Cell electrotransfection reagent and application thereof

Abstract

The invention relates to a method for preparing cells overexpressing exogenous genes through electrotransfection, which comprises the following steps: treating the cells with a cell electrotransfection pretreatment reagent before electrotransfection, and the cell electrotransfection pretreatment reagent contains cholesterol derivatives. The invention also relates to the exogenous gene overexpressed cell prepared by the method, application of the cell electrotransfection pretreatment reagent in preparation of the exogenous gene overexpressed cell, and a cell electrotransfection kit. The cell electrotransfection kit comprises a cell electrotransfection medium component and a cell treatment component. The cell treatment component comprises a cholesterol derivative. The method provided by the invention can greatly improve the survival rate of cells after electrotransfection and the proportion and quantity of cells with positive exogenous gene expression.

Description

Technical Field

[0001] This invention relates to cell electroporation and culture, and specifically to a cell electroporation reagent and its application. Background Technology

[0002] Electroporation (or electroporation) is a well-established method in some areas of medicine and is increasingly being applied in biotechnology. By momentarily applying a high electric field pulse to cells or tissues, a permeability barrier is created at the cell membrane surface, allowing charged molecules to enter the cell. Classical electroporation enhances transmembrane transport and alters electrical conductivity. In electroporation-based transgenic manipulation, exogenous DNA is reversibly introduced into the cell via electroporation, and the exogenous gene is expressed in its new host cell and inherited with cell division. Combining electroporation with non-viral gene modification systems capable of inducing stable transgene expression, such as transposon systems, is another effective method for modifying immune effector cells besides viral vector systems, showing high efficiency in T cells. Currently, commercially available electroporation instruments and associated buffers (such as Lonza Nucleofactor) are readily available.

[0003] Compared to viral vector systems, electroporation combined with transposon systems offers advantages such as simple operation, low genotoxicity, and low safety risks. However, it also has significant drawbacks: instantaneous high voltage can easily cause excessive cell death, and transfection efficiency is specifically related to cell type and electroporation conditions (including voltage, waveform, pulse duration, and electroporation buffer composition). This is particularly true for immune effector cells, such as PBMCs and TILs, which exhibit strong immune responses to directly introduced exogenous DNA or RNA into the cytoplasm, making electroporation relatively more challenging. Although relatively mature electroporation instruments and supporting buffer systems are available on the market, the high mortality rate of immune effector cells after electroporation and the low percentage of cells expressing positive exogenous genes remain prominent issues. Therefore, a method to improve the survival rate of immune effector cells after electroporation and the positive rate of exogenous gene expression is still needed. Summary of the Invention

[0004] The first aspect of the present invention provides a method for preparing cells overexpressing a foreign gene by electroporation, comprising the following steps: treating the cells with a cell electroporation pretreatment reagent before electroporation, the cell electroporation pretreatment reagent comprising a cholesterol derivative.

[0005] In one or more embodiments, the cholesterol derivative comprises cholesterol sulfate.

[0006] In one or more embodiments, the cell electroporation pretreatment reagent further comprises a reducing agent. In one or more embodiments, the reducing agent comprises reduced glutathione.

[0007] In one or more embodiments, the method further includes the step of treating the cells with a reducing agent after electroporation. In one or more embodiments, the reducing agent comprises reduced glutathione.

[0008] In one or more embodiments, the method includes the following steps:

[0009] 1) Incubate the cells to be electroporated with the cell pretreatment reagent to separate the cells;

[0010] 2) Introduce exogenous genes into cells after incubation by electrotransfer 1).

[0011] In one or more embodiments, the cells are immune effector cells. Optionally, the immune effector cells include any one or more of T cells, TILs, NK cells, NKT cells, CAR-T cells, CIK cells, CTLs, TCR-T cells, LAK cells, DCs, and γδT cells. Preferably, they are T cells or TILs.

[0012] In one or more embodiments, the cell electroporation pretreatment reagent in step 1) comprises a cholesterol derivative; the cholesterol derivative preferably comprises cholesterol sulfate. Optionally, the cell electroporation treatment reagent further comprises a reducing agent; preferably, the reducing agent comprises reduced glutathione.

[0013] In one or more embodiments, the cholesterol derivative in step 1) comprises cholesterol sulfate at a final concentration of 1-100 μM; optionally, 10-50 μM; optionally, 25-50 μM.

[0014] In one or more embodiments, the reducing agent in step 1) comprises reduced glutathione at a final concentration of 1-50 mM; optionally, 1-25 mM; optionally, 5-10 mM.

[0015] In one or more embodiments, the co-incubation temperature in step 1) is 25-42°C; optionally, 30-37°C; optionally, 30°C.

[0016] In one or more embodiments, the CO2 concentration for co-incubation in step 1) is 1-10 v / v%; preferably, it is 5 v / v%.

[0017] In one or more embodiments, the co-incubation duration in step 1) is 1-20 hours; optionally, 2-20 hours; optionally, 8-16 hours; optionally, 12-16 hours.

[0018] In one or more embodiments, the cells to be electroporated in step 1) are resuspended in a cell culture medium; optionally, the cell culture medium comprises a basal culture medium; optionally, the basal culture medium comprises CTS TM Serum-free cell culture medium, DMEM medium, RPMI 1640 medium, and X-VIVO 15, or any one or more of these.

[0019] In one or more embodiments, the cell culture medium further comprises cytokines; optionally, the cytokines include any one or more of IL-2, IL-7, IL-12, IL-15, IL-21, TNF-α, and IFN-γ. Preferably, the cytokines include IL-2; preferably, the final concentration of IL-2 is 100-5000 IU / mL; more preferably, the final concentration of IL-2 is 200-3000 IU / mL.

[0020] In one or more embodiments, the cell culture medium is X-VIVO 15 medium containing 500 IU / mL IL-2.

[0021] In one or more embodiments, the cell density of the cells to be electroporated in step 1) resuspended in cell culture medium is 1 × 10⁻⁶ cells / year. 6 -1×10 7 / mL; optionally, 2×10 6 -8×10 6 / mL; optionally, 3×10 6 -6×10 6 / mL; optionally, 4×10 6 / mL.

[0022] In one or more embodiments, the cell separation in step 1) is achieved by centrifugation. Optionally, the centrifugation speed is 100-1000g; optionally, 300-800g; optionally, 800g. Optionally, the centrifugation time is 1-20 minutes; optionally, the centrifugation time is 3-8 minutes.

[0023] In one or more embodiments, the electroporation conditions described in step 2) are suitable for the electroporation of immune cells; optionally, they are suitable for electroporation of any one or more selected from T cells, TILs, NK cells, NKT cells, CAR-T cells, CIK cells, CTLs, TCR-Ts, LAKs, DCs, and γδT cells; optionally, they are suitable for the electroporation of T cells or TILs.

[0024] In one or more embodiments, the conditions for electroporation in step 2) are any one of the Lonza Nucleofector series electroporators T-020, T023, V-024, U-014 and U-017.

[0025] In one or more embodiments, the vector of the exogenous gene in step 2) is a DNA vector or an RNA vector; optionally, the DNA vector is single-stranded DNA or double-stranded DNA; optionally, the DNA vector is a plasmid vector.

[0026] In one or more embodiments, the method further includes: 3) continuing to culture the cells in 2) in which the exogenous gene was introduced via electroporation.

[0027] In one or more embodiments, the culture in step 3) is performed in a cell culture medium; optionally, the cell culture medium comprises a basal culture medium; optionally, the basal culture medium is selected from... CTS TM Any one or more of serum-free cell culture medium, DMEM medium, RPMI 1640 medium and X-VIVO 15 medium.

[0028] In one or more embodiments, the cell culture medium further comprises cytokines; optionally, the cytokines include any one or more of IL-2, IL-7, IL-12, IL-15, IL-21, TNF-α, and IFN-γ.

[0029] In one or more embodiments, the cell culture medium is X-VIVO 15 medium containing 500 IU / mL IL-2.

[0030] In one or more embodiments, the cell culture medium further comprises a reducing agent; optionally, the reducing agent is reduced glutathione. In one or more embodiments, the final concentration of the reduced glutathione is 1-100 mM; optionally, 1-50 mM; optionally, 5-10 mM.

[0031] In one or more embodiments, the culture temperature in step 3) is 25-42°C; optionally, 30-37°C; optionally, 37°C.

[0032] In one or more embodiments, the CO2 concentration for cultivation in step 3) is 1-10 v / v%; preferably, it is 5 v / v%.

[0033] In one or more embodiments, the culture time described in step 3) is 2 hours to 30 days; optionally, it is 2 hours to 15 days.

[0034] In one or more embodiments, the initial cell density for culture in step 3) is 1 × 10⁻⁶. 6 -1×10 7 / mL; optionally, 2×10 6 -8×10 6 / mL; optionally, 2.5×10 6 -5×10 6 / mL; optionally, 2.5×10 6 / mL.

[0035] A second aspect of the present invention provides cells overexpressing exogenous genes prepared by the above method.

[0036] In one or more embodiments, the cells are immune effector cells; preferably, the immune effector cells include any one or more of T cells, TILs, NK cells, NKT cells, CAR-T cells, CIK cells, CTLs, TCR-Ts, LAKs, DCs, and γδT cells.

[0037] A third aspect of the present invention provides the application of a cell electroporation pretreatment reagent in the preparation of cells overexpressing exogenous genes, wherein the cell electroporation pretreatment reagent includes a cholesterol derivative.

[0038] In one or more embodiments, the cells are immune effector cells; preferably, the immune effector cells include any one or more of T cells, TILs, NK cells, NKT cells, CAR-T cells, CIK cells, CTLs, TCR-Ts, LAKs, DCs, and γδT cells.

[0039] In one or more embodiments, the cholesterol derivative comprises cholesterol sulfate.

[0040] In one or more embodiments, the cell electroporation pretreatment reagent further comprises a reducing agent; preferably, the reducing agent comprises reduced glutathione.

[0041] A fourth aspect of the present invention provides a cell electroporation kit, which includes a cell electroporation medium component and a cell treatment component; the cell treatment component includes a cholesterol derivative.

[0042] In one or more embodiments, the cholesterol derivative includes cholesterol sulfate.

[0043] In one or more embodiments, the cell treatment component further includes a reducing agent; preferably, the reducing agent includes reduced glutathione.

[0044] In one or more embodiments, the cell electroporation medium component includes a cell electroporation buffer.

[0045] In one or more embodiments, the cell electroporation buffer contains Na + K + Ca 2+ Mg 2+ and Zn 2+ Any one or more of them.

[0046] In one or more embodiments, the cell electroporation buffer contains Cl - SO4 2- PO4 3- HPO4 2- H2PO4 - HEPES and NO3 - Any one or more of them.

[0047] In one or more embodiments, the cell electroporation buffer is selected from Lonza human T cells. Solution ( Solution for Human T Cells), Maxcyte Electroporation Buffer, ThermoFisher CTS TM Xenon TM Electroporation buffer (CTS) TM Xenon TM Electroporation Buffer), Miltenyi Electroporation buffer ( One or more of the following: Electroporation Buffer, BTXpress Electroporation Solution, and Bio-rad Gene Pulser Electroporation Buffer.

[0048] In one or more embodiments, the cell electroporation kit comprises: Lonza human T cells The solution includes Lonza electroporation supplement and cholesterol sulfate. In one or more embodiments, the cell electroporation kit also includes reduced glutathione.

[0049] This invention has the following advantages: When introducing nucleic acid vectors containing exogenous genes, such as DNA vectors, into immune cells, especially T cells and TILs, using common electroporation procedures, the instantaneous high voltage can easily cause excessive cell death. Furthermore, the naked nucleic acids, such as naked DNA, entering the cytoplasm through cell membrane perforations formed under instantaneous high voltage can trigger a strong immune response in T cells or TILs, leading to further mass death of exogenous gene-positive immune cells after electroporation. In the method for preparing cells overexpressing exogenous genes by electroporation provided by this invention, pretreatment of immune cells, such as T cells or TILs, with cholesterol derivatives or cholesterol derivatives + reducing agents before electroporation significantly improves the survival rate of electroporated immune cells, as well as the proportion and number of exogenous gene-positive immune cells. Adding a reducing agent, such as reduced glutathione, during the continued cell culture after electroporation can further significantly improve these indicators. Attached Figure Description

[0050] Figure 1 The number of viable cells after peripheral blood T cells were treated with different electroporation methods;

[0051] Figure 2 The percentage of viable cells in peripheral blood T cells after treatment with different electroporation methods;

[0052] Figure 3 The proportion of viable cells positive for exogenous gene EGFP expression after peripheral blood T cells were treated with different electroporation methods;

[0053] Figure 4 : Number of viable cells after TIL treatment using different electroporation methods;

[0054] Figure 5 The proportion of viable cells after TIL treatment using different electroporation methods;

[0055] Figure 6 The proportion of live cells positive for exogenous gene EGFP expression after TIL treatment by different electroporation methods. Detailed Implementation

[0056] Unless otherwise defined, all technical terms, symbols, and other technical and scientific terms or proprietary vocabulary used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In some instances, terms having a conventionally understood meaning are defined herein for clarification and / or ease of reference; such definitions should not be construed as indicating a significant difference from the conventional understanding in the art. Section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter.

[0057] All publications mentioned in this invention, including patent documents, academic papers, and databases, are incorporated herein by reference in their entirety to the same extent that each individual publication is incorporated by reference as if independently. If the definitions presented herein differ from or are otherwise inconsistent with those presented in patents, publications, and other publications incorporated herein by reference, the definitions presented herein shall prevail with respect to those presented in the documents incorporated herein by reference.

[0058] Terminology Definition

[0059] As used herein, the singular forms “a,” “an,” and “the” include plural references unless otherwise explicitly stated in the text. For example, “an” or “a” means “at least one or more” or “one or more.”

[0060] When a range of values ​​is provided, it should be understood that every intermediate value between the upper and lower limits of the range, as well as any other stated or intermediate values ​​within the range, is included within the claimed subject matter. The smaller range may independently include the upper and lower limits of these smaller ranges, which also fall within the scope of the claimed subject matter, unless the upper and lower limits of the range are explicitly excluded.

[0061] As used in this invention, the term "about" refers to the general range of error for values ​​that is readily known to those skilled in the art. References to "about" values ​​or parameters herein include (and describe) implementations that refer to that value or parameter itself.

[0062] The term "International Unit" (IU) used in this invention refers to the specific unit definition for various cytokines, interferons, or other factors established by the WHO Expert Committee on Standardization of Biological Agents under the World Health Organization (WHO).

[0063] The "electroporation" described in this invention, also known as "electroporation" or "electroporation," refers to the process by which a transient electric field is applied to a cell, creating temporary pores on the cell membrane surface. Extracellular genetic material (such as DNA and RNA), protein molecules, or other molecules that need to enter the cell then pass through these pores into the cytoplasm to exert their functions. The electroporation in this invention is reversible. That is, the temporary pores created on the cell membrane surface by the transient electric field applied during electroporation do not persist for long. After the electric field is removed, the pores close quickly, the cell membrane returns to its normal state, and the exogenous substances that entered the cytoplasm through the temporarily formed pores are trapped inside the cell and perform their biological functions.

[0064] The "cell pretreatment" described in this invention refers to pretreating cells with a special reagent before applying a transient electric field. Pretreated cells have better survival rates during subsequent electroporation and exhibit better absorption and tolerance to exogenous molecules such as nucleic acids, proteins, and other molecules that need to enter the cells. After pretreatment, the special reagent used for cell pretreatment can be separated from the cells using conventional methods in the art, and the separated cells can then be used for the next electroporation operation. Alternatively, the reagent can be used directly in the next electroporation operation without separation from the cells after pretreatment, as long as the reagent does not negatively affect the cell survival rate or the efficiency of exogenous substance introduction after electroporation.

[0065] In this invention, "derivative" refers to a compound whose structure is similar to that of a parent compound, formed by substituting one or more atoms in the parent compound. This substitution can occur through a substantial chemical reaction or simply through imaginary substitution. The substantial chemical reaction can be a one-step direct reaction or a multi-step reaction, as long as the final result of the reaction is to replace one or more specific atoms in the parent compound.

[0066] In this invention, the term "reducing agent" refers to a reactant that loses electrons to other reactants in a redox reaction, thereby reducing those reactants by giving them electrons. In this invention, the reducing agent contains atoms with low oxidation numbers, and it itself is oxidized in the redox reaction, while the reactants that gain electrons are reduced.

[0067] The "reduced glutathione" mentioned in this invention refers to the reduced form of glutathione, which has a different structure from its oxidized form. In this tripeptide, the sulfhydryl group of cysteine ​​is in a reduced state, and the sulfur atom is bonded to a hydrogen atom, rather than to the sulfur atom of the sulfhydryl group of the other cysteine, forming two tripeptides linked by a disulfide bond. The reduced glutathione has a reducing agent function.

[0068] In this invention, "DNA vector" refers to any DNA fragment carrying a foreign gene coding sequence or a fragment thereof. The DNA fragment can be double-stranded DNA, such as plasmid DNA with or without genome integration function, viral vector DNA modified based on a viral genome, or microcircular DNA. The DNA can also be single-stranded DNA, such as circular single-stranded DNA (cssDNA), which is converted into double-stranded DNA by the intracellular DNA replication machinery after entering the cell. The DNA fragment can be circular DNA or linearized DNA, such as a DNA fragment linearized by enzymatic digestion of a plasmid.

[0069] In this invention, "RNA vector" refers to any RNA fragment carrying a foreign gene coding sequence or a fragment thereof. The RNA fragment can be a linear RNA fragment, such as mRNA containing a foreign gene coding sequence. The mRNA can be natural, unmodified mRNA, or mRNA containing chemical modifications or uncommon nucleotide substitutions to achieve a longer intracellular half-life. The RNA fragment can also be a circular RNA fragment, such as single-stranded circular RNA (circRNA). The RNA can be single-stranded RNA or double-stranded RNA (dsRNA), such as double-stranded RNA from dsRNA viruses.

[0070] In this invention, "cellular electrotransfer medium" refers to a medium capable of mediating a potential difference across the cell membrane. The presence of this potential difference causes temporary pores to form on the cell membrane surface, thereby inducing extracellular substances to enter the cytoplasm through these pores. The medium typically includes different types of anions and cations, and may also contain certain balanced salt solutions and sugars such as sucrose to maintain a certain conductivity and osmotic pressure.

[0071] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as embodiments) can be combined with each other to form preferred technical solutions.

[0072] When introducing exogenous genes into immune effector cells, particularly T cells and tumor-infiltrating lymphocytes (TILs), via electroporation, in addition to the potential cell death caused by the electrical pulses themselves, the nucleic acids (DNA or RNA) containing the exogenous gene introduced during electroporation are directly exposed in the cytoplasm without any encapsulation. This triggers an immune response that leads to further mass death of exogenous gene-positive cells. If the tolerance of immune effector cells to electrical stimulation can be increased while simultaneously reducing their immune response to naked DNA or RNA present in the cytoplasm, the survival rate of cells after electroporation and the proportion of exogenous gene-positive cells can be significantly improved. Through extensive experiments, the inventors of this invention discovered that when cells, especially immune effector cells, are pretreated with a cell electroporation pretreatment reagent containing cholesterol derivatives before electroporation to introduce exogenous genes, the sensitivity of cells to the introduced nucleic acid vector containing the exogenous gene is significantly reduced, resulting in a substantial increase in cell survival rate and the proportion of exogenous gene-positive cells after electroporation. This led to the completion of this invention.

[0073] The present invention provides a method for preparing cells overexpressing exogenous genes by electroporation, comprising the following steps: treating the cells with a cell electroporation pretreatment reagent before electroporation, wherein the cell electroporation pretreatment reagent contains a cholesterol derivative.

[0074] In some embodiments, the method further includes the step of treating the cells with a reducing agent after electroporation.

[0075] In this invention, the cholesterol derivative can be a molecule formed by replacing one or more specific atoms on a cholesterol molecule with other atoms or substituent groups. The molecular formula of the cholesterol is C10. 27 H 46 O, chemical formula as shown in Formula 1:

[0076]

[0077] In this invention, the cholesterol derivative is preferably cholesterol sulfate, with the molecular formula C10. 27 H 46 O4S, chemical formula as shown in Formula 2:

[0078]

[0079] In this invention, the cell electroporation pretreatment reagent may further include a reducing agent. The reducing agent can be any common substance in the art capable of donating electrons in a redox reaction, as long as it can effectively reduce the harmful oxidative effects on the cells caused by the application of a transient electric field during electroporation. For example, the reducing agent can be any one or more of organic salts, inorganic salts, organic acids, sugars, and polypeptides with reducing power. In some embodiments, the reducing agent is a reducing polypeptide. In some embodiments, the reducing polypeptide is reduced glutathione with the molecular formula C2. 10 H 17 N3O6S, chemical formula as shown in Formula 3:

[0080]

[0081] In this invention, the reduced glutathione is the reduced form of the tripeptide glutathione, that is, the cysteine ​​sulfhydryl group in the tripeptide is in a reduced form that binds to a hydrogen atom, rather than forming a disulfide bond with the sulfur atom of the cysteine ​​sulfhydryl group on another glutathione.

[0082] In some embodiments, the method for preparing cells overexpressing a foreign gene by electroporation includes the following steps:

[0083] 1) Incubate the cells to be electroporated with the cell pretreatment reagent to separate the cells;

[0084] 2) Introduce exogenous genes into cells after incubation by electrotransfer 1).

[0085] In some embodiments, the cells are immune effector cells, which include any cells capable of playing a specific role in an immune response, including but not limited to, T cells, TILs, NK cells, NKT cells, CAR-T cells, CIK cells, CTLs, TCR-Ts, LAKs, DCs, and γδT cells.

[0086] T cells: T cells are immune cells produced in the thymus and capable of immune killing or regulation through cell-cell interactions. The T cells involved in this invention include peripheral blood-derived T cells and solid tissue-derived T cells, wherein the solid tissue-derived T cells include T cells derived from solid tumor tissue, all of which are positive for CD3 expression on their cell surface. Based on CD4 and CD8 expression, T cells can be further divided into CD4+ T cells and CD8+ T cells.

[0087] LAK (lymphokine-activated killer cells): LAK cells are killer cells formed by inducing NK cells or T cells with high doses of cytokines such as IL-2 during in vitro culture. They are capable of killing NK-insensitive tumor cells and have broad-spectrum anti-tumor effects. LAK cell adoptive immunotherapy can be combined with direct injection of cytokines such as IL-2 to treat tumors.

[0088] DC (dendritic cell) therapy involves culturing and inducing the generation of DCs from the patient's own mononuclear cells in vitro, then loading them with corresponding tumor antigens to create tumor antigen-loaded dendritic cells. These dendritic cells are then injected into the body to stimulate the proliferation of tumor-killing lymphocytes, thereby exerting long-term tumor surveillance and tumor-killing effects to achieve the goal of eliminating tumors.

[0089] CIK: CIK (cytokine-induced killer) or NK cell-like T lymphocytes are killer cells induced by a variety of cytokines. They combine the strong anti-tumor activity of T lymphocytes with the non-MHC-restricted tumor killing advantage of NK cells.

[0090] CTLs (cytotoxic T lymphocytes) are CD8+ T lymphocytes with the ability to directly kill other cells. CTLs induce apoptosis in target cells that are antigen-specific to the target cells through direct contact with them, using an MHC-restricted mechanism. CTLs play important roles in tumor immunity and antiviral infection control. In clinically significant viral infections such as HIV-1, CTLs are considered a key defense mechanism. CTLs are also considered an important component of antitumor immunity.

[0091] CAR-T: CAR-T (Chimeric Antigen Receptor T-Cell) cells express chimeric antigen receptors on the surface of T cells. Chimeric antigen receptors contain extracellular polypeptides that recognize tumor antigens, hinge regions, transmembrane regions, and one or more intracellular signaling regions. CAR-T activates ITAM signaling via extracellular single-chain antibody fragments that specifically recognize tumor antigens, activating the intracellular signaling pathway of CD3ζ or FcεRIγ. However, first-generation CAR receptors lack co-stimulatory signals for T cells, resulting in transient T cell effects, short lifespan in vivo, and low cytokine secretion. Second- and third-generation CARs combine the two signals required for T cell activation, directly linking the second signal (e.g., the intracellular signaling region of CD28 and / or 4-1BB) to the CD3ζ molecule.

[0092] TCR-T: TCR-T (T Cell Receptor T-Cell) cell therapy is similar to CAR-T cell therapy. It activates and guides T cells to kill cancer cells by inducing them to express novel T cell receptors (TCRs) that recognize cancer cells. TCR-T can be used for solid tumors, targeting targets that normal cells generally do not express but tumor cells may. TCR-T uses novel artificial TCRs to recognize target antigen peptides presented by MHC proteins on tumor cells. For a specific target-specific TCR, a specific MHC (HLA typing) is required for adaptation. Therefore, TCR-T is MHC-restricted. However, unlike CAR-T, which targets are limited to tumor membrane proteins, TCR-T can target any "non-self" protein, including intracellular proteins. The mechanism of TCR-T is closer to the natural mechanism of T cells, resulting in relatively lower toxicity.

[0093] NK cells: NK (natural killer) cells can directly and non-specifically kill tumor cells. This natural killer activity is not MHC-restricted, does not depend on antibodies, and does not require antigen sensitization. Therapeutic NK cells can be used for adoptive transfer therapy for cancer. Unlike T cells, NK cells do not release large amounts of inflammatory proteins, leading to a cytokine storm. Another advantage of NK cells is their versatility; they can be used from healthy individuals or umbilical cord blood, thus reducing patient waiting time and treatment costs.

[0094] NKT cells: NKT (Natural Killer T) cells are a heterogeneous group of cells that possess characteristics of both NK cells and T cells. Many NKT cells can recognize an atypical MHC molecule, CD-1d. Unlike conventional MHC molecules that bind peptides, CD-1d typically binds to cellular or exogenous lipid or glycolipid molecules. Therefore, NKT cells play an important role in recognizing glycolipids of microbial origin, such as those found in bacterial cell walls. NKT cells express αβ T cell receptors and are classified into type I and type II NKT cells. NKT cells can possess markers of both NK cells and T cells; their cell surface can simultaneously express CD3, CD16, and CD56.

[0095] CAR-NK: Similar to CAR-T, CAR-NK uses NK cells instead of T cells, combining the high affinity and targeting of CARs with the safety and versatility of NK cells. CAR-NK cell therapy has reported an objective response rate of 73% and has not caused similar complications to CAR-T therapy.

[0096] TILs (tumor-infiltrating lymphocytes) are infiltrating T cells that emerge during tumor formation. TILs are an important component of tumor cell immunotherapy. The preparation of TIL cells involves: isolating a small number of TIL cells from the patient's tumor tissue, optionally screening for tumor-specific TIL cells, and expanding the TIL cell count to a certain level (e.g., 10⁻⁶). 10 (The above) is reinfused into the tumor patient's body.

[0097] γδT cells: γδT cells are a type of T cell that differs from conventional αβT cells, possessing a heterodimer composed of one γ and one δ chain. Unlike αβT cells, γδT cell activation does not depend on antigen-presenting cells (APCs) and is not restricted by the major histocompatibility complex (MHC), allowing for direct antigen recognition and response. γδT cells are typically classified as innate immune cells in cell classification. γδT cells are CD3+, with the vast majority being CD4-CD8- double-negative cells, and only a minority being CD8+ cells.

[0098] In some embodiments, the cells to be electroporated in step 1) can be any type of cell, as long as electroporation allows the introduction of exogenous molecules into the cell. In some embodiments, the cells to be electroporated are immune effector cells. In some embodiments, the immune effector cells can be any one or more selected from T cells, TILs, NK cells, NKT cells, CAR-T cells, CIK cells, CTLs, TCR-T cells, LAK cells, DCs, and γδT cells. In some embodiments, the immune effector cells are T cells or TILs. In some implementations, the TIL can be derived from any solid tumor tissue, including but not limited to TILs derived from breast, prostate, lung, and colon cancer or epithelial cancer, such as breast cancer, colon cancer, prostate cancer, head and neck cancer, skin cancer, melanoma; genitourinary tract cancers, such as ovarian cancer, endometrial cancer, cervical cancer, endometrial stromal sarcoma, poorly differentiated pelvic adenocarcinoma, kidney cancer, bladder cancer, prostate cancer; lung cancer, gastric cancer, gastrointestinal stromal tumor, small intestine cancer, liver cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, esophageal cancer, salivary gland cancer, thyroid cancer, and osteosarcoma.

[0099] In some embodiments, the cell electroporation pretreatment reagent in step 1) comprises a cholesterol derivative, preferably cholesterol sulfate. In some embodiments, the concentration of cholesterol sulfate in the co-incubation system in step 1) can be any suitable concentration, as long as it does not produce any significant side effects on the cell state. For example, the concentration of cholesterol sulfate in the co-incubation system can be 1-100 μM, optionally 1-50 μM. For example, the concentration of cholesterol sulfate can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25, 27, 30, 33, 35, 37, 40, 42, 44, 47, 50, 52, 55, 57, 60, 65, 70, 75, 80, 85, 90, 95, or 100 μM.

[0100] In some embodiments, the cell electroporation pretreatment reagent in step 1) further comprises reduced glutathione. In some embodiments, the concentration of reduced glutathione can be any suitable concentration, as long as it does not produce any significant side effects on the state of the cells. For example, the concentration of reduced glutathione can be 1-50 mM, optionally 1-25 mM. For example, the concentration of reduced glutathione in the co-incubation system can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mM.

[0101] In some embodiments, the co-incubation temperature in step 1) can be any suitable temperature, as long as it does not affect the state of the cells or the effect of the cell electroporation pretreatment reagent on the cells. In some embodiments, the co-incubation temperature can be 25-42°C, optionally 30-37°C. For example, the co-incubation temperature can be 25, 26, 27, 28, 29, 30, 31, 32, 33, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42°C.

[0102] In some embodiments, the CO2 concentration used for co-incubation in step 1) can be any suitable concentration, as long as it does not affect the state of the cells or excessively alter the pH of the co-incubation system. In some embodiments, the CO2 concentration used for co-incubation can be 1-10 v / v%, optionally 1-5 v / v%. In some embodiments, the CO2 concentration used for co-incubation can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 v / v%.

[0103] In some embodiments, the co-incubation time in step 1) can be any suitable duration, as long as it does not affect the state of the cells or the effect of the pretreatment reagents on the cells. In some embodiments, the co-incubation time can be 1-20 hours, optionally 2-20 hours. In some embodiments, the co-incubation time can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 hours.

[0104] In some embodiments, the cells to be electroporated in step 1) can be resuspended in any suitable culture medium, which may contain any suitable basal medium, as long as the basal medium can provide sufficient nutrition for the cells to maintain their growth and preserve their good biological state before electroporation. In some embodiments, the basal medium may be selected from... CTS TM Serum-free cell culture medium, DMEM medium, RPMI 1640 medium, and X-VIVO 15, or any one or more of these.

[0105] In some embodiments, the cell culture medium may further contain serum. The serum may be any suitable animal-derived serum or human serum, as long as it can maintain and / or activate the survival and proliferation of the cells. In some embodiments, the serum is calf serum or fetal bovine serum. In some embodiments, the serum is human AB serum. In some embodiments, the serum may be present in any proportion in the culture medium, as long as it can maintain and / or activate the survival and proliferation of the cells without having a side effect on the cell state. In some embodiments, the serum may be present in a proportion of 1-10 v / v% in the culture medium, optionally 1-5 v / v%. In some embodiments, the serum may be present in a proportion of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 v / v% in the culture medium.

[0106] In some embodiments, the cell culture medium may further contain cytokines. The cytokines may be any suitable cytokines, as long as they can maintain and / or stimulate the survival, activation, and / or proliferation of the cells in the culture medium. In some embodiments, the cytokines may be any one or more selected from IL-2, IL-7, IL-12, IL-15, IL-21, TNF-α, and IFN-γ.

[0107] In some embodiments, the concentration of the cytokine in the culture medium can be any suitable concentration, as long as it can maintain and / or activate the survival and / or proliferation of the cells without producing any side effects on the state of the cells. In some embodiments, the cytokine includes IL-2, the concentration of which in the culture medium can be 100-5000 IU / mL, preferably 200-3000 IU / mL. In some embodiments, the concentration of IL-2 in the culture medium can be 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, or 3000 IU / mL.

[0108] In some embodiments, the culture medium comprises basal medium X-VIVO 15 and IL-2 at 500 IU / mL.

[0109] In some embodiments, the culture includes basal culture medium X-VIVO 15, 500 IU / mL IL-2, 20 ng / mL IL-7, and 25 ng / mL IL-21.

[0110] In some embodiments, the culture medium includes a basal culture medium. CTS TM Serum-free cell culture medium, 2000 IU / mL IL-2, 50 ng / mL IL-7 and 50 ng / mL IL-15.

[0111] In some embodiments, the culture medium comprises basal medium X-VIVO 15, 1000 IU / mL IL-2, 25 ng / mL IL-7, 30 ng / mL IL-15, 10 pg / mL TNF-α, and 1000 IU / mL IFN-γ.

[0112] In some embodiments, the culture medium includes basal DMEM medium, 500 IU / mL IL-2, 100 ng / mL IL-7, 50 ng / mL IL-12, 25 ng / mL IL-21, 20 pg / mL TNF-α, and 1000 IU / mL IFN-γ.

[0113] In some embodiments, the cell density of the cells to be electroporated resuspended in the cell culture medium in step 1) can be any suitable cell density, as long as this cell density value does not produce any significant side effects on the pretreatment of the cells to be electroporated with the cell pretreatment reagent. That is, below this cell density, after the cells to be electroporated are treated with the cell pretreatment reagent, the cells to be electroporated are in a state that is readily accepting exogenous molecules such as DNA or RNA and do not show a significant decrease in cell morphology, cell viability, and other key biological indicators. In some embodiments, the cell density of the cells to be electroporated resuspended in the cell culture medium is 1 × 10⁻⁶. 6 -1×10 7 / mL, optionally 2×10 6 -8×10 6 / mL. In some embodiments, the cell density of the cells to be electroporated, resuspended in cell culture medium, can be 1×10⁻⁶ cells / mL. 6 2×10 6 3×10 6 4×10 6 5×10 6 6×10 6 7×10 6 8×10 6 9×10 6 Or 1×10 7 / mL.

[0114] In some embodiments, the cell separation in step 1) refers to the process of separating cells pretreated with the cell electroporation pretreatment reagent from the cell electroporation pretreatment reagent and the culture medium that may be used to resuspend the cells to be electroporated. After the cell separation operation, the cells pretreated with the cell electroporation pretreatment reagent are no longer in contact with the cell electroporation pretreatment reagent or the cell culture medium to minimize their influence on the subsequent electroporation process. In some embodiments, the cell separation is performed by centrifugation. In some embodiments, the centrifugation speed can be any suitable speed, as long as the cells can be well separated from the cell electroporation pretreatment reagent and the cell culture medium at that speed and the cell viability is not significantly affected. In some embodiments, the centrifugation speed is 100-1000g, optionally 300-1000g. In some embodiments, the centrifugation speed is 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000g. In some embodiments, the centrifugation time can be any suitable duration, as long as this duration, combined with the centrifugation speed, allows for effective separation of the cells from the pretreatment reagents for electroporation and the cell culture medium. In some embodiments, the centrifugation time can be 1-20 minutes, optionally 3-8 minutes. In some embodiments, the centrifugation time can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes.

[0115] In some embodiments, the electroporation conditions in step 2) can be any different electroporation conditions suitable for different cell types, as long as the electroporation conditions match the specific cell type to be electroporated. In some embodiments, the electroporation conditions are suitable for the electroporation of immune effector cells, optionally suitable for one or more of T cells, TILs, NK cells, NKT cells, CAR-T cells, CIK cells, CTLs, TCR-Ts, LAKs, DCs, and γδT cells. In some embodiments, the electroporation conditions are suitable for the electroporation of T cells or TILs. In some embodiments, the electroporation conditions are any one of the Lonza Nucleofector series electroporators T-020, T023, V-024, U-014, and U-017.

[0116] In some embodiments, the vector for the exogenous gene in step 2) can be any suitable vector, as long as it can carry the exogenous gene and transport it into the cell. In some embodiments, the vector can be a viral vector or a non-viral vector, optionally a non-viral vector. In some embodiments, the vector can be a DNA vector or an RNA vector, optionally a DNA vector. In some embodiments, the DNA vector can be a double-stranded DNA vector, optionally a non-viral DNA vector, such as a non-viral DNA vector with integration capability or a non-viral DNA vector that is free from genomic DNA and lacks integration capability. In some embodiments, the non-viral DNA vector with integration capability can be a DNA vector based on a transposon system, such as a DNA vector based on the Sleeping Beauty transposon system or the PiggyBac transposon system. In some embodiments, the DNA vector can be a single-stranded DNA vector, preferably a circular single-stranded DNA (cssDNA) vector. In some embodiments, the cssDNA can be cssDNA with genome integration capability or cssDNA without genome integration capability. In some embodiments, the CSSDNA vector with genome integration capability contains a sense strand or an antisense strand of the foreign gene coding sequence. Preferably, the 5' and 3' ends of the foreign gene coding sequence in the CSSDNA vector with genome integration capability further contain sequence pairs homologous to sequences near the genome integration site. These sequence pairs are capable of mediating the integration of the foreign gene coding sequence in the CSSDNA vector into a specific site in the genome.

[0117] In some embodiments, the method further includes: 3) continuing to culture the cells in 2) in which the exogenous gene was introduced via electroporation.

[0118] In some embodiments, the culture in step 3) is performed in a cell culture medium. The cell culture medium may contain any suitable basal medium, as long as it provides sufficient nutrition to sustain the cells' growth and maintain their good biological state after electroporation. In some embodiments, the basal medium may be selected from... CTS TM Serum-free cell culture medium, DMEM medium, RPMI 1640 medium, and X-VIVO 15, or any one or more of these.

[0119] In some embodiments, the cell culture medium may further contain serum. The serum may be any suitable animal-derived serum or human serum, as long as it can maintain and / or activate the survival and proliferation of the cells. In some embodiments, the serum is calf serum or fetal bovine serum. In some embodiments, the serum is human AB serum. In some embodiments, the serum may be present in any proportion in the culture medium, as long as it can maintain and / or activate the survival and proliferation of the cells without having a side effect on the cell state. In some embodiments, the serum may be present in a proportion of 1-10 v / v% in the culture medium, optionally 1-5 v / v%. In some embodiments, the serum may be present in a proportion of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 v / v% in the culture medium.

[0120] In some embodiments, the cell culture medium may further contain cytokines. The cytokines may be any suitable cytokines, as long as they can maintain and / or stimulate the survival, activation, and / or proliferation of the cells in the culture medium. In some embodiments, the cytokines may be any one or more selected from IL-2, IL-7, IL-12, IL-15, IL-21, TNF-α, and IFN-γ.

[0121] In some embodiments, the concentration of the cytokine in the culture medium can be any suitable concentration, as long as it can maintain and / or activate the survival and / or proliferation of the cells without producing any side effects on the state of the cells. In some embodiments, the cytokine includes IL-2, the concentration of which in the culture medium can be 100-5000 IU / mL, preferably 200-3000 IU / mL. In some embodiments, the concentration of IL-2 in the culture medium can be 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, or 3000 IU / mL. In some embodiments, the cytokine includes IL-7, which has a concentration in the culture medium of 1-100 ng / mL, preferably 5-50 ng / mL. In some embodiments, the concentration of IL-7 in the culture medium can be 5, 6, 7, 8, 7, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 ng / mL. In some embodiments, the cytokine includes IL-12, which has a concentration in the culture medium of 1-100 ng / mL, preferably 5-50 ng / mL. In some embodiments, the concentration of IL-12 in the culture medium may be 5, 6, 7, 8, 7, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 ng / mL. In some embodiments, the cytokine includes IL-15, the concentration of which in the culture medium may be 1-100 ng / mL, preferably 5-50 ng / mL. In some embodiments, the concentration of IL-15 in the culture medium may be 5, 6, 7, 8, 7, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 ng / mL.In some embodiments, the cytokine includes IL-21, which has a concentration in the culture medium of 1-100 ng / mL, preferably 5-50 ng / mL. In some embodiments, the concentration of IL-21 in the culture medium can be 5, 6, 7, 8, 7, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 ng / mL. In some embodiments, the cytokine includes TNF-α, which has a concentration in the culture medium of 1-100 pg / mL, preferably 2-50 pg / mL. In some embodiments, the concentration of TNF-α in the culture medium may be 2, 3, 4, 5, 6, 7, 8, 7, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 ng / mL. In some embodiments, the cytokine includes IFN-γ, the concentration of which in the culture medium may be 50-5000 IU / mL, preferably 100-3000 IU / mL. In some embodiments, the concentration of IFN-γ in the culture medium can be 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, or 3000 IU / mL.

[0122] In some embodiments, the cell culture medium in step 3) may further contain a reducing agent. In some embodiments, the reducing agent is reduced glutathione (GSSH). In some embodiments, the concentration of reduced glutathione in the cell culture medium can be any suitable concentration, as long as it does not have a negative effect on the viability and state of the cells through electroporation of the exogenous gene. In some embodiments, the concentration of reduced glutathione can be 1-100 mM, optionally 1-50 mM. In some embodiments, the concentration of the reduced glutathione can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 mM.

[0123] In some embodiments, the culture medium comprises basal medium X-VIVO 15 and IL-2 at 500 IU / mL.

[0124] In some embodiments, the culture medium comprises basal medium X-VIVO 15, 500 IU / mL IL-2 and 5 mM reduced glutathione.

[0125] In some embodiments, the culture medium comprises basal medium X-VIVO 15, 500 IU / mL IL-2 and 10 mM reduced glutathione.

[0126] In some embodiments, the culture medium comprises basal medium X-VIVO 15, 500 IU / mL IL-2 and 25 mM reduced glutathione.

[0127] In some embodiments, the culture includes basal culture medium X-VIVO 15, 500 IU / mL IL-2, 20 ng / mL IL-7, and 25 ng / mL IL-21.

[0128] In some embodiments, the culture medium includes a basal culture medium. CTS TM Serum-free cell culture medium, 2000 IU / mL IL-2, 50 ng / mL IL-7 and 50 ng / mL IL-15.

[0129] In some embodiments, the culture medium comprises basal medium X-VIVO 15, 1000 IU / mL IL-2, 25 ng / mL IL-7, 30 ng / mL IL-15, 10 pg / mL TNF-α, and 1000 IU / mL IFN-γ.

[0130] In some embodiments, the culture medium includes basal DMEM medium, 500 IU / mL IL-2, 100 ng / mL IL-7, 50 ng / mL IL-12, 25 ng / mL IL-21, 20 pg / mL TNF-α, and 1000 IU / mL IFN-γ.

[0131] In some embodiments, the culture temperature in step 3) can be any suitable temperature, as long as it does not affect the state of the cells or the effect of the pretreatment reagents on the cells. In some embodiments, the culture temperature can be 25-42°C, optionally 30-37°C. For example, the culture temperature can be 25, 26, 27, 28, 29, 30, 31, 32, 33, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42°C.

[0132] In some embodiments, the CO2 concentration used in step 3) can be any suitable concentration, as long as it does not affect the state of the cells or drastically alter the pH of the cell culture medium. In some embodiments, the CO2 concentration used in the culture can be 1-10 v / v%, optionally 1-5 v / v%. In some embodiments, the CO2 concentration used in the culture can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 v / v%.

[0133] In some embodiments, the culture time in step 3) can be any suitable duration, as long as it does not affect the state of the cells or the effect of the pretreatment reagents on the cells. In some embodiments, the culture time can be 2 hours to 30 days, optionally 2 hours to 15 days. In some embodiments, the culture time can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. In some embodiments, the culture time can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days.

[0134] In some embodiments, the initial cell density for culture in step 3) can be any suitable cell density, as long as it does not negatively affect the viability and growth status of the cells after electroporation. In some embodiments, the cell density in step 3) is 1 × 10⁻⁶. 6 -1×10 7 / mL; optionally, 2×10 6 -8×10 6 / mL. In some embodiments, the cell density can be 1×10⁶. 6 2×10 6 3×10 6 4×10 6 5×10 6 6×10 6 7×10 6 8×10 6 9×10 6 Or 1×10 7 / mL.

[0135] In some embodiments, the method includes the following steps: 1) resuspending peripheral blood T cells in X-VIVO15 medium containing 500 IU / mL IL-2 until the cell density is 4 × 10⁻⁶ cells / mL. 6 / mL, add cholesterol sulfate to a final concentration of 10μM, incubate at 30℃ for 20h, centrifuge at 300g for 8 minutes, wash the cell pellet with physiological saline and resuspend to obtain pretreated T cells; 2) Select the T-020 program of the Lonza Nucleofector electroporator to introduce a transposon-based non-viral vector containing the exogenous gene into the pretreated T cells in step 1), to obtain T cells overexpressing the exogenous gene. Optionally, the method further includes: 3) culturing the T cells overexpressing the exogenous gene in step 2) in X-VIVO 15 medium containing 500 IU / mL IL-2 at 37℃ and 5v / v% CO2.

[0136] In some embodiments, the method includes the following steps: 1) resuspending peripheral blood T cells in X-VIVO15 medium containing 500 IU / mL IL-2 until the cell density is 1×10⁻⁶ cells / mL. 6 / mL, add cholesterol sulfate to a final concentration of 5μM, incubate at 37℃ for 8h, centrifuge at 300g for 8 minutes, wash the cell pellet with physiological saline and resuspend to obtain pretreated T cells; 2) Select the U-014 program of the Lonza Nucleofector electroporator to introduce a transposon-based non-viral vector containing the exogenous gene into the pretreated T cells in step 1), to obtain T cells overexpressing the exogenous gene. Optionally, the method further includes: 3) culturing the T cells overexpressing the exogenous gene in step 2) in X-VIVO 15 medium containing 500 IU / mL IL-2 and 5mM reduced glutathione at 37℃ and 5v / v% CO2.

[0137] In some embodiments, the method includes the following steps: 1) resuspending peripheral blood T cells in X-VIVO15 medium containing 500 IU / mL IL-2 until the cell density is 2 × 10⁻⁶ cells / mL. 6 / mL, add cholesterol sulfate to a final concentration of 10μM and reduced glutathione to a final concentration of 10mM, incubate at 30℃ for 16h, centrifuge at 300g for 8 minutes, wash the cell pellet with physiological saline and resuspend to obtain pretreated T cells; 2) Select the T-020 program of the Lonza Nucleofector electroporator to introduce a transposon-based non-viral vector containing the exogenous gene into the pretreated T cells in step 1), to obtain T cells overexpressing the exogenous gene. Optionally, the method further includes: 3) culturing the T cells overexpressing the exogenous gene in step 2) in X-VIVO 15 medium containing 500 IU / mL IL-2 at 37℃ and 5v / v% CO2.

[0138] In some embodiments, the method includes the following steps: 1) resuspending peripheral blood T cells in X-VIVO15 medium containing 500 IU / mL IL-2 until the cell density is 5 × 10⁻⁶ cells / mL. 6 / mL, add cholesterol sulfate to a final concentration of 50μM, incubate at 30℃ for 20h, centrifuge at 300g for 8 minutes, wash the cell pellet with physiological saline and resuspend to obtain pretreated T cells; 2) Select the T-020 program of the Lonza Nucleofector electroporator to introduce a transposon-based non-viral vector containing the exogenous gene into the pretreated T cells in step 1), to obtain T cells overexpressing the exogenous gene. Optionally, the method further includes: 3) culturing the T cells overexpressing the exogenous gene in step 2) in X-VIVO 15 medium containing 500 IU / mL IL-2 and 10mM reduced glutathione at 37℃ and 5v / v% CO2.

[0139] In some embodiments, the method includes the following steps: 1) resuspending peripheral blood T cells in X-VIVO15 medium containing 500 IU / mL IL-2 until the cell density is 6 × 10⁻⁶. 6 / mL, add cholesterol sulfate to a final concentration of 100μM, incubate at 37℃ for 4h, centrifuge at 300g for 8 minutes, wash the cell pellet with physiological saline and resuspend to obtain pretreated T cells; 2) Select the T-020 program of the Lonza Nucleofector electroporator to introduce a transposon-based non-viral vector containing the exogenous gene into the pretreated T cells in step 1), to obtain T cells overexpressing the exogenous gene. Optionally, the method further includes: 3) culturing the T cells overexpressing the exogenous gene in step 2) in X-VIVO 15 medium containing 500 IU / mL IL-2 and 25mM reduced glutathione at 37℃ and 5v / v% CO2.

[0140] In some embodiments, the method includes the following steps: 1) resuspending TILs in X-VIVO15 medium containing 500 IU / mL IL-2 until the cell density is 3 × 10⁻⁶ cells / mL. 6 / mL, add cholesterol sulfate to a final concentration of 50μM, incubate at 30℃ for 20h, centrifuge at 300g for 8 minutes, wash the cell pellet with physiological saline and resuspend to obtain pretreated T cells; 2) Select the T-020 program of the Lonza Nucleofector electroporator to introduce a transposon-based non-viral vector containing the exogenous gene into the pretreated T cells in step 1), to obtain T cells overexpressing the exogenous gene. Optionally, the method further includes: 3) culturing the T cells overexpressing the exogenous gene in step 2) in X-VIVO 15 medium containing 500 IU / mL IL-2 at 37℃ and 5v / v% CO2.

[0141] In some embodiments, the method includes the following steps: 1) resuspending TILs in X-VIVO15 medium containing 500 IU / mL IL-2 until the cell density is 4 × 10⁻⁶ cells / mL. 6 / mL, add cholesterol sulfate to a final concentration of 1μM, incubate at 30℃ for 16h, centrifuge at 300g for 8 minutes, wash the cell pellet with physiological saline and resuspend to obtain pretreated T cells; 2) Select the T-020 program of the Lonza Nucleofector electroporator to introduce a transposon-based non-viral vector containing the exogenous gene into the pretreated T cells in step 1), to obtain T cells overexpressing the exogenous gene. Optionally, the method further includes: 3) culturing the T cells overexpressing the exogenous gene in step 2) in X-VIVO 15 medium containing 500 IU / mL IL-2 and 5mM reduced glutathione at 37℃ and 5v / v% CO2.

[0142] In some embodiments, the method includes the following steps: 1) resuspending peripheral blood T cells in X-VIVO15 medium containing 500 IU / mL IL-2 until the cell density is 8 × 10⁻⁶ cells / mL. 6 / mL, add cholesterol sulfate to a final concentration of 50μM and reduced glutathione to a final concentration of 10mM, incubate at 30℃ for 12h, centrifuge at 300g for 8 minutes, wash the cell pellet with physiological saline and resuspend to obtain pretreated T cells; 2) Select the T-020 program of the Lonza Nucleofector electroporator to introduce a transposon-based non-viral vector containing the exogenous gene into the pretreated T cells in step 1), to obtain T cells overexpressing the exogenous gene. Optionally, the method further includes: 3) culturing the T cells overexpressing the exogenous gene in step 2) in X-VIVO15 medium containing 500IU / mL IL-2 and 25mM reduced glutathione at 37℃ and 5v / v% CO2.

[0143] In some embodiments, the method includes the following steps: 1) resuspending peripheral blood T cells in X-VIVO15 medium containing 500 IU / mL IL-2 until the cell density is 9 × 10⁻⁶ cells / mL. 6 / mL, add cholesterol sulfate to a final concentration of 25μM and reduced glutathione to a final concentration of 25mM, incubate at 37℃ for 8h, centrifuge at 300g for 8 minutes, wash the cell pellet with physiological saline and resuspend to obtain pretreated T cells; 2) Select the T-020 program of the Lonza Nucleofector electroporator to introduce a transposon-based non-viral vector containing the exogenous gene into the pretreated T cells in step 1), to obtain T cells overexpressing the exogenous gene. Optionally, the method further includes: 3) culturing the T cells overexpressing the exogenous gene in step 2) in X-VIVO 15 medium containing 500 IU / mL IL-2 and 25mM reduced glutathione at 37℃ and 5v / v% CO2.

[0144] In some embodiments, the method includes the following steps: 1) resuspending peripheral blood T cells in X-VIVO15 medium containing 500 IU / mL IL-2 until the cell density is 1×10⁻⁶ cells / mL. 7 / mL, add cholesterol sulfate to a final concentration of 10μM, incubate at 30℃ for 20h, centrifuge at 300g for 8 minutes, wash the cell pellet with physiological saline and resuspend to obtain pretreated T cells; 2) Select the T-020 program of the Lonza Nucleofector electroporator to introduce a transposon-based non-viral vector containing the exogenous gene into the pretreated T cells in step 1), to obtain T cells overexpressing the exogenous gene. Optionally, the method further includes: 3) culturing the T cells overexpressing the exogenous gene in step 2) in X-VIVO 15 medium containing 500 IU / mL IL-2 and 10mM reduced glutathione at 37℃ and 5v / v% CO2.

[0145] Another aspect of the present invention provides a cell electroporation kit, which includes a cell electroporation medium component and a cell treatment component; the cell treatment component includes a cholesterol derivative.

[0146] In this invention, the main function of the cell electroporation medium component in the cell electroporation kit is to mediate the formation of an electric field around the cell when a transient high voltage is applied, causing temporary pores to form on the cell membrane surface. To achieve this effect, the cell electroporation medium component needs to contain cations and / or anions. In some embodiments, the cell electroporation medium component includes a cell electroporation buffer. In some embodiments, the cell electroporation buffer includes any suitable anions and cations, as long as the anions and cations can normally mediate the formation of an electric field around the cell and mediate a potential difference across the cell membrane. In some embodiments, the potential difference across the cell membrane mediated by the anions and / or cations can form temporary pores on the cell membrane surface, and this process is reversible, i.e., the cell membrane can return to normal after the high voltage applied to the cell is removed. In some embodiments, the cell electroporation buffer contains anions including Cl... - SO4 2- PO4 3- HPO4 2- H2PO4 - HEPES and NO3 - Any one or more of the following. In some embodiments, the cell electroporation buffer contains cations including Na+. + K + Ca 2+ Mg 2+ and Zn 2+ Any one or more of them.

[0147] In some embodiments, the cell electroporation buffer can be any suitable homemade electroporation buffer or any suitable commercially available electroporation buffer, as long as it can achieve the aforementioned effects on cells under instantaneous high voltage. In some embodiments, the cell electroporation buffer can be a commercially available electroporation buffer that can be used with its respective brand of electroporator and the conditions and electroporation programs matched to different cell types within that brand of electroporator. In some embodiments, the commercially available electroporation buffer can be used across brands with other brands of electroporators and the corresponding electroporation programs within that brand of electroporator. In some embodiments, the commercially available cell electroporation buffer can be selected from Lonza human T cells. Solution ( Solution for Human T Cells), Maxcyte Electroporation Buffer, ThermoFisher CTS TM Xenon TM Electroporation buffer (CTS) TM Xenon TM Electroporation Buffer), Miltenyi Electroporation buffer ( One or more of the following: Electroporation Buffer, BTXpress Electroporation Solution, and Bio-rad Gene Pulser Electroporation Buffer.

[0148] In this invention, the cholesterol derivative may include cholesterol sulfate.

[0149] In this invention, the cell treatment component further includes a reducing agent. In some embodiments, the reducing agent may include reduced glutathione.

[0150] In some embodiments, the cholesterol derivative and the reducing agent may be present in the cell electroporation kit at any suitable dosage ratio, as long as the ratio of the two can be combined with the other components of the kit and the corresponding electroporation program to complete the electroporation of cells. In some embodiments, the molar ratio of the cholesterol derivative to the reducing agent included in the cell electroporation kit is 1:100000 to 100000:1.

[0151] In some embodiments, the cell electroporation kit may include the inherent components of a commercially available cell electroporation kit, the cholesterol derivative, and the reducing agent.

[0152] In some embodiments, the inherent components of the mature, commercially available electroporation kits in the cell electroporation kit may be inherent components of Lonza, Maxcyte, ThermoFisher, Miltenyi, BTX, and Bio-rad-related electroporation kits.

[0153] In some embodiments, the cell electroporation kit comprises: Lonza human T cells The solution includes Lonza electroporation supplement and cholesterol sulfate. In some embodiments, the cell electroporation kit also includes reduced glutathione.

[0154] In some embodiments, the cell electroporation kit includes Maxcyte electroporation buffer and cholesterol sulfate. In some embodiments, the cell electroporation kit also includes reduced glutathione.

[0155] In some embodiments, the cell electroporation kit includes: ThermoFisher CTS TM Xenon TM Electroporation buffer (CTS) TM Xenon TM Electroporation Buffer and cholesterol sulfate. In some embodiments, the cell electroporation kit also includes reduced glutathione.

[0156] In some embodiments, the cell electroporation kit includes: Miltenyi Electroporation buffer ( Electroporation Buffer and cholesterol sulfate. In some embodiments, the cell electroporation kit also includes reduced glutathione.

[0157] In some embodiments, the cell electroporation kit includes BTXpress electroporation solution and cholesterol sulfate. In some embodiments, the cell electroporation kit also includes reduced glutathione.

[0158] In some embodiments, the cell electroporation kit includes Bio-rad Gene Pulser Electroporation Buffer and cholesterol sulfate. In some embodiments, the cell electroporation kit also includes reduced glutathione.

[0159] The pKB20-EGFP used in the following examples is the one described in Example 1 of WO2022078310A1, the entire contents of which are incorporated herein by reference.

[0160] The small molecule compounds cholesterol sulfate and reduced glutathione used in the following examples were purchased from MCE. All other reagents used were available through conventional commercial channels.

[0161] Example 1: Preparation of human activated T cells

[0162] Coat six-well plates 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 removing the coating solution, wash the plates 1-3 times with physiological saline. Add X-VIVO 15 medium containing 5% human AB serum for later use. Resuscitate human peripheral blood PBMCs (purchased from ALLCELLS) at 37°C and culture them in an adherent environment for 2-4 hours. The non-adherent suspension cells are the initial T cells. Collect the suspension cells into 15ml centrifuge tubes, centrifuge at 1200 rpm for 3 minutes, discard the supernatant, add physiological saline, centrifuge at 1200 rpm for 3 minutes, discard the physiological saline, and repeat this step. Then transfer the washed initial T cells to antibody-coated wells containing the culture medium to be used, and culture at 37°C and 5% CO2 for 3-4 days before proceeding with subsequent experiments.

[0163] Example 2: Preparation of TIL seed cells from solid tumor tissue

[0164] TIL seed cells for melanoma were prepared using the following method:

[0165] 1) Prepare physiological saline containing penicillin at a final concentration of 100 U / mL, streptomycin at a final concentration of 100 μg / mL, and gentamicin at a final concentration of 50 μg / mL for later use;

[0166] 2) In a sterile environment in a biosafety cabinet, the freshly isolated tumor tissue samples from the tumor patients were washed in a 10cm culture dish containing 30mL of physiological saline prepared in step 1), and then transferred to a new 10cm culture dish containing 30mL of physiological saline prepared in step 1). This washing process was repeated 3 times.

[0167] 3) Using a sterile scalpel blade, remove adipose and necrotic tissue, and cut the tumor tissue into pieces with a diameter of 3×3×3mm. 3 Forty-two randomly selected tumor tissue blocks were placed in two G-REX100 culture jars (purchased from Wilsonwolf). Seed cell culture medium was added to each jar, and the components of the seed culture medium included: 3000 IU / mL IL-2, 20 ng / mL IL-7, 20 ng / mL IL-15, 500 U / mL GM-CSF, 1000 IU / mL IFN-γ, 3 μg / mL anti-CD137 mAb, 3 μg / mL anti-CD28 mAb, 3 μg / mL anti-PD-1 mAb, 10 ng / mL TNF-α, 5% v / v human AB serum, 1×PS double antibody, and X-VIVO 15 basal medium to the final volume. Excess tumor tissue blocks were cryopreserved in liquid nitrogen using a programmed freezing system with CryoStor10 cryopreservation solution (purchased from BioLifeSolutions).

[0168] 4) After adding 1L of the above seed cell culture medium to the G-REX100 culture vessel containing tumor tissue blocks in 3), the tumor tissue blocks were cultured at 37℃ and 5v / v%CO2. Every 4 days, half the volume of old seed cell culture medium was removed and half the volume of fresh seed cell culture medium was added. On the 12th day, the TIL seed cells were harvested by centrifugation and the total number of cells and the viability were counted.

[0169] Example 3: Electroporation preparation of T cells overexpressing EGFP

[0170] Electroporate the plasmid pKB20-EGFP, which expresses EGFP, into activated T cells using the following methods.

[0171] Method 1:

[0172] 1) The peripheral blood T cells obtained in Example 1 were resuspended in X-VIVO 15 medium containing 500 IU / mL IL-2 to a concentration of 4 × 10⁻⁶. 6 / mL, add cholesterol sulfate to a final concentration of 10μM, and incubate at 30℃ for 20h;

[0173] 2) Add 2 mL of X-VIVO 15 medium containing 500 IU / mL IL-2 to each well of a 12-well plate, and then transfer it to a cell culture incubator to preheat at 37°C and 5v / v% CO2 for 1 hour.

[0174] 3) Prepare the electro-spreading fluid ratio for each well using the following table:

[0175] 100 μL Nucleocuvette TM Strip (μL) Nucleofector TM volume of the solution 82 Electroporation replenishment solution 18

[0176] 4) Take the cell suspension processed in 1), centrifuge at 300g for 8 minutes, discard the supernatant, then resuspend the cells in physiological saline, and add the cells to EP tubes, adding 5×10⁶ cells to each EP tube. 6 For each cell, repeat the centrifugation process and wash the cell pellet twice with physiological saline.

[0177] 5) Add 5 μg of plasmid pKB20-EGFP to the electroporation buffer prepared in step 3), and then let it stand at room temperature for no more than 30 minutes;

[0178] 6) Resuspend the cell pellet from step 4) in the plasmid-containing electroporation buffer prepared in step 5), 100 μL per tube. Carefully transfer the cell resuspended buffer into a LONZA 100 μL electroporation cuvette and place the cuvette into a LONZA Nucleofector. TM Inside the 2b electro-spinning tank, start the electro-spinning program and select T-020;

[0179] 7) After electroporation, carefully remove the electroporation cup, aspirate the cell suspension and transfer it to EP tubes. Add 200 μL of preheated X-VIVO 15 medium to each tube, then transfer the solution to the wells in the 12-well plate from step 1) containing preheated 500 IU / mL IL-2X-VIVO 15 medium. Incubate at 37°C and 5 v / v % CO2. Replace half the medium with fresh medium every two days.

[0180] The procedure was repeated three times. After 5 days of culture, the number of cells and cell viability were measured using a cell counter, and the average value was taken. Simultaneously, a portion of the cells was subjected to flow cytometry to determine the percentage of EGFP-positive cells.

[0181] Method 2:

[0182] 1) The peripheral blood T cells obtained in Example 1 were resuspended in X-VIVO 15 medium containing 500 IU / mL IL-2 to a concentration of 1×10⁻⁶. 6 / mL, add cholesterol sulfate to a final concentration of 5μM, and incubate at 37℃ for 8h;

[0183] 2) Add 2 mL of X-VIVO 15 medium containing 500 IU / mL IL-2 to each well of a 12-well plate, and then transfer it to a cell culture incubator to preheat at 37°C and 5% CO2 for 1 hour.

[0184] 3) Prepare the electro-spreading fluid ratio for each well using the following table:

[0185] 100 μL Nucleocuvette TM Strip (μL)] Nucleofector TM volume of the solution 82 Electroporation replenishment solution 18

[0186] 4) Take the cell suspension processed in 1), centrifuge at 300g for 8 minutes, discard the supernatant, then resuspend the cells in physiological saline, and add the cells to EP tubes, adding 5×10⁶ cells to each EP tube.6 For each cell, repeat the centrifugation process and wash the cell pellet twice with physiological saline.

[0187] 5) Add 5 μg of plasmid pKB20-EGFP to the electroporation buffer prepared in step 3), and then let it stand at room temperature for no more than 30 minutes;

[0188] 6) Resuspend the cell pellet from step 4) in the plasmid-containing electroporation buffer prepared in step 5), 100 μL per tube. Carefully transfer the cell resuspended buffer into a LONZA 100 μL electroporation cuvette and place the cuvette into a LONZA Nucleofector. TM Inside the 2b electro-rotation tank, start the electro-rotation program and select program U-014;

[0189] 7) After electroporation, carefully remove the electroporation cup, aspirate the cell suspension and transfer it to EP tubes. Add 200 μL of preheated X-VIVO 15 medium to each tube, then transfer it to the wells of the 12-well plate from step 1) containing preheated 500 IU / mL IL-2X-VIVO 15 medium. Add reduced glutathione to a final concentration of 5 mM and incubate at 37°C and 5% CO2. Replace half the medium with fresh medium every two days.

[0190] The procedure was repeated three times. After 5 days of culture, the number of cells and cell viability were measured using a cell counter, and the average value was taken. Simultaneously, a portion of the cells was subjected to flow cytometry to determine the percentage of EGFP-positive cells.

[0191] Method 3:

[0192] 1) The peripheral blood T cells obtained in Example 1 were resuspended in X-VIVO 15 medium containing 500 IU / mL IL-2 to a concentration of 2×10⁻⁶. 6 / mL, add cholesterol sulfate to a final concentration of 10μM and reduced glutathione to a final concentration of 10mM, and incubate at 30℃ for 16h.

[0193] 2) Add 2 mL of X-VIVO 15 medium containing 500 IU / mL IL-2 to each well of a 12-well plate, and then transfer it to a cell culture incubator to preheat at 37°C and 5% CO2 for 1 hour.

[0194] 3) Prepare the electro-spreading fluid ratio for each well using the following table:

[0195] 100 μL Nucleocuvette TM Strip (μL) Nucleofector TM volume of the solution 82 Electroporation replenishment solution 18

[0196] 4) Take the cell suspension processed in 1), centrifuge at 300g for 8 minutes, discard the supernatant, then resuspend the cells in physiological saline, and add the cells to EP tubes, adding 5×10⁶ cells to each EP tube. 6For each cell, repeat the centrifugation process and wash the cell pellet twice with physiological saline.

[0197] 5) Add 5 μg of plasmid pKB20-EGFP to the electroporation buffer prepared in step 3), and then let it stand at room temperature for no more than 30 minutes;

[0198] 6) Resuspend the cell pellet from step 4) in the plasmid-containing electroporation buffer prepared in step 5), 100 μL per tube. Carefully transfer the cell resuspended buffer into a LONZA 100 μL electroporation cuvette and place the cuvette into a LONZA Nucleofector. TM Inside the 2b electro-spinning tank, start the electro-spinning program and select T-020;

[0199] 7) After electroporation, carefully remove the electroporation cup, aspirate the cell suspension and transfer it to EP tubes. Add 200 μL of preheated X-VIVO 15 medium to each tube, then transfer it to the wells of the 12-well plate from step 1) containing preheated 500 IU / mL IL-2X-VIVO 15 medium. Incubate at 37°C and 5% CO2. Replace half the medium with fresh medium every two days.

[0200] The procedure was repeated three times. After 5 days of culture, the number of cells and cell viability were measured using a cell counter, and the average value was taken. Simultaneously, a portion of the cells was subjected to flow cytometry to determine the percentage of EGFP-positive cells.

[0201] Method 4:

[0202] 1) The peripheral blood T cells obtained in Example 1 were resuspended in X-VIVO 15 medium containing 500 IU / mL IL-2 to a concentration of 5 × 10⁻⁶. 6 / mL, add cholesterol sulfate to a final concentration of 50μM, and incubate at 30℃ for 20h;

[0203] 2) Add 2 mL of X-VIVO 15 medium containing 500 IU / mL IL-2 to each well of a 12-well plate, and then transfer it to a cell culture incubator to preheat at 37°C and 5% CO2 for 1 hour.

[0204] 3) Prepare the electro-spreading fluid ratio for each well using the following table:

[0205] 100 μL Nucleocuvette TM Strip (μL)] Nucleofector TM volume of the solution 82 Electroporation replenishment solution 18

[0206] 4) Take the cell suspension processed in 1), centrifuge at 300g for 8 minutes, discard the supernatant, then resuspend the cells in physiological saline, and add the cells to EP tubes, adding 5×10⁶ cells to each EP tube. 6 For each cell, repeat the centrifugation process and wash the cell pellet twice with physiological saline.

[0207] 5) Add 5 μg of plasmid pKB20-EGFP to the electroporation buffer prepared in step 3), and then let it stand at room temperature for no more than 30 minutes;

[0208] 6) Resuspend the cell pellet from step 4) in the plasmid-containing electroporation buffer prepared in step 5), 100 μL per tube. Carefully transfer the cell resuspended buffer into a LONZA 100 μL electroporation cuvette and place the cuvette into a LONZA Nucleofector. TM Inside the 2b electro-spinning tank, start the electro-spinning program and select T-020;

[0209] 7) After electroporation, carefully remove the electroporation cup, aspirate the cell suspension and transfer it to EP tubes. Add 200 μL of preheated X-VIVO 15 medium to each tube, then transfer it to the wells of the 12-well plate from step 1) containing preheated 500 IU / mL IL-2X-VIVO 15 medium. Add reduced glutathione to a final concentration of 10 mM and incubate at 37°C and 5% CO2. Replace half the medium with fresh medium every two days.

[0210] The procedure was repeated three times. After 5 days of culture, the number of cells and cell viability were measured using a cell counter, and the average value was taken. Simultaneously, a portion of the cells was subjected to flow cytometry to determine the percentage of EGFP-positive cells.

[0211] Method 5:

[0212] 1) The peripheral blood T cells obtained in Example 1 were resuspended in X-VIVO 15 medium containing 500 IU / mL IL-2 to a concentration of 6 × 10⁻⁶. 6 / mL, add cholesterol sulfate to a final concentration of 100μM, and incubate at 37℃ for 4h;

[0213] 2) Add 2 mL of X-VIVO 15 medium containing 500 IU / mL IL-2 to each well of a 12-well plate, and then transfer it to a cell culture incubator to preheat at 37°C and 5% CO2 for 1 hour.

[0214] 3) Prepare the electro-spreading fluid ratio for each well using the following table:

[0215] 100 μL Nucleocuvette TM Strip (μL) Nucleofector TM volume of the solution 82 Electroporation replenishment solution 18

[0216] 4) Take the cell suspension processed in 1), centrifuge at 300g for 8 minutes, discard the supernatant, then resuspend the cells in physiological saline, and add the cells to EP tubes, adding 5×10⁶ cells to each EP tube. 6 For each cell, repeat the centrifugation process and wash the cell pellet twice with physiological saline.

[0217] 5) Add 5 μg of plasmid pKB20-EGFP to the electroporation buffer prepared in step 3), and then let it stand at room temperature for no more than 30 minutes;

[0218] 6) Resuspend the cell pellet from step 4) in the plasmid-containing electroporation buffer prepared in step 5), 100 μL per tube. Carefully transfer the cell resuspended buffer into a LONZA 100 μL electroporation cuvette and place the cuvette into a LONZA Nucleofector. TM Inside the 2b electro-spinning tank, start the electro-spinning program and select T-020;

[0219] 7) After electroporation, carefully remove the electroporation cup, aspirate the cell suspension and transfer it to EP tubes. Add 200 μL of preheated X-VIVO 15 medium to each tube, then transfer it to the wells of the 12-well plate from step 1) containing preheated 500 IU / mL IL-2X-VIVO 15 medium. Add reduced glutathione to a final concentration of 25 mM and incubate at 37°C and 5% CO2. Replace half the medium with fresh medium every two days.

[0220] The procedure was repeated three times. After 5 days of culture, the number of cells and cell viability were measured using a cell counter, and the average value was taken. Simultaneously, a portion of the cells was subjected to flow cytometry to determine the percentage of EGFP-positive cells.

[0221] Compare with method 1:

[0222] 1) Add 2 mL of X-VIVO 15 medium containing 500 IU / mL IL-2 to each well of a 12-well plate, and then transfer it to a cell culture incubator to preheat at 37°C and 5% CO2 for 1 hour.

[0223] 2) Prepare the electro-spreading fluid ratio for each well using the following table:

[0224] 100 μL Nucleocuvette TM Strip (μL) <![CDATA[Nucleofector TM Volume of solution 82 Electroporation replenishment solution 18

[0225] 3) Take the peripheral blood T cell suspension to be electroporated, centrifuge at 300g for 8 minutes, discard the supernatant, then resuspend the cells in physiological saline, and add the cells to EP tubes, adding 5×10⁻⁶ cells to each EP tube. 6 One peripheral blood T cell was collected, and the centrifugation process was repeated. The cell pellet was then washed twice with physiological saline.

[0226] 4) Add 5 μg of plasmid pKB20-EGFP to the electroporation buffer prepared in step 2), and then let it stand at room temperature for no more than 30 minutes;

[0227] 5) Resuspend the cell pellet from step 4) in the plasmid-containing electroporation buffer prepared in step 4), 100 μL per tube. Carefully transfer the cell resuspended solution into a LONZA 100 μL electroporation cuvette and place the cuvette into a LONZA Nucleofector. TM Inside the 2b electro-spinning tank, start the electro-spinning program and select T-020;

[0228] 6) After electroporation, carefully remove the electroporation cup, aspirate the cell suspension and transfer it to EP tubes. Add 200 μL of preheated X-VIVO 15 medium to each tube, then transfer it to the wells of the 12-well plate from step 1) containing preheated 500 IU / mL IL-2X-VIVO 15 medium. Incubate at 37°C and 5% CO2. Replace half the medium with fresh medium every two days.

[0229] The procedure was repeated three times. After 5 days of culture, the number of cells and cell viability were measured using a cell counter, and the average value was taken. Simultaneously, a portion of the cells was subjected to flow cytometry to determine the percentage of EGFP-positive cells.

[0230] Compare with method 2:

[0231] In step 1) of method 4, replace cholesterol sulfate with cholesterol, and the rest is the same as in method 4.

[0232] The results are as follows Figures 1-3 As shown, compared with the T cells overexpressing EGFP prepared by control methods 1 and 2, the T cells overexpressing EGFP prepared by methods 1-5 had significantly higher numbers of viable cells, viable cell ratio, and percentage of EGFP-positive viable cells.

[0233] Example 4: Electroporation preparation of TILs overexpressing EGFP

[0234] Electroporation of the plasmid pKB20-EGFP, which overexpresses EGFP, into TIL seed cells was performed using the following methods.

[0235] Method 6:

[0236] 1) The TIL seed cells obtained in Example 2 were resuspended in X-VIVO 15 medium containing 500 IU / mL IL-2 to a concentration of 3 × 10⁻⁶. 6 / mL, add cholesterol sulfate to a final concentration of 50μM, and incubate at 30℃ for 20h;

[0237] 2) Add 2 mL of X-VIVO 15 medium containing 500 IU / mL IL-2 to each well of a 12-well plate, and then transfer it to a cell culture incubator to preheat at 37°C and 5% CO2 for 1 hour.

[0238] 3) Prepare the electro-spreading fluid ratio for each well using the following table:

[0239] 100 μL Nucleocuvette TM Strip (μL) <![CDATA[Nucleofector TM Volume of solution 82 Electroporation replenishment solution 18

[0240] 4) Take the cell suspension processed in 1), centrifuge at 300g for 8 minutes, discard the supernatant, then resuspend the cells in physiological saline, and add the cells to EP tubes, adding 5×10⁶ cells to each EP tube. 6For each cell, repeat the centrifugation process and wash the cell pellet twice with physiological saline.

[0241] 5) Add 5 μg of plasmid pKB20-EGFP to the electroporation buffer prepared in step 3), and then let it stand at room temperature for no more than 30 minutes;

[0242] 6) Resuspend the cell pellet from step 4) in the plasmid-containing electroporation buffer prepared in step 5), 100 μL per tube. Carefully transfer the cell resuspended buffer into a LONZA 100 μL electroporation cuvette and place the cuvette into a LONZA Nucleofector. TM Inside the 2b electro-spinning tank, start the electro-spinning program and select T-020;

[0243] 7) After electroporation, carefully remove the electroporation cup, aspirate the cell suspension and transfer it to EP tubes. Add 200 μL of preheated X-VIVO 15 medium to each tube, then transfer it to the wells of the 12-well plate from step 1) containing preheated 500 IU / mL IL-2X-VIVO 15 medium. Incubate at 37°C and 5% CO2. Replace half the medium with fresh medium every two days.

[0244] The procedure was repeated three times. After 5 days of culture, the number of cells and cell viability were measured using a cell counter, and the average value was taken. Simultaneously, a portion of the cells was subjected to flow cytometry to determine the percentage of EGFP-positive cells.

[0245] Method 7:

[0246] 1) The TIL seed cells obtained in Example 2 were resuspended in X-VIVO 15 medium containing 500 IU / mL IL-2 to a concentration of 4 × 10⁻⁶. 6 / mL, add cholesterol sulfate to a final concentration of 1μM, and incubate at 30℃ for 16h;

[0247] 2) Add 2 mL of X-VIVO 15 medium containing 500 IU / mL IL-2 to each well of a 12-well plate, and then transfer it to a cell culture incubator to preheat at 37°C and 5% CO2 for 1 hour.

[0248] 3) Prepare the electro-spreading fluid ratio for each well using the following table:

[0249] 100 μL Nucleocuvette TM Strip (μL) Nucleofector TM volume of the solution 82 Electroporation replenishment solution 18

[0250] 4) Take the cell suspension processed in 1), centrifuge at 300g for 8 minutes, discard the supernatant, then resuspend the cells in physiological saline, and add the cells to EP tubes, adding 5×10⁶ cells to each EP tube. 6 For each cell, repeat the centrifugation process and wash the cell pellet twice with physiological saline.

[0251] 5) Add 5 μg of plasmid pKB20-EGFP to the electroporation buffer prepared in step 3), and then let it stand at room temperature for no more than 30 minutes;

[0252] 6) Resuspend the cell pellet from step 4) in the plasmid-containing electroporation buffer prepared in step 5), 100 μL per tube. Carefully transfer the cell resuspended buffer into a LONZA 100 μL electroporation cuvette and place the cuvette into a LONZA Nucleofector. TM Inside the 2b electro-spinning tank, start the electro-spinning program and select T-020;

[0253] 7) After electroporation, carefully remove the electroporation cup, aspirate the cell suspension and transfer it to EP tubes. Add 200 μL of preheated X-VIVO 15 medium to each tube, then transfer it to the wells of the 12-well plate from step 1) containing preheated 500 IU / mL IL-2X-VIVO 15 medium. Add reduced glutathione to a final concentration of 5 mM and incubate at 37°C and 5% CO2. Replace half the medium with fresh medium every two days.

[0254] The procedure was repeated three times. After 5 days of culture, the number of cells and cell viability were measured using a cell counter, and the average value was taken. Simultaneously, a portion of the cells was subjected to flow cytometry to determine the percentage of EGFP-positive cells.

[0255] Method 8:

[0256] 1) The TIL seed cells obtained in Example 2 were resuspended in X-VIVO 15 medium containing 500 IU / mL IL-2 to a concentration of 8 × 10⁻⁶. 6 / mL, add cholesterol sulfate to a final concentration of 50μM and reduced glutathione to a final concentration of 10mM, and incubate at 30℃ for 12h.

[0257] 2) Add 2 mL of X-VIVO 15 medium containing 500 IU / mL IL-2 to each well of a 12-well plate, and then transfer it to a cell culture incubator to preheat at 37°C and 5% CO2 for 1 hour.

[0258] 3) Prepare the electro-spreading fluid ratio for each well using the following table:

[0259]

[0260]

[0261] 4) Take the cell suspension processed in 1), centrifuge at 300g for 8 minutes, discard the supernatant, then resuspend the cells in physiological saline, and add the cells to EP tubes, adding 5×10⁶ cells to each EP tube. 6 For each cell, repeat the centrifugation process and wash the cell pellet twice with physiological saline.

[0262] 5) Add 5 μg of plasmid pKB20-EGFP to the electroporation buffer prepared in step 3), and then let it stand at room temperature for no more than 30 minutes;

[0263] 6) Resuspend the cell pellet from step 4) in the plasmid-containing electroporation buffer prepared in step 5), 100 μL per tube. Carefully transfer the cell resuspended buffer into a LONZA 100 μL electroporation cuvette and place the cuvette into a LONZA Nucleofector. TM Inside the 2b electro-spinning tank, start the electro-spinning program and select T-020;

[0264] 7) After electroporation, carefully remove the electroporation cup, aspirate the cell suspension and transfer it to EP tubes. Add 200 μL of preheated X-VIVO 15 medium to each tube, then transfer it to the wells of the 12-well plate from step 1) containing preheated 500 IU / mL IL-2X-VIVO 15 medium. Incubate at 37°C and 5% CO2. Replace half the medium with fresh medium every two days.

[0265] The procedure was repeated three times. After 5 days of culture, the number of cells and cell viability were measured using a cell counter, and the average value was taken. Simultaneously, a portion of the cells was subjected to flow cytometry to determine the percentage of EGFP-positive cells.

[0266] Method 9:

[0267] 1) The TIL seed cells obtained in Example 2 were resuspended in X-VIVO 15 medium containing 500 IU / mL IL-2 to a concentration of 9 × 10⁻⁶. 6 / mL, add cholesterol sulfate to a final concentration of 25μM and reduced glutathione to a final concentration of 25mM, and incubate at 37℃ for 8h.

[0268] 2) Add 2 mL of X-VIVO 15 medium containing 500 IU / mL IL-2 to each well of a 12-well plate, and then transfer it to a cell culture incubator to preheat at 37°C and 5% CO2 for 1 hour.

[0269] 3) Prepare the electro-spreading fluid ratio for each well using the following table:

[0270] 100 μL Nucleocuvette TM Strip (μL) Nucleofector TM volume of the solution 82 Electroporation replenishment solution 18

[0271] 4) Take the cell suspension processed in 1), centrifuge at 300g for 8 minutes, discard the supernatant, then resuspend the cells in physiological saline, and add the cells to EP tubes, adding 5×10⁶ cells to each EP tube. 6 For each cell, repeat the centrifugation process and wash the cell pellet twice with physiological saline.

[0272] 5) Add 5 μg of plasmid pKB20-EGFP to the electroporation buffer prepared in step 3), and then let it stand at room temperature for no more than 30 minutes;

[0273] 6) Resuspend the cell pellet from step 4) in the plasmid-containing electroporation buffer prepared in step 5), 100 μL per tube. Carefully transfer the cell resuspended buffer into a LONZA 100 μL electroporation cuvette and place the cuvette into a LONZA Nucleofector. TM Inside the 2b electro-spinning tank, start the electro-spinning program and select T-020;

[0274] 7) After electroporation, carefully remove the electroporation cup, aspirate the cell suspension and transfer it to EP tubes. Add 200 μL of preheated X-VIVO 15 medium to each tube, then transfer it to the wells of the 12-well plate from step 1) containing preheated 500 IU / mL IL-2X-VIVO 15 medium. Incubate at 37°C and 5% CO2. Replace half the medium with fresh medium every two days.

[0275] The procedure was repeated three times. After 5 days of culture, the number of cells and cell viability were measured using a cell counter, and the average value was taken. Simultaneously, a portion of the cells was subjected to flow cytometry to determine the percentage of EGFP-positive cells.

[0276] Method 10:

[0277] 1) The TIL seed cells obtained in Example 2 were resuspended in X-VIVO 15 medium containing 500 IU / mL IL-2 to a concentration of 1×10⁻⁶. 7 / mL, add cholesterol sulfate to a final concentration of 10μM, and incubate at 30℃ for 20h;

[0278] 2) Add 2 mL of X-VIVO 15 medium containing 500 IU / mL IL-2 to each well of a 12-well plate, and then transfer it to a cell culture incubator to preheat at 37°C and 5% CO2 for 1 hour.

[0279] 3) Prepare the electro-spreading fluid ratio for each well using the following table:

[0280] 100 μL Nucleocuvette TM Strip (μL)] <![CDATA[Nucleofector TM Volume of solution 82 Electroporation replenishment solution 18

[0281] 4) Take the cell suspension processed in 1), centrifuge at 300g for 8 minutes, discard the supernatant, then resuspend the cells in physiological saline, and add the cells to EP tubes, adding 5×10⁶ cells to each EP tube. 6 For each cell, repeat the centrifugation process and wash the cell pellet twice with physiological saline.

[0282] 5) Add 5 μg of plasmid pKB20-EGFP to the electroporation buffer prepared in step 3), and then let it stand at room temperature for no more than 30 minutes;

[0283] 6) Resuspend the cell pellet from step 4) in the plasmid-containing electroporation buffer prepared in step 5), 100 μL per tube. Carefully transfer the cell resuspended buffer into a LONZA 100 μL electroporation cuvette and place the cuvette into a LONZA Nucleofector. TM Inside the 2b electro-spinning tank, start the electro-spinning program and select T-020;

[0284] 7) After electroporation, carefully remove the electroporation cup, aspirate the cell suspension and transfer it to EP tubes. Add 200 μL of preheated X-VIVO 15 medium to each tube, then transfer it to the wells of the 12-well plate from step 1) containing preheated 500 IU / mL IL-2X-VIVO 15 medium. Add reduced glutathione to a final concentration of 10 mM and incubate at 37°C and 5% CO2. Replace half the medium with fresh medium every two days.

[0285] The procedure was repeated three times. After 5 days of culture, the number of cells and cell viability were measured using a cell counter, and the average value was taken. Simultaneously, a portion of the cells was subjected to flow cytometry to determine the percentage of EGFP-positive cells.

[0286] Compare with method 1:

[0287] 1) Add 2 mL of X-VIVO 15 medium containing 500 IU / mL IL-2 to each well of a 12-well plate, and then transfer it to a cell culture incubator to preheat at 37°C and 5% CO2 for 1 hour.

[0288] 2) Prepare the electro-spreading fluid ratio for each well using the following table:

[0289] 100 μL Nucleocuvette TM Strip (μL) <![CDATA[Nucleofector TM Volume of solution 82 Electroporation replenishment solution 18

[0290] 3) Take the TIL seed cell suspension to be electroporated, centrifuge at 300g for 8 minutes, discard the supernatant, then resuspend the cells with physiological saline, and add the cells to EP tubes, adding 5×10⁶ cells to each EP tube. 6 Seed TIL cells, repeat the centrifugation steps, and wash the cell pellet twice with physiological saline.

[0291] 4) Add 5 μg of plasmid pKB20-EGFP to the electroporation buffer prepared in step 2), and then let it stand at room temperature for no more than 30 minutes;

[0292] 5) Resuspend the cell pellet from step 4) in the plasmid-containing electroporation buffer prepared in step 4), 100 μL per tube. Carefully transfer the cell resuspended solution into a LONZA 100 μL electroporation cuvette and place the cuvette into a LONZA Nucleofector. TM Inside the 2b electro-spinning tank, start the electro-spinning program and select T-020;

[0293] 6) After electroporation, carefully remove the electroporation cup, aspirate the cell suspension and transfer it to EP tubes. Add 200 μL of preheated X-VIVO 15 medium to each tube, then transfer it to the wells of the 12-well plate from step 1) containing preheated 500 IU / mL IL-2X-VIVO 15 medium. Incubate at 37°C and 5% CO2. Replace half the medium with fresh medium every two days.

[0294] The procedure was repeated three times. After 5 days of culture, the number of cells and cell viability were measured using a cell counter, and the average value was taken. Simultaneously, a portion of the cells was subjected to flow cytometry to determine the percentage of EGFP-positive cells.

[0295] Compare with method 3:

[0296] In step 1) of Method 6, replace cholesterol sulfate with cholesterol; otherwise, follow the same procedure as in Method 6.

[0297] Compare with method 4:

[0298] In step 1) of Method 10, cholesterol sulfate is not added for incubation; otherwise, the process is the same as in Method 10.

[0299] Compare with method 5:

[0300] In step 1) of Method 9, the addition of cholesterol sulfate to a final concentration of 25 μM and reduced glutathione to a final concentration of 25 mM is changed to adding only reduced glutathione to a final concentration of 25 mM, while the rest remains the same as in Method 9.

[0301] The results are as follows Figures 4-6 As shown, compared with the TILs overexpressing EGFP prepared by methods 1, 3, 4, and 5, the TILs overexpressing EGFP prepared by methods 6-10 had significantly higher numbers of live cells, a higher proportion of live cells, and a higher percentage of EGFP-positive live cells.

[0302] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing description of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for preparing cells overexpressing a foreign gene by electroporation, characterized in that, It includes the following steps: Before electroporation, the cells are treated with a cell electroporation pretreatment reagent, which contains a cholesterol derivative; preferably, the cholesterol derivative contains cholesterol sulfate.

2. The method according to claim 1, characterized in that, The cell electroporation pretreatment reagent further includes a reducing agent; preferably, the reducing agent includes reduced glutathione.

3. The method according to claim 1 or 2, characterized in that, The method further includes the following steps: after electroporation of the cells, the cells are further treated with a reducing agent; preferably, the reducing agent comprises reduced glutathione.

4. The method according to claim 1, characterized in that, It includes the following steps: 1) Incubate the cells to be electroporated with the cell pretreatment reagent to separate the cells; 2) Introducing exogenous genes into cells after incubation with the CCP via electrotransfer; Preferably, the cells in the method are immune effector cells; optionally, the immune effector cells include any one or more of T cells, TILs, NK cells, NKT cells, CAR-T cells, CIK cells, CTLs, TCR-Ts, LAKs, DCs, and γδT cells. Preferably, the cell electroporation pretreatment reagent in step 1) includes a cholesterol derivative; the cholesterol derivative preferably includes cholesterol sulfate; optionally, the cell electroporation treatment reagent further includes a reducing agent; preferably, the reducing agent includes reduced glutathione. Preferably, the reducing agent in step 1) comprises reduced glutathione, with a final concentration of 1-50 mM; Preferably, the CO2 concentration during co-incubation in step 1) is 1-10 v / v%. Preferably, the co-incubation time in step 1) is 1-20 hours; Preferably, the cells to be electroporated in step 1) are resuspended in a cell culture medium; optionally, the cell culture medium comprises a basal culture medium; optionally, the basal culture medium comprises AIM- CTS TM Serum-free cell culture medium, DMEM medium, RPMI 1640 medium, and X-VIVO 15, or any one or more of these. Preferably, the cell culture medium further comprises cytokines; optionally, the cytokines include any one or more of IL-2, IL-7, IL-12, IL-15, IL-21, TNF-α, and IFN-γ; preferably, the cytokines include IL-2; preferably, the final concentration of IL-2 is 100-5000 IU / mL. Preferably, in step 1), the cell density of the cells to be electroporated resuspended in the cell culture medium is 1 × 10⁻⁶ cells / year. 6 -1×10 7 / mL; Preferably, the cell separation in step 1) is achieved by centrifugation; Preferably, the electroporation conditions described in step 2) are suitable for the electroporation of immune cells; optionally, they are suitable for electroporation conditions selected from any one or more of T cells, TILs, NK cells, NKT cells, CAR-T cells, CIK cells, CTLs, TCR-Ts, LAKs, DCs, and γδT cells. Preferably, the vector for the exogenous gene in step 2) is a DNA vector or an RNA vector; optionally, the DNA vector is single-stranded DNA or double-stranded DNA.

5. The method according to claim 4, characterized in that, It also includes: 3) continuing to culture the cells in 2) where exogenous genes were introduced via electroporation; Preferably, the culture in step 3) is carried out in a cell culture medium; optionally, the cell culture medium includes a basal culture medium; optionally, the basal culture medium is selected from... AIM- CTS TM Any one or more of serum-free cell culture medium, DMEM medium, RPMI 1640 medium, and X-VIVO 15 medium; Preferably, the cell culture medium further comprises cytokines; optionally, the cytokines include any one or more of IL-2, IL-7, IL-12, IL-15, IL-21, TNF-α, and IFN-γ. Preferably, the cell culture medium further comprises a reducing agent; optionally, the reducing agent is reduced glutathione; preferably, the final concentration of the reduced glutathione is 1-100 mM. Preferably, the culture temperature in step 3) is 25-42℃; Preferably, the CO2 concentration during cultivation in step 3) is 1-10 v / v%. Preferably, the culture time in step 3) is 2 hours to 30 days; Preferably, the initial cell density for culture in step 3) is 1 × 10⁻⁶. 6 -1×10 7 / mL.

6. Cells overexpressing exogenous genes prepared by the method according to any one of claims 1-5; preferably, the cells are immune effector cells; preferably, the immune effector cells include any one or more of T cells, TILs, NK cells, NKT cells, CAR-T cells, CIK cells, CTLs, TCR-Ts, LAKs, DCs, and γδT cells.

7. Application of cell electroporation pretreatment reagents in the preparation of cells overexpressing exogenous genes, wherein the cell electroporation pretreatment reagents include cholesterol derivatives; Preferably, the cells are immune effector cells; preferably, the immune effector cells include any one or more of T cells, TILs, NK cells, NKT cells, CAR-T cells, CIK cells, CTLs, TCR-Ts, LAKs, DCs, and γδT cells. Preferably, the cholesterol derivative comprises cholesterol sulfate.

8. The application according to claim 7, characterized in that, The cell electroporation pretreatment reagent further includes a reducing agent; preferably, the reducing agent includes reduced glutathione.

9. A cell electroporation kit, characterized in that, It includes a cell electroporation mediator component and a cell treatment component; the cell treatment component includes cholesterol derivatives; Preferably, the cholesterol derivative includes cholesterol sulfate; Preferably, the cell treatment component further includes a reducing agent; preferably, the reducing agent includes reduced glutathione. Preferably, the cell electroporation medium component includes a cell electroporation buffer; Preferably, the cell electroporation buffer contains Na. + K + Ca 2+ Mg 2+ and Zn 2+ any one or more of them; Preferably, the cell electroporation buffer contains Cl. - SO4 2- PO4 3- HPO4 2- H2PO4 - HEPES and NO3 - any one or more of them; Preferably, the cell electroporation buffer is selected from Lonza human T cells. Solution ( Solution for Human T Cells), Maxcyte Electroporation Buffer, ThermoFisher CTS TM Xenon TM Electroporation buffer (CTS) TM Xenon TM Electroporation Buffer), Miltenyi Electroporation buffer ( One or more of the following: Electroporation Buffer, BTXpress Electroporation Solution, and Bio-rad Gene Pulser Electroporation Buffer.

10. The cell electroporation kit according to claim 9, characterized in that, It includes: Lonza human T cells The solution, Lonza electroporation supplement solution, and cholesterol sulfate; preferably, the cell electroporation kit also includes reduced glutathione.

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