CN116875545A - Preparation method of immune effector cell expressing exogenous gene - Google Patents

Preparation method of immune effector cell expressing exogenous gene Download PDF

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CN116875545A
CN116875545A CN202310864727.3A CN202310864727A CN116875545A CN 116875545 A CN116875545 A CN 116875545A CN 202310864727 A CN202310864727 A CN 202310864727A CN 116875545 A CN116875545 A CN 116875545A
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金华君
许馥慧
黄晨
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Shanghai Junsai Biotechnology Co ltd
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Abstract

The application relates to a preparation method of immune effector cells expressing exogenous genes, in particular to a method for preparing cells expressing exogenous genes by electrotransformation, which comprises the following steps: 1) Treating the cells with a calcium ion chelating agent; 2) Introducing a nucleic acid construct comprising a gene encoding said exogenous gene into said cell of 1) by electrosteering. The method remarkably improves electrotransformation efficiency and cell survival rate.

Description

Preparation method of immune effector cell expressing exogenous gene
Technical Field
The present application relates to the field of biotechnology. In particular to a preparation method of immune effector cells expressing exogenous genes.
Background
Immune cell therapy has become a very important class of tumor treatment. In a variety of engineered immune effector cells, including CAR-T cells, designed to kill tumors, it is highly dependent on efficient, stable, safe transgene platforms for different purposes, such as tumor targeting, immune cell activation, etc., and it is necessary to integrate and express different exogenous transgenes. Techniques for introducing exogenous transgenes into immune cells of interest for integrated expression mainly include both viral-based and non-viral-based delivery methods. In the example of CAR-T cells, virus-based delivery expresses the CAR gene by using a retroviral vector or a lentiviral vector, the CAR gene is introduced into immune effector cells by packaged viral particles, and integrated into the cell genome by the integration system of the retrovirus or lentivirus itself. Although the virus-based delivery method can efficiently and stably introduce exogenous genes into target cells, such as immune effector cells, and integrate the exogenous genes into the genome of the target cells, the virus-based delivery method has a plurality of defects, such as high cost, time and labor consumption in the preparation and production process, long-term monitoring after the exogenous genes are returned into human bodies, and certain potential safety hazards and the like.
Among the non-viral based delivery methods, electroporation (or electroporation) has been a well established method in a part of the medical field, and its application in biotechnology has begun to emerge in recent years. Compared with a viral vector delivery system, the method for introducing the exogenous gene into the cell by using electrotransformation has the advantages of simple operation, low cost, low safety risk and the like. However, the electrotransport itself has significant problems including the tendency to cause excessive cell death at transient high voltages. In addition, for immune effector cells such as PBMC (PBMC), TIL (t-cell line) cells and the like, the death rate of the cells after DNA is directly introduced into the cells through electrotransformation is usually very high, and the preparation and application of the transgenic immune effector cells are seriously influenced. Although there are mature electrotransfer devices and matched buffer solution systems in the market at present, the problem of high death rate of immune effector cells after electrotransfer is still outstanding, and a method capable of effectively improving the survival rate of cells after electrotransfer of exogenous genes and guaranteeing a certain exogenous gene expression positive rate is still urgently required to be developed for the immune effector cells at present.
Disclosure of Invention
In a first aspect the present application provides a cell treatment composition for treating cells to be electrotransformed, thereby improving the efficiency of electrotransformation and cell viability, the treatment composition comprising a matrix and a calcium ion chelating agent.
In one or more embodiments, the calcium ion chelating agent is EGTA or EDTA.
In one or more embodiments, the cell is an immune effector cell. Optionally, the immune effector cell is any one or more selected from the group consisting of a T cell, DN T cell, NK cell, NKT cell, CAR-T cell, TCR-T cell, TIL, CTL, LAK cell, and CIK cell. The cells are preferably T cells or TIL cells. The T cells are alpha beta T cells or gamma delta T cells. The αβ T cells are preferably, for example, cd3+cd8+ T cells or cd3+cd4+ T cells.
In one or more embodiments, the T cells are activated. Preferably, the T cells are activated by an anti-CD 3 antibody and/or an anti-CD 28 antibody.
In one or more embodiments, the final concentration of the calcium ion chelating agent in the cell treatment fluid is from 0.1 to 50mM, for example from 0.2 to 30mM, preferably from 0.5 to 5.0mM, more preferably from 1.0 to 2.0mM.
In one or more embodiments, the matrix comprises a cell culture medium or buffer. The cell culture medium is AIM-V, X-VIVO, DMEM or RPMI1640, preferably AIM-V or X-VIVO. Such buffers include, but are not limited to, PB, PBS, TBS, tris-HCl, tris-TBS, HEPES buffer, and cacodylic acid buffer, preferably phosphate-based buffers such as PBS.
The present application also provides a cell processing kit for processing cells to be electrotransformed to improve transduction efficiency and cell survival, the kit comprising a calcium ion chelating agent. The kit optionally further comprises a cell culture medium and/or a buffer.
In one or more embodiments, the calcium ion chelating agent is EGTA or EDTA.
In one or more embodiments, the cell processing kit is an electrotransport kit further comprising one or more of electrotransport fluids.
In one or more embodiments, the cell culture medium is AIM-V, X-VIVO, DMEM or RPMI1640, preferably AIM-V or X-VIVO.
In one or more embodiments, the buffer includes, but is not limited to, PB, PBS, TBS, tris-HCl, tris-TBS, HEPES buffer, cacodylic acid buffer, preferably phosphate-based buffers, such as PBS.
In one or more embodiments, the electrotransfer fluid comprises a Nucleofector solution and an electrotransfer make-up solution.
In one or more embodiments, the kit further comprises a T cell activating reagent, such as an anti-CD 3 antibody and/or an anti-CD 28 antibody.
In one or more embodiments, the cell is an immune effector cell. Optionally, the immune effector cell is any one or more selected from the group consisting of a T cell, DN T cell, NK cell, NKT cell, CAR-T cell, TCR-T cell, TIL, CTL, LAK cell, and CIK cell. The cells are preferably T cells or TIL cells. The T cells are alpha beta T cells or gamma delta T cells. The αβ T cells are preferably, for example, cd3+cd8+ T cells or cd3+cd4+ T cells.
The application also provides a method of treating cells to be electrotransformed, thereby improving the efficiency and viability of the cells, the method comprising treating the cells with a calcium ion chelating agent prior to electrotransformation.
In one or more embodiments, the calcium ion chelating agent is EGTA or EDTA.
In one or more embodiments, the treatment time period is from 0.5h to 2h.
In one or more embodiments, the treatment is performed in a buffer or cell culture medium.
In one or more embodiments, the concentration of the calcium ion chelating agent in the treated system is from 0.5 to 5mM; preferably 1.0-2.0mM.
In one ofOr in various embodiments, the concentration of cells in the treated system is 1X 10 6 Up to 1X 10 10 Per mL, e.g. 1X 10 7 Up to 1X 10 8 Per mL, preferably 1X 10 7 And/300. Mu.L.
The present application also provides a method for preparing a cell expressing a foreign gene by electrotransformation, comprising: 1) Treating the cells with a calcium ion chelating agent; 2) A nucleic acid construct containing a gene encoding the exogenous gene is introduced by electrotransduction cells.
In one or more embodiments, the calcium ion chelating agent is EGTA or EDTA.
In one or more embodiments, 1) the treatment is performed prior to electrotransformation.
In one or more embodiments, 1) the treatment is performed in a buffer or cell culture medium.
In one or more embodiments, 1) the treatment duration is from 0.5h to 2h.
In one or more embodiments, 1) the treatment temperature is from 30 ℃ to 37 ℃.
In one or more embodiments, 1) the concentration of the calcium ion chelating agent in the treated system is from 0.5 to 5mM; preferably 1.0-2.0mM.
In one or more embodiments, 1) the concentration of cells in the treated system is 1X 10 6 Up to 1X 10 10 Per mL, e.g. 1X 10 7 Up to 1X 10 8 Per mL, preferably 1X 10 7 And/300. Mu.L.
In one or more embodiments, the cell is an immune effector cell. Optionally, the immune effector cell is any one or more selected from T cells, DN T cells, NK cells, NKT cells, CAR-T cells, TCR-T cells, TIL, CTL, LAK cells, and CIK cells. The cells are preferably T cells or TIL cells. The T cells are alpha beta T cells or gamma delta T cells. The αβ T cells are preferably, for example, cd3+cd8+ T cells or cd3+cd4+ T cells.
In one or more embodiments, the cell is a T cell, and the method further comprises the step of activating the T cell prior to electrotransformation. The step of activating T cells comprises: t cells are activated using anti-CD 3 antibodies and/or anti-CD 28 antibodies.
In one or more embodiments, the nucleic acid construct is DNA or RNA, such as a vector or mRNA.
The application also provides a cell expressing the exogenous gene obtained by the method for preparing the cell expressing the exogenous gene by electrotransformation. In one or more embodiments, the cell is an immune effector cell. Optionally, the immune effector cell is any one or more selected from T cells, DN T cells, NK cells, NKT cells, CAR-T cells, TCR-T cells, TIL, CTL, LAK cells, and CIK cells. The cells are preferably T cells or TIL cells. The T cells are alpha beta T cells or gamma delta T cells. The αβ T cells are preferably, for example, cd3+cd8+ T cells or cd3+cd4+ T cells.
The application also provides the use of a calcium ion chelating agent in the treatment of cells to be electrotransformed.
In one or more embodiments, the calcium ion chelating agent is EGTA or EDTA.
In one or more embodiments, the cell is an immune effector cell. Optionally, the immune effector cell is any one or more selected from the group consisting of a T cell, DN T cell, NK cell, NKT cell, CAR-T cell, TCR-T cell, TIL, CTL, LAK cell, and CIK cell.
In one or more embodiments, the cell is a T cell or a TIL cell. The T cells are alpha beta T cells or gamma delta T cells. The αβ T cells are preferably, for example, cd3+cd8+ T cells or cd3+cd4+ T cells.
The application has the advantages that: before exogenous genes are introduced into cells, particularly immune effector cells, by using an electrotransformation method, the cells to be electrotransformed are pretreated by using a calcium ion chelating agent, so that the survival rate and the number of living cells of the cells after electrotransformation can be obviously improved, and meanwhile, the proportion and the number of cells positive in exogenous gene expression can be improved.
Drawings
Fig. 1: survival rate of TIL of electrotransport EGFP expression vector after different concentrations of EGTA treatment.
Fig. 2: number of living cells in TIL of electrotransport EGFP expression vector after different concentrations of EGTA treatment.
Fig. 3: EGFP-expressing positive cell ratios in TIL of electrotransport EGFP expression vectors after treatment with different concentrations of EGFA.
Fig. 4: number of cells positive for EGFP expression in TIL of the electrotransport EGFP expression vector after EGFA treatment at different concentrations.
Fig. 5: survival rate of T cells electrotransport EGFP expression vectors following different concentrations of EGTA treatment.
Fig. 6: number of viable cells in T cells of electrotransport EGFP expression vectors following different concentrations of EGTA treatment.
Fig. 7: EGFP-expressing positive cell ratios in T cells of the electrotransport EGFP expression vector after EGFA treatment at different concentrations.
Fig. 8: number of cells positive for EGFP expression in T cells of the electrotransport EGFP expression vector following treatment with different concentrations of EGFA.
Detailed Description
It is understood that within the scope of the present application, the above-described technical features of the present application and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute a preferred technical solution.
The inventor finds that the survival rate of cells after electrotransformation and the positive rate of exogenous gene expression can be effectively improved by pretreatment of the immune effector cells with a calcium ion chelating agent before electrotransformation of exogenous nucleic acid.
Accordingly, the present application provides a method for preparing a cell expressing a foreign gene by electrotransformation, comprising: treating the cells with a calcium ion chelating agent and electrotransferring the treated cells into a nucleic acid construct comprising a gene encoding the exogenous gene.
In the present application, immune effector cells have the meaning well known in the art and refer to cells involved in or associated with an immune response, including lymphocytes, dendritic cells, monocytes/macrophages, granulocytes, mast cells, and the like. Lymphocytes include T lymphocytes, DN T cells, tumor Infiltrating Lymphocytes (TIL), B lymphocytes, NK lymphocytes, NKT cells, CAR-T cells, TCR-T cells, CTLs, LAK cells, and CIK cells. Immune effector cells suitable for use in the present application include, inter alia, those typically used in adoptive cell therapy for tumors. T (lymphocytes) as described herein include alpha beta T cells and gamma delta T cells. The αβ T cells are preferably, for example, cd3+cd8+ T cells or cd3+cd4+ T cells.
As used herein, the term "calcium ion chelating agent" includes any agent that selectively chelates calcium ions, such as BAPTA, EGTA, or EDTA. The calcium ion chelating agents described herein also include derivatives of their corresponding acid or base forms.
Herein, the duration of the treatment of the cells with the calcium ion chelating agent is not limited, for example, 0.2 to 5 hours. Typically, the time for the calcium ion chelating agent to treat the cells is from 0.5h to 2h, such as 0.5h, 1.0h, 1.5h, 2.0h, or a range between any two of the foregoing.
The temperature of the cells treated with the calcium ion chelating agent is not limited, for example, 30℃to 37 ℃. The temperature at which the cells are usually treated with the calcium ion chelating agent is, for example, 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃ or 37 ℃.
The concentration of the calcium ion chelating agent in the system (typically a solution) in which the cells are treated is 0.5-5mM, e.g., 0.5mM, 1.0mM, 1.5mM, 2.0mM, 2.5mM, 3.0mM, 3.5mM, 4.0mM, 4.5mM, 5.0mM or a range between any two of the foregoing. The concentration of cells in the treatment system was 1X 10 6 Up to 1X 10 10 Per mL, e.g. 1X 10 7 Up to 1X 10 8 Per mL, preferably 1X 10 7 And/300. Mu.L.
The system for treating the cells may be a cell culture solution responsive to the cells or a buffer capable of maintaining cell viability. For immune effector cells, particularly T cells or TIL cells, the cell culture medium is AIM-V, X-VIVO, DMEM or RPMI1640, preferably AIM-V or X-VIVO; such buffers include, but are not limited to, PB, PBS, TBS, tris-HCl, tris-TBS, HEPES buffer, and cacodylic acid buffer, preferably phosphate-based buffers such as PBS.
In a specific embodiment, the step of treating the cells comprises: 1) Preheating a cell culture medium (e.g., AIM-V medium); 2) Cells (e.g., TIL, activated or non-activated T cells) are plated at 1X 10 6 Up to 1X 10 10 personal/mLThe treated cells were obtained by incubating the supernatant with 0.5-5mM calcium ion chelating agent for 0.2-5h in PBS and centrifuging the supernatant.
The electrotransformation referred to in the methods herein may be any electrotransformation method known in the art for transferring nucleic acid constructs into cells. The corresponding procedures, reagents required, parameters are within the knowledge of those skilled in the art, as long as the cells to be electrotransformed in the procedure are replaced with the cells treated with the calcium ion chelating agent as described herein.
The nucleic acid construct required for the electrotransformation process may be a DNA or RNA, such as a plasmid vector or mRNA, carrying the coding sequence of the exogenous gene of interest and the required components for its expression in the cell (integrated expression or plasmid expression), such as a promoter, terminator or transposon.
In a specific embodiment, the step of electrotransferring the cell comprises: according to the electrotransformation method, preparing electrotransformation solution, adding nucleic acid construct (such as plasmid) and treated cells, and selecting proper electrotransformation program in electrotransformation instrument for electrotransformation. Illustratively, a LONZA Nucleofector 2b electrotransport machine and its associated electrotransport fluid were used, with an electrotransport program of T-020.
In one or more embodiments, the methods of making cells expressing an exogenous gene described herein further comprise the step of culturing the electrotransformed cells with a cell culture medium. The culture medium is as described elsewhere herein, and the steps of culturing the cells are routine to those skilled in the art.
The application also provides a cell expressing the exogenous gene obtained by the method for preparing the cell expressing the exogenous gene by electrotransformation. Preferably, the cell is an immune effector cell, as described elsewhere herein.
The above system for treating cells can be made into a cell treatment composition for treating cells to be electrotransformed, thereby improving electrotransformation efficiency and cell viability, the treatment composition comprising a calcium ion chelating agent and a cell culture medium and/or buffer. The final concentration of the calcium ion chelating agent in the cell treatment liquid is 0.1 to 50mM, for example, 0.2 to 30mM, preferably 0.5 to 5.0mM, more preferably 1.0 to 2.0mM.
The reagents for treating or electrotransferring cells described above may be prepared into a cell treatment or electrotransferring kit for treating cells to be electrotransferred to thereby improve electrotransfer efficiency and cell viability, the kit comprising a calcium ion chelating agent as described herein. The kit optionally further comprises a cell culture medium (e.g., AIM-V or X-VIVO), a buffer (e.g., PBS), an electrotransfer solution (e.g., comprising a Nucleofector solution and an electrotransfer make-up solution).
The application will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.
The EGFP expression vector pKB20-EGFP used in the examples below was constructed as described in example 1 on page 21 of the specification of PCT application WO2022078310A1, the entire contents of which are incorporated herein by reference.
Example 1: isolated culture of human liver cancer tissue-derived TIL cells
Freshly excised human liver cancer tissue specimens were collected and immediately processed under sterile conditions. The liver cancer tissue of this example was treated and cultured to obtain TIL according to the medium described in example 1 of WO2022111571A1 and the tumor sample treatment method and TIL culture method described in example 2. WO2022111571A1 is incorporated by reference in its entirety.
The method comprises the following steps:
1) Preparing physiological saline containing 100U/mL penicillin, 100 mug/mL streptomycin and 50 mug/mL gentamicin for later use;
2) Placing the obtained tumor tissue sample of the freshly isolated tumor patient in a 10cm culture dish added with 30mL of the physiological saline prepared in the step 1) in a sterile environment in a secondary biosafety cabinet for washing, transferring the tumor tissue sample to a new 10cm dish added with 30mL of the physiological saline prepared in the step 1) for washing, and repeating the washing for 3 times;
3) Removing fat tissue and necrotic tissue with a sterile scalpel, cutting tumor tissue becomes a diameter of 3X 3mm 3 Is used for the production of the small blocks,2G-REX 100 culture tanks (purchased from Wilsonwolf) were taken, 42 randomly selected tumor tissue pieces were placed in each G-REX100 culture tank, and seed cell culture medium was added to the culture tank, and the seed culture medium had the following composition: 3000IU/mL IL-2, 20ng/mL IL-7, 20ng/mL IL-15, 500U/mL GM-CSF, 1000IU/mL IFN-gamma, 3 μg/mL anti-CD 137mAb, 3 μg/mL anti-CD 28 mAb, 3 μg/mL anti-PD-1 mAb, 10ng/mL TNF-alpha, 5% v/v human AB serum, 1 XPS diabody, and a final volume of X-VIVO 15 basal medium; the redundant tumor tissue blocks are frozen and stored by a cryo-Stor 10 (purchased from BioLifeSolons) frozen solution through a program cooling instrument liquid nitrogen;
4) 3) adding 1L of the seed cell culture medium into a G-REX100 culture tank containing tumor tissue blocks, and adding 5% CO at 37 ℃ to the tumor tissue blocks 2 Culturing, removing half of old seed cell culture medium every 4 days, supplementing half of fresh seed cell culture medium, centrifuging at 12 th day, and counting total number and activity rate of cells after harvesting TIL seed cells;
5) Taking the seed cells harvested in 4), re-suspending to 5.0X10 s with an expanding medium containing 500IU/mL IL-2, 7ng/mL IL-7, 30ng/mL IL-15, 5% v/v human AB serum, 1 XPS diabody and a final volume of X-VIVO 15 basal medium 5 Per mL, in a cell culture vessel pretreated with anti-CD 3 mAb, anti-CD 28 mAb and anti-CD 137mAb coating, 37℃5% CO 2 After 2 days of activation, the activated cells were collected by centrifugation and inoculated into a G-REX500M culture tank to which an expansion medium which had been preheated in advance was added, and the expansion medium in the G-REX500M culture tank was identical to the expansion medium described above. The volume of the expansion medium in each G-REX500M was 5L. Activated seed cells were grown according to 2.5X10 5 /cm 2 Inoculation density inoculation of (C) 37℃ 5% CO 2 Culturing, removing half volume of old expansion medium after cell count every 4 days, and supplementing half volume of fresh expansion medium until total cell count in each G-REX500M tank reaches 1.0X10 10 Afterwards, the flasks were separated at a ratio of 1:2, and each flask was supplemented with fresh expansion medium to 5L and then continued to culture. Cells were harvested after a total of 12 days of culture in an enlarged medium of a G-REX500M culture tank before and after culturing to obtain TIL.
Example 2: preparation of human activated T cells
Using an anti-CD 3 antibody (CD 3 Monoclonal Antibody (OKT 3), eBioscience containing 5. Mu.g/ml TM Cat.No. 14-0037-82, thermoFisher) and 5 μg/ml of anti-CD 28 antibody (CD 28 Monoclonal Antibody (CD 28.2), eBioscience TM 14-0289-82, thermoFisher) coating six-well plate at room temperature for 2-4 hours, sucking the coating liquid, washing the well plate with physiological saline for 1-3 times, and adding 2% FBS AIM-V medium for use; resuscitating human peripheral blood PBMC (purchased from Chimaphila) in a water bath at 37 ℃ and culturing the PBMC for 2-4 hours in an adherence manner, wherein non-adherence suspension cells are initial T cells, collecting the suspension cells into a 15ml centrifuge tube, centrifuging for 3min at 1200rmp, discarding the supernatant, adding physiological saline, centrifuging for 3min at 1200rmp, discarding the physiological saline, and repeating the steps; transferring the washed initial T cells into antibody coated holes containing culture medium at 37deg.C and 5% CO 2 After 3-4 days of culture, the subsequent experiments are carried out.
Example 3: use of calcium ion chelating agent EGTA in electrotransformation for preparing TIL cell over-expressing EGFP
1) X-VIVO is preformed TM 15 (Lonza, cat. No.: 02-053Q) medium was added to 4 wells in a 12-well plate, 2mL each, followed by transfer into a cell incubator, 5% CO at 30 ℃ 2 Preheating for 1 hour;
2) TIL cells prepared in example 1 were divided into 4 groups of 1X 10 cells each 7 Individual cells, resuspended in medium preheated at 30℃in 4 wells of 1), respectively, and IL-2 (R) was added&Dsystems, cat.No.: BT-002) to a final concentration of 500IU/mL, 5% CO at 30 ℃C 2 Standing the cell incubator for 8 hours;
3) After the stationary treatment at 30℃for 8 hours, EGTA was added to the medium of each well to a final concentration of 0mM, 1mM, 1.5mM and 2mM, respectively, and the stationary treatment at 30℃was continued for 2 hours;
4) TIL cells treated with 0mM, 1mM, 1.5mM and 2mM EGTA were centrifuged for 3min at 800g, and the cell pellet was resuspended in PBS and centrifuged for 3min at 800g again, and the supernatant was discarded;
5) The ratio of the electrotransport liquid with single dosage per hole is carried out according to the following table:
100μL Nucleocuvette TM Strip(μL)
Nucleofector TM volume of solution 82
Electrolysis supplementary solution 18
6) Adding plasmid pKB20-EGFP 5 mug into the prepared electrotransfer solution, re-suspending 4) the prepared plasmid-containing electrotransfer solution to obtain 4-tube TIL cell sediment, carefully sucking 100 mug of each tube, transferring the cell re-suspension into a LONZA 100 mug electrotransfer cup, and placing the electrotransfer cup into LONZA Nucleofector TM 2b, starting an electric transfer program in the electric transfer groove, wherein the electric transfer program selects T-020; X-VIVO was previously performed 1 hour before electrotransformation TM 15 medium was added to 4 wells in a 12-well plate, 2mL each, and then transferred into a cell incubator at 37℃with 5% CO 2 Preheating for 1 hour;
7) After completion of the electrotransfer, the electrotransfer cup was carefully removed, the cell suspension was aspirated and transferred to EP tubes, and each tube was filled with pre-warmed X-VIVO containing 5% v/v human AB serum (Sigmaaldrich, cat. No.: H4522) at 37 ℃ TM 15 medium 200. Mu.L followed by transfer to a 12 well plate in 6) pre-warmed X-VIVO TM 15 medium wells, IL-2 was added to each well to a final concentration of 500IU/mL,37℃and 5% CO 2 Culturing.
The above EGTA concentration treatment conditions were further provided with 3 parallel sub-wells, and the cell viability and viable cell number in each well after 13 days of culture were measured by a cell counter after trypan blue staining, and the average value was obtained. The proportion (cell positive rate) and number (positive cell number) of cells positive for EGFP expression after electrotransformation were examined by flow cytometry.
The results are shown in FIGS. 1-4. FIG. 1 shows that the survival rate of cells after each group of TIL electrotransport EGFP expression vectors is higher, more than 95%; FIG. 2 shows that the total number of living cells in each group is substantially equivalent; FIGS. 3 and 4 show that the proportion of EGFP-expressing positive cells and the total number of EGFP-expressing positive cells in TILs of 1mM, 1.5mM and 2mM EGTA treated groups were significantly higher than in the 0mM group. This shows that the transfer efficiency of the exogenous gene can be obviously improved and the quantity of the transgenic TIL can be improved on the premise of not influencing the survival level of the TIL after the TIL is pretreated with EGTA before electrotransformation.
Example 4: use of calcium ion chelating agent EGTA in electrotransformation for preparing T cells over-expressing EGFP
1) X-VIVO is preformed TM 15 medium was added to 4 wells in a 12-well plate, 2mL each, followed by transfer to a cell incubator at 37℃with 5% CO 2 Preheating for 1 hour;
2) The activated T cells prepared in example 2 were divided into 4 groups of 2X 10 cells 7 Individual cells, resuspended in 1) 4 wells of medium preheated at 37℃and IL-2 (R) added&Dsystems, cat.No.: BT-002) to a final concentration of 500IU/mL, 5% CO at 37 ℃ 2 The cell incubator was left to stand for 4 hours,
3) After the stationary treatment at 37℃for 4 hours, EGTA was added to the medium of each well to a final concentration of 0mM, 1mM, 1.5mM and 2mM, respectively, and the stationary treatment at 37℃was continued for 2 hours;
4) T cells treated with 0mM, 1mM, 1.5mM and 2mM EGTA were centrifuged for 3min at 800g, and the cell pellet was resuspended in PBS and centrifuged for 3min at 800g again, and the supernatant was discarded;
5) The ratio of the electrotransport liquid with single dosage per hole is carried out according to the following table:
100μL Nucleocuvette TM Strip(μL)
Nucleofector TM volume of solution 82
Electrolysis supplementary solution 18
6) Adding plasmid pKB20-EGFP 5 mug into the prepared electrotransfer solution, re-suspending the prepared plasmid-containing electrotransfer solution by 4 pipe T cell sediment obtained in 4), 100 mug each pipe, carefully sucking the cell re-suspension and transferring into LONZA 100 mug electrotransfer cup, placing the electrotransfer cup into LONZA Nucleofector TM 2b, starting an electric transfer program in the electric transfer groove, wherein the electric transfer program selects T-020; X-VIVO was previously performed 1 hour before electrotransformation TM 15 medium was added to 4 wells in a 12-well plate, 2mL each, and then transferred into a cell incubator at 37℃with 5% CO 2 Preheating for 1 hour;
7) After completion of the electrotransfer, the electrotransfer cup was carefully removed, the cell suspension was aspirated and transferred to EP tubes, and each tube was filled with pre-warmed X-VIVO containing 5% v/v human AB serum (Sigmaaldrich, cat. No.: H4522) at 37 ℃ TM 15 medium 200. Mu.L followed by transfer to a 12 well plate in 6) pre-warmed X-VIVO TM 15 medium wells, IL-2 was added to each well to a final concentration of 500IU/mL,37℃and 5% CO 2 Culturing.
The above EGTA concentration treatment conditions were further provided with 3 parallel sub-wells, and the cell viability and viable cell number in each well after 13 days of culture were measured by a cell counter after trypan blue staining, and the average value was obtained. The proportion (cell positive rate) and number (positive cell number) of cells positive for EGFP expression after electrotransformation were examined by flow cytometry.
The results are shown in FIGS. 5-8. FIG. 5 shows that the survival rate of cells exceeds 90% after electrotransformation of EGFP expression vectors by each group of T cells, and FIG. 6 shows that the total number of living cells is basically equivalent; FIG. 7 and FIG. 8 show that the proportion of EGFP-expressing positive cells and the total number of EGFP-expressing positive cells in T cells of the 1mM, 1.5mM and 2mM EGTA treated groups were significantly higher than in the 0mM group. This shows that the transfer efficiency of the exogenous gene can be obviously improved and the number of the transgenic T cells can be improved on the premise of not influencing the survival level of TIL after the T cells are pretreated with EGTA before electrotransformation.
Although specific embodiments of the application have been described in detail, those skilled in the art will appreciate. Numerous modifications and substitutions of details are possible in light of all the teachings disclosed, and such modifications are contemplated as falling within the scope of the present application. The full scope of the application is given by the appended claims and any equivalents thereof.

Claims (11)

1. A method of electrotransformation for preparing a cell expressing a foreign gene, comprising:
1) Treating the cells with a calcium ion chelating agent; and
2) A nucleic acid construct containing a gene encoding the exogenous gene is introduced into the cell by electrosteering.
2. The method of claim 1, wherein the calcium ion chelating agent is EGTA or EDTA,
preferably, the concentration of the calcium ion chelating agent in the treated system of step 1) is between 0.5 and 5mM, more preferably between 1.0 and 2.0mM.
3. The method of claim 1 or 2, wherein the cell is an immune effector cell; preferably, the immune effector cell is any one or more selected from T cells, DN T cells, NK cells, NKT cells, CAR-T cells, TCR-T cells, TIL, CTL, LAK cells and CIK cells,
preferably, the concentration of cells in the treatment system of step 1) is 1X 10 6 Up to 1X 10 10 And each mL.
4. The method according to claim 1 or 2, wherein the treatment of step 1) is performed in a buffer or in a cell culture medium,
preferably, the method comprises the steps of,
the cell culture medium comprises: AIM-V, X-VIVO, DMEM or RPMI1640,
the buffer solution comprises: phosphate-based buffers, TBS, tris-HCl, tris-TBS, HEPES buffer or cacodylic buffer.
5. The method of claim 1 or 2, wherein,
the treatment duration of step 1) is 0.5h-2h, and/or
Step 1) the treatment temperature is 30-37 ℃, and/or
The nucleic acid construct is DNA or RNA, such as a vector or mRNA.
6. The method of claim 1 or 2, wherein the cells are T cells, the method further comprising the step of activating the T cells prior to electrotransformation,
preferably, the step of activating T cells comprises: t cells are activated using anti-CD 3 antibodies and/or anti-CD 28 antibodies.
7. A method for treating cells to be electrotransformed, thereby improving the electrotransformation efficiency and cell viability, characterized in that the method comprises treating the cells with a calcium ion chelating agent prior to electrotransformation,
preferably, the method comprises the steps of,
the calcium ion chelating agent is EGTA or EDTA, and/or
The treatment is carried out in a buffer or in a cell culture medium, and/or
The treatment time is 0.5h-2h, and/or
The treatment temperature is 30-37 ℃, and/or
The cell is an immune effector cell and,
more preferably, the process is carried out,
the cell culture medium comprises: AIM-V, X-VIVO, DMEM or RPMI1640, and/or
The buffer solution comprises: phosphate-based buffers, TBS, tris-HCl, tris-TBS, HEPES buffer or cacodylic buffer, and/or
The concentration of the calcium ion chelating agent in the treated system is 0.5-5mM, and/or
The concentration of cells in the treated system was 1X 10 6 Up to 1X 10 10 personal/mL, and/or
The immune effector cells are any one or more selected from T cells, DN T cells, NK cells, NKT cells, CAR-T cells, TCR-T cells, TIL, CTL, LAK cells and CIK cells.
8. A cell treatment composition for treating cells to be electrotransformed to improve electrotransformation efficiency and cell viability, the treatment composition comprising a substrate and a calcium ion chelating agent,
preferably, the method comprises the steps of,
the calcium ion chelating agent is EGTA or EDTA, and/or
The matrix comprises a cell culture medium or buffer, and/or
The cell is an immune effector cell and,
more preferably, the process is carried out,
the final concentration of the calcium ion chelating agent in the cell treatment liquid is 0.1-50mM, and/or
The immune effector cell is any one or more selected from T cells, DN T cells, NK cells, NKT cells, CAR-T cells, TCR-T cells, TIL, CTL, LAK cells and CIK cells, and/or
The cell culture medium comprises AIM-V, X-VIVO, DMEM or RPMI1640, and/or
The buffer includes phosphate-based buffer, TBS, tris-HCl, tris-TBS, HEPES buffer or cacodylic buffer.
9. A cell treatment kit for treating cells to be electrotransformed to improve transduction efficiency and cell survival, the kit comprising a calcium ion chelating agent, the kit optionally further comprising a cell culture medium and/or a buffer,
preferably, the method comprises the steps of,
the calcium ion chelating agent is EGTA or EDTA, and/or
The cell culture medium comprises: AIM-V, X-VIVO, DMEM or RPMI1640, and/or
The buffer solution comprises: phosphate-based buffers, TBS, tris-HCl, tris-TBS, HEPES buffer, cacodylic buffer, and/or
The cell is an immune effector cell and,
more preferably, the process is carried out,
the immune effector cell is any one or more selected from T cells, DN T cells, NK cells, NKT cells, CAR-T cells, TCR-T cells, TIL, CTL, LAK cells and CIK cells, and/or
The cell processing kit is an electrotransport kit, further comprising an electrotransport fluid; preferably, the electrotransport liquid comprises a Nucleofector solution and an electrotransport replenishment solution, and/or
The kit further comprises T cell activating agents, such as anti-CD 3 antibodies and/or anti-CD 28 antibodies.
10. A cell expressing a foreign gene obtained by the method of any one of claim 1 to 6,
preferably, the cell is an immune effector cell; more preferably, the immune effector cell is any one or more selected from the group consisting of T cells, DN T cells, NK cells, NKT cells, CAR-T cells, TCR-T cells, TIL, CTL, LAK cells, and CIK cells.
11. The use of a calcium ion chelating agent for the treatment of cells to be electrotransformed,
preferably, the calcium ion chelating agent is EGTA or EDTA,
preferably, the cell is an immune effector cell; more preferably, the immune effector cell is any one or more selected from the group consisting of T cells, DN T cells, NK cells, NKT cells, CAR-T cells, TCR-T cells, TIL, CTL, LAK cells, and CIK cells.
CN202310864727.3A 2022-07-14 2023-07-14 Preparation method of immune effector cell expressing exogenous gene Pending CN116875545A (en)

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