331] 7) After the electroporation is completed, carefully remove the electroporation cup, aspirate the cell suspension and transfer it to the EP tube, add 200 μL of the preheated expansion medium described in Example 1 to each tube, and then transfer it to the wells of the 6-well plate containing the preheated expansion medium in 2), and culture at 37°C, 5% v / v CO2 for 24 hours; during this period, pre-coat a new 6-well plate with antibody OKT-3, add 1 mL of 5 μg / μL OKT-3 antibody coating solution to each well, incubate at 37°C for 4 hours, discard the antibody coating solution, and wash 3 times with PBS for later use;

[0332] 8) After culturing at 37°C and 5% v / v CO2 for 24 hours, the expansion culture medium suspension containing each cell was transferred to the wells of the OKT-3-coated six-well plate and cultured for another 24 hours under the stimulation of the antibody OKT-3;

[0333] 9) Each cell was centrifuged at 800 g for 10 min, the supernatant was discarded, the cell pellet was resuspended with the expansion medium described in Example 1 and then transferred to the wells of a new six-well plate for continued culture. Half the volume of the old medium was removed every two days, and half the volume of fresh expansion medium was added. After continued culture for 10 days, cell counting and flow cytometry were performed on each cell of Group 1 and Group 2, and the average values ​​of the survival rate, number of live cells, and proportion and number of EGFP-positive cells of three samples of each of Group 1 and Group 2 were calculated.

[0334] The results showed that compared with the untreated group, the number of live cells, the proportion of EGFP-positive cells and the number of EGFP-positive cells in the CaCl2+NaCl electroporation group were significantly lower, and there was no significant difference in cell survival rate.

[0335] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims. At the same time, all the documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference separately.

Claims

1. A method for preparing cells expressing exogenous genes by electroporation, comprising the following steps: Treating the cells with divalent cations before electroporation, and / or electroporating the cells under conditions containing monovalent cations; Preferably, the method comprises the steps of: 1) treating the cells with a pretreatment solution before electroporation; and 2) introducing a nucleic acid construct encoding the exogenous gene into the cell by electroporation, Alternatively, the method comprises the steps of: 1) introducing a nucleic acid construct encoding the exogenous gene into the cell by electroporation in an electroporation solution, Alternatively, the method comprises the steps of: 1) treating the cells with a pretreatment solution before electroporation; and 2) In an electroporation solution, introducing a nucleic acid construct encoding the exogenous gene into the cells by electroporation, wherein the pretreatment solution contains divalent cations and the electroporation solution contains monovalent cations.

2. The method according to claim 1, characterized in that The divalent cation is selected from Ca 2+ Mg 2+ , Mn 2+ Any one, any two or all of; preferably, the concentration of the divalent cation in the pretreatment solution is 0.1-90, 0.2-80, 0.3-70, 0.4-60, 0.5-50, 1-50, 1-25, 1-20, 0.5-20, 0.5-15, 0.5-10, 1-10 or 2-10 mM; The monovalent cation is selected from Na + , K + , Li + Any one, any two or all of; preferably, the concentration of the monovalent cation in the electrotransfer solution is 0.2-500, 0.5-500, 0.5-400, 0.5-300, 0.5-200, 0.5-150, 0.5-100, 1-500, 1-250, 1-200, 1-100, 1-50, 2-50 or 5-50 mM.

3. The method according to claim 1 or 2, characterized in that The pretreatment solution further comprises cytokines; preferably, the cytokines comprise one or more selected from the following: interleukins, tumor necrosis factor (TNF), interferon (IFN), colony stimulating factor (CSF) and tumor growth factor (TGF); more preferably, the concentration of IL-2 in the pretreatment solution is 200-6000 IU / mL, preferably 200-950, 200-850, 200-750, 200-650, 200-550 or 200-500 IU / mL; and / or The pretreatment solution further contains a cell culture medium; preferably, the cell culture medium includes AIM-V, X-VIVO, DMEM, RPMI1640; and / or The electrotransfer solution further comprises an electrotransfer buffer; and / or The cells are immune cells; preferably, the immune cells are selected from any one or more of T cells, DN T cells, NK cells, NKT cells, CAR-T cells, TCR-T cells, TIL, CTL, LAK cells and CIK cells; more preferably, they are T cells or TIL cells.

4. The method according to claim 1 or 2, characterized in that: The method further comprises one or more features selected from the following: (A) The treatment in step 1) is incubation; preferably, the incubation is carried out in an incubator; (B) the treatment time in step 1) is at least 0.5 hours; preferably at least 1 hour; (C) The treatment temperature in step 1) is 25°C-40°C; preferably 30°C-40°C; (D) The concentration of cells in the system treated in step 1) is 1×10 5 Up to 1×10 10 / mL, preferably 1×10 5 Up to 1×10 7 Pieces / mL; (E) The nucleic acid construct in step 2) is DNA or RNA; preferably a vector or mRNA; more preferably any one or more of a plasmid, an adenoviral vector, a lentiviral vector, an adeno-associated viral vector and a non-viral vector; further preferably, the non-viral vector is a non-viral vector based on a transposon system.

5. The method according to claim 1 or 2, characterized in that: The method comprises the steps of: 1) incubating the cells in a pretreatment solution for at least 0.5 hours, and 2) introducing a nucleic acid construct encoding the exogenous gene into the cells by electroporation; preferably, the pretreatment solution contains Ca 2+ Mg 2+ , Mn 2+ Any one, any two or all of The method comprises the steps of: 1) incubating the cells in an electroporation solution for at least 0.1 hour, wherein the electroporation solution contains a nucleic acid construct encoding the exogenous gene and a monovalent cation, and 2) introducing the nucleic acid construct into the cells by electroporation in the electroporation solution; preferably, the electroporation solution contains Na + , K + , Li + Any one, any two or all of The method comprises the steps of: 1) incubating the cells in a pretreatment solution for at least 0.5 hours, 2) incubating the cells in an electroporation solution for at least 0.1 hours, wherein the electroporation solution contains a nucleic acid construct encoding the exogenous gene and monovalent cations, and 3) introducing the nucleic acid construct into the cells by electroporation in the electroporation solution; preferably, the pretreatment solution contains Ca 2+ Mg 2+ , Mn 2+ Any one, any two or all of the following, wherein the electrotransfer solution contains Na + , K + , Li + Any one, any two or all of the following.

6. A cell expressing a foreign gene prepared by the method according to any one of claims 1 to 5; Preferably, the cell is an immune cell; More preferably, the immune cells are selected from any one or more of T cells, DN T cells, NK cells, NKT cells, CAR-T cells, TCR-T cells, TIL, CTL, LAK cells and CIK cells.

7. Use of the cell expressing the exogenous gene as claimed in claim 6 in preparing a drug for treating a disease; Preferably, the disease is a disease suitable for cell therapy; More preferably, the disease comprises a tumor; Even more preferably, the tumor comprises: Prostate cancer, breast cancer, lung cancer, colorectal cancer, stomach cancer, liver cancer, pancreatic cancer, bladder cancer, skin cancer, kidney cancer, melanoma, cervical cancer, ovarian cancer, head and neck tumors.

8. An electrotransfer solution combination, comprising one or both of a pretreatment solution for treating cells to be electrotransformed before electrotransformation and an electrotransfer solution for electrotransformation during electrotransformation; wherein: The pretreatment solution includes divalent ions, and / or the electrotransfer solution includes monovalent cations; Preferably, the pretreatment solution and electro-conversion solution are as described in any one of claims 1-5.

9. An electroporation kit for improving transduction efficiency and cell survival, the kit comprising one or both of a pretreatment solution for treating cells to be electroporated before electroporation and an electroporation solution for electroporation; wherein: The pretreatment solution includes divalent cations, and the electrotransfer solution includes monovalent cations. Preferably, the pretreatment solution and the electro-conversion solution are as described in any one of claims 1 to 5. Preferably, the kit optionally further comprises any one or more of a cell culture medium, a cytokine, and an electroporation buffer.

10. Use of the pretreatment solution and / or electrotransfer solution as described in any one of claims 1 to 5, the electrotransfer solution combination as described in claim 8, or the electrotransfer kit as described in claim 9 in improving the transduction efficiency of electrotransfer, the survival rate of electrotransfer cells, the number of live cells after electrotransfer, the proportion of electrotransfer positive cells, the number of electrotransfer positive cells and / or the survival of electrotransfer cells.

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