CN117925535A - Culture method of CAR-T cells - Google Patents

Abstract

The invention provides a culture method of CAR-T cells, which comprises the following steps: s1: coating the cell culture container; s2: preparing a culture medium, wherein the culture medium comprises autologous plasma, alyS505,505N 0 serum-free cell culture medium and Gomesin; s3: adjusting the density of the PBMCs with a medium containing zoledronic acid, and then adding the PBMCs into a cell culture container; the cell culture vessel was placed in an incubator for culture. S4: adding a lentivirus of Polybrene and Mesothelin CAR to a cell culture vessel; placing into an incubator for culturing. S5: adding the culture medium into a cell culture container, and placing the cell culture container into an incubator for culture; s6: suspending cells and collecting cell suspension, transferring the cell suspension into a 15mL centrifuge tube, centrifuging, removing supernatant, suspending cell pellet with the culture medium, placing cells in a new cell culture container, and culturing in a culture box; s7: the cell density is adjusted by adopting a mode of half liquid exchange or liquid supplement, and the cells are harvested after 12 days of culture.

Description

Culture method of CAR-T cells

Technical Field

The application relates to the technical field of biology, in particular to a culture method of CAR-T cells.

Background

CAR-T cell immunotherapy is chimeric antigen Receptor T cell immunotherapy, english full-scale CHIMERIC ANTIGEN Receptor T-Cell Immunotherapy. The novel accurate targeted therapy for treating tumors has a good effect on clinical tumor treatment through optimization and improvement in recent years, and is a novel tumor immunotherapy method which is very promising, can be accurate, rapid and efficient and can possibly cure cancers; more specifically, it transduces genes encoding Chimeric Antigen Receptors (CARs) onto T cells of the patient themselves by genetic engineering and cell culture techniques, allowing the T cells to acquire new functions of recognizing and killing tumors. After in vitro activation and proliferation, a large number of CAR-T cells are returned to the body of a patient, and can be accurately positioned at the tumor part to play an anti-tumor role. The in vitro activation refers to the introduction of chimeric antigen receptor genes into T cells using viral or non-viral vectors to express chimeric antigen receptors. CAR-T cell therapy has shown powerful clinical efficacy in the field of hematological tumors, especially lymphomas.

The advantage of CAR-T cell therapy is that it combines the advantages of cell therapy and gene therapy, enabling precise programming and control of immune cell function. The CAR structure comprises a tumor antigen recognition domain and a T cell activation domain, and can redirect T cells to combine with specific tumor cell subsets, thereby playing a killing role. Meanwhile, the CAR-T cells also retain the proliferation capability of the T cells, and can be amplified in vivo to generate a durable anti-tumor immune response. Through the technological accumulation of nearly 30 years, CAR-T cell engineering preparation platforms have matured day by day.

Disclosure of Invention

The invention provides a culture method of CAR-T cells, which comprises the following steps:

S1: coating the cell culture container;

S2: preparing a culture medium, wherein the culture medium comprises autologous plasma, alyS505,505N 0 serum-free cell culture medium and Gomesin, and the amino acid sequence of Gomesin is shown as SEQ ID NO. 1;

s3: the PBMCs density was adjusted with a medium containing 5. Mu.M zoledronic acid and then added to a cell culture vessel; placing the cell culture container in an incubator, and culturing for 18h under a first culture condition;

S4: adding a lentivirus of Polybrene and Mesothelin CAR to a cell culture vessel; culturing in an incubator under a second culture condition for 48 hours;

s5: adding the culture medium into a cell culture container, and culturing for 24 hours under a third culture condition in an incubator;

s6: suspending cells with a disposable pipette, collecting cell suspension, transferring the cell suspension into a 15mL centrifuge tube, centrifuging, removing supernatant, suspending cell sediment by using the culture medium, placing the cells in a new cell culture container, continuing culturing in a culture box, and culturing under a fourth culture condition;

s7: the cell density is adjusted by adopting a mode of half liquid exchange or liquid supplement, and the cells are harvested after 12 days of culture.

Optionally, the concentration of Gomesin in the culture medium is 40-80 μg/ml.

Alternatively, the cell culture vessel is a 6-well plate.

Optionally, coating the cell culture container with a coating solution; the coating liquid adopts one of polylysine, fibrin and gelatin.

Optionally, the coating treatment of the cell culture vessel comprises the following steps:

S11, diluting the retroNectin to 100 mug/mL by using PBS buffer solution to obtain retroNectin diluent;

s12, adding the retroNectin diluent solution into a 6-hole plate, and adding 2mL of the retroNectin diluent solution into each hole;

S13, then placing a 6-well plate containing the retroNectin diluent in an incubator. Standing for 8h at 35 ℃.

Alternatively, the medium contains 10% autologous plasma, alyS N-0 serum-free cell culture broth of 600U/mL IL2, and 60 μg/mL Gomesin.

Alternatively, the PBMCs density is adjusted with a medium containing 5. Mu.M zoledronic acid, and then added to a cell culture vessel comprising the steps of:

PBMCs were density adjusted to 2X 10 ⁵ cells/mL medium with 5. Mu.M zoledronic acid and then added to the 6-well plate at 2mL per well.

Optionally, the first, second, third, and fourth culture conditions are the same.

Optionally, the first culture condition is that the temperature in the incubator is 35-38 ℃, and the concentration of CO ₂ in the incubator is 4-8%; and/or

The second culture condition is that the temperature in the incubator is 35-38 ℃, and the concentration of CO ₂ in the incubator is 4-8%; and/or;

The third culture condition is that the temperature in the incubator is 35-38 ℃, and the concentration of CO ₂ in the incubator is 4-8%; and/or;

The fourth culture condition is that the temperature in the incubator is 35-38 ℃, and the concentration of CO ₂ in the incubator is 4-8%.

Alternatively, lentiviruses of the Mesothelin CAR were added in one well at a ratio of moi=10.

Compared with the prior art, the culture method of the CAR-T cells can effectively improve the killing efficiency of the CAR-T cells on tumor cells.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and together with the description, serve to explain the principles of the invention.

In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, and it will be obvious to a person skilled in the art that other drawings can be obtained from these drawings without inventive effort.

FIG. 1 is an amplification curve of a cell expansion rate experiment in the present invention.

FIG. 2 shows the results of an in vitro killing experiment of target cells by CAR-T cells according to the present invention.

FIG. 3 is a flow chart of one embodiment of a method of culturing CAR-T cells according to the present invention.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present application more apparent, the technical solutions of the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present application, and it is apparent that the described embodiments are some embodiments of the present application, but not all embodiments of the present application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.

It should be noted that in this document, relational terms such as "first" and "second" and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, elements defined by the statement "include one … …" are not excluded from the package.

The invention provides a culture method of CAR-T cells, which comprises the following steps:

S1: coating the cell culture container;

S2: preparing a culture medium, wherein the culture medium comprises autologous plasma, alyS505,505N 0 serum-free cell culture medium and Gomesin, and the amino acid sequence of Gomesin is shown as SEQ ID NO. 1;

s3: the PBMCs density was adjusted with a medium containing 5. Mu.M zoledronic acid and then added to a cell culture vessel; placing the cell culture container in an incubator, and culturing for 18h under a first culture condition;

S4: adding a lentivirus of Polybrene and Mesothelin CAR to a cell culture vessel; culturing in an incubator under a second culture condition for 48 hours;

s5: adding the culture medium into a cell culture container, and culturing for 24 hours under a third culture condition in an incubator;

s6: suspending cells with a disposable pipette, collecting cell suspension, transferring the cell suspension into a 15mL centrifuge tube, centrifuging, removing supernatant, suspending cell sediment by using the culture medium, placing the cells in a new cell culture container, continuing culturing in a culture box, and culturing under a fourth culture condition;

s7: the cell density is adjusted by adopting a mode of half liquid exchange or liquid supplement, and the cells are harvested after 12 days of culture.

Optionally, the concentration of Gomesin in the culture medium is 40-80 μg/ml.

Alternatively, the cell culture vessel is a 6-well plate.

Optionally, coating the cell culture container with a coating solution; the coating liquid adopts one of polylysine, fibrin and gelatin.

Optionally, the coating treatment of the cell culture vessel comprises the following steps:

S11, diluting the retroNectin to 100 mug/mL by using PBS buffer solution to obtain retroNectin diluent;

s12, adding the retroNectin diluent solution into a 6-hole plate, and adding 2mL of the retroNectin diluent solution into each hole;

S13, then placing a 6-well plate containing the retroNectin diluent in an incubator. Standing for 8h at 35 ℃.

Alternatively, the medium contains 10% autologous plasma, alyS N-0 serum-free cell culture broth of 600U/mL IL2, and 60 μg/mL Gomesin.

Alternatively, the PBMCs density is adjusted with a medium containing 5. Mu.M zoledronic acid, and then added to a cell culture vessel comprising the steps of:

PBMCs were density adjusted to 2X 10 ⁵ cells/mL medium with 5. Mu.M zoledronic acid and then added to the 6-well plate at 2mL per well.

Optionally, the first, second, third, and fourth culture conditions are the same.

Optionally, the first culture condition is that the temperature in the incubator is 35-38 ℃, and the concentration of CO ₂ in the incubator is 4-8%; and/or

The second culture condition is that the temperature in the incubator is 35-38 ℃, and the concentration of CO ₂ in the incubator is 4-8%; and/or;

The third culture condition is that the temperature in the incubator is 35-38 ℃, and the concentration of CO ₂ in the incubator is 4-8%; and/or;

The fourth culture condition is that the temperature in the incubator is 35-38 ℃, and the concentration of CO ₂ in the incubator is 4-8%.

Alternatively, lentiviruses of the Mesothelin CAR were added in one well at a ratio of moi=10.

In the present invention, the terms "tumor" and "cancer" are interchangeable terms that refer to the growth or proliferation of any abnormal cell or tissue in an animal. "tumor" as used herein encompasses both solid tumors and hematological tumors, and also encompasses malignant, premalignant, and benign growths, such as dysplasia.

Non-limiting examples of solid tumors include lung cancer (small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC)), urinary tract cancer, squamous cell carcinoma, pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma, lung adenocarcinoma, lung squamous carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer (including endometrial cancer), salivary gland cancer, kidney cancer, prostate cancer, vulval cancer, thyroid cancer, brain cancer, testicular cancer, gall bladder cancer, cholangiocarcinoma, gastric cancer, oral cancer, neuroblastoma, multiple squamous cell carcinoma, chondrosarcoma, melanoma, nasopharyngeal carcinoma, thymus cancer, mucous sarcoma, melanoma, lymphoid malignancies, fibrosarcoma, mesothelioma, and various types of head and neck cancer. Non-limiting examples of hematological neoplasms include acute leukemia, chronic leukemia, polycythemia vera, lymphoma, hodgkin's disease, non-hodgkin's lymphoma, multiple myeloma, waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, spinal cord dysplasia.

"CAR-T cells" as used herein generally refer to T cells that have been recombinantly modified to express a CAR (also known as a "chimeric antigen receptor").

The "chimeric antigen receptor" and "CAR" described herein are used interchangeably. The CAR (chimeric antigen receptor) is a fusion protein that confers novel capabilities to immune cells, on the one hand, to target specific antigen proteins and, on the other hand, to transmit activation signals into cells. CARs generally comprise three main parts: extracellular domain, transmembrane domain and intracellular domain. The extracellular domain is capable of recognizing antigens, essentially determining the target cells of the CAR-T cell. The primary function of the transmembrane domain is on the one hand to anchor the CAR molecule on the cell membrane and on the other hand to link the extracellular domain of the CAR molecule to the intracellular domain and to transduce extracellular recognition signals into the cell, which have an important role in the stable expression of the CAR molecule. Intracellular domains may include signal transduction domains (CSD) and co-stimulatory domains.

The term "vector" in the context of the present invention may also be a "CAR-expressing vector", which is used interchangeably herein, and is a replicon, such as a plasmid, phage, virus, cosmid, BAC or YAC, or the like, to replicate and/or express an attached nucleic acid molecule in a cell. "vector" includes episomal (e.g., plasmid) or non-episomal vectors. The term "vector" includes viral and non-viral means for introducing a nucleic acid molecule into a cell in vitro, in vivo or ex vivo. The term "vector" may include synthetic vectors. The vector may be introduced into the desired host cell by well known methods including, but not limited to, transfection, cell fusion and lipofection.

In some embodiments, the vector is derived from or comprises a non-viral vector. Advantages of non-viral vectors include ease of production and relatively low cost of sufficient amounts required to meet the entire patient population, stability during storage, and lack of immunogenicity. Non-limiting examples of transposon systems suitable for use in the vectors of the present technology are Mariner-like transposons such as sleeping beauty transposons, piggyBac-like transposons or hAT family transposons such as TcBuster transposons.

In the context of the present invention, viruses "include, but are not limited to, lentiviruses, retroviruses, adenoviruses and adeno-associated viruses. It will be apparent that viral vectors according to the present disclosure are not necessarily limited to specific viral components. The viral vectors may comprise components derived from two or more different viruses, and may also comprise synthetic components.

Gomesin in the present invention is an active compound extracted from spider venom, which is a polypeptide containing 18 amino acids, having a molecular weight of 2292.7Da and an amino acid sequence of ZCRRLCYKQRCVTYCRGR (SEQ ID NO. 1). Gomesin have a broad spectrum of biological activity, such as their bactericidal effect against a wide variety of bacteria, fungi and parasites.

Example 1

Culture of CAR-T cells

RetroNectin was diluted to 100. Mu.g/mL using PBS buffer, and the addition of RetroNectin diluent was 2mL per well in a 6-well plate. And placing the 6-hole plate with the retroNectin diluent in an incubator at 35 ℃ for standing for 8 hours, removing the retroNectin diluent in the 6-hole plate before use, and washing 1-2 times by using 1mL of PBS. Peripheral blood was collected, and the blood was equally distributed to two 50mL sterile centrifuge tubes and centrifuged at 800g at room temperature for 10min. The plasma was transferred to a new 50mL sterile centrifuge tube using a disposable sterile pipette and placed in a thermostatic incubator at 55 ℃ for 30min to inactivate the complement. Adding physiological saline into the centrifuged blood cells to make the final volume of the blood cells 15mL, uniformly mixing, slowly moving the diluted blood cells into a 50mL centrifuge tube containing 15mL lymphocyte separation liquid according to the proportion of 1:1 (volume ratio) by adopting a disposable sterile pipette, gently moving the centrifuge tube into the centrifuge, and centrifuging at 400g for 30min at room temperature. The centrifuge tube was removed, placed in a biosafety cabinet, and the middle PBMCs layer was aspirated using a 10mL disposable sterile pipette and placed into a new 50mL centrifuge tube. Then adding physiological saline to 45mL, uniformly mixing, and centrifuging at 450g for 8min at room temperature. The wash was repeated once. The medium contained 10% autologous plasma, alyS N-0 serum-free cell culture medium of 600U/mL IL2 and 60 μg/mL Gomesin. The amino acid sequence of Gomesin is shown as SEQ ID NO. 1; PBMCs were density adjusted to 2X 10 ⁶ cells/mL medium with 5. Mu.M zoledronic acid, then added to a 6-well plate with 2mL each, and then incubated in an incubator at 35℃with 6% CO ₂ for 18h. Polybrene was added at 8 μg/mL per well in 6-well plates, and lentivirus (CAR-T) of Mesothelin CAR was added to each well at a ratio of MOI=10, and centrifuged at 25℃for 30min; the 6-well plate was taken out, placed at 35℃and cultured in a 6% CO ₂ incubator for 48 hours. Adding 2mL of the culture medium into each hole of a 6-hole plate, and continuously placing the mixture into a 35 ℃ and 6% CO ₂ incubator for culturing for 24 hours; suspending cells by a disposable pipette, collecting cell suspension into a 15mL centrifuge tube, centrifuging at 450g at room temperature for 5min, discarding supernatant in the centrifuge tube, suspending cell sediment by using 3mL of the culture medium, placing the cells into a new 6-hole plate, and continuously culturing in a 6% CO ₂ incubator at 35 ℃; the cell density was adjusted to 2X 10 ⁶ cells/mL medium by half-volume exchange or replacement and placed in a 35℃6% CO ₂ incubator for culture. Harvesting cells on day 12

Example 2

RetroNectin was diluted to 100. Mu.g/mL using PBS buffer, and the addition of RetroNectin diluent was 2mL per well in a 6-well plate. And placing the 6-hole plate with the retroNectin diluent in an incubator at 35 ℃ for standing for 8 hours, removing the retroNectin diluent in the 6-hole plate before use, and washing 1-2 times by using 1mL of PBS. Peripheral blood was collected, and the blood was equally distributed to two 50mL sterile centrifuge tubes and centrifuged at 800g at room temperature for 10min. The plasma was transferred to a new 50mL sterile centrifuge tube using a disposable sterile pipette and placed in a thermostatic incubator at 55 ℃ for 30min to inactivate the complement. Adding physiological saline into the centrifuged blood cells to make the final volume of the blood cells 15mL, uniformly mixing, slowly moving the diluted blood cells into a 50mL centrifuge tube containing 15mL lymphocyte separation liquid according to the proportion of 1:1 (volume ratio) by adopting a disposable sterile pipette, gently moving the centrifuge tube into the centrifuge, and centrifuging at 400g for 30min at room temperature. The centrifuge tube was removed, placed in a biosafety cabinet, and the middle PBMCs layer was aspirated using a 10mL disposable sterile pipette and placed into a new 50mL centrifuge tube. Then adding physiological saline to 45mL, uniformly mixing, and centrifuging at 450g for 8min at room temperature. The wash was repeated once. The medium contained 10% autologous plasma, alyS N-0 serum-free cell culture broth of 600U/mL IL 2. PBMCs were density adjusted to 2X10 ⁶ cells/mL medium with 5. Mu.M zoledronic acid, then added to a 6-well plate with 2mL each, and then incubated in an incubator at 35℃with 6% CO ₂ for 18h. Polybrene was added at 8 μg/mL per well in 6-well plates, and lentivirus (CAR-T) of Mesothelin CAR was added to each well at a ratio of MOI=10, and centrifuged at 25℃for 30min; the 6-well plate was taken out, placed at 35℃and cultured in a 6% CO ₂ incubator for 48 hours. Adding 2mL of the culture medium into each hole of a 6-hole plate, and continuously placing the mixture into a 35 ℃ and 6% CO ₂ incubator for culturing for 24 hours; suspending cells by a disposable pipette, collecting cell suspension into a 15mL centrifuge tube, centrifuging at 450g at room temperature for 5min, discarding supernatant in the centrifuge tube, suspending cell sediment by using 3mL of the culture medium, placing the cells into a new 6-hole plate, and continuously culturing in a 6% CO ₂ incubator at 35 ℃; the cell density was adjusted to 2X10 ⁶ cells/mL medium by half-volume exchange or replacement and placed in a 35℃6% CO ₂ incubator for culture. Harvesting cells on day 12

Cell expansion rate experiments:

the fold expansion of the cells in examples 1 and 2 relative to the initial cell number was determined and an expansion curve was drawn on days 3,6,9, 12, respectively; the amplification curve is plotted as shown in FIG. 1.

The specific test mode is that 2 holes are randomly taken from a 6-hole plate in each test, the expansion times of cells in two holes are calculated respectively, and the average value of the expansion times of the two holes is calculated.

The experimental results show that example 1 expands faster than example 2, indicating that increasing Gomesin in the medium is beneficial to increasing the expansion rate of the cells.

Cell killing activity assay:

SKOV3 cells expressing Mesothelin were labeled with Calcein-AM.

Experimental group: the CAR-T cells obtained in example 1 were incubated with Calcein-AM labeled SKOV3 cells at a ratio of 10:1 for 4h.

Comparison group: the CAR-T cells obtained in example 2 were incubated with Calcein-AM labeled SKOV3 cells at a ratio of 10:1 for 4h.

Blank group: the obtained T cells were incubated with Calcein-AM-labeled SKOV3 cells at a ratio of 10:1 for 4h.

In the experiment, the in vitro killing of the CAR-T cells on the target cells is calculated by detecting the release of Calcein after the target cells are killed. The experimental results are shown in FIG. 2.

Experimental results show that CAR-T cells cultured in example 1 have stronger killing activity than the control group. It is demonstrated that the addition of Gomesin to the culture medium can effectively improve the killing activity of the CAR-T cells.

The foregoing is only a specific embodiment of the invention so that those skilled in the art may understand or practice the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (10)

1. A method of culturing CAR-T cells, comprising the steps of:

S1: coating the cell culture container;

S2: preparing a culture medium, wherein the culture medium comprises autologous plasma, alyS505,505N 0 serum-free cell culture medium and Gomesin, and the amino acid sequence of Gomesin is shown as SEQ ID NO. 1;

s3: the PBMCs density was adjusted with a medium containing 5. Mu.M zoledronic acid and then added to a cell culture vessel; placing the cell culture container in an incubator, and culturing for 18h under a first culture condition;

S4: adding a lentivirus of Polybrene and Mesothelin CAR to a cell culture vessel; culturing in an incubator under a second culture condition for 48 hours;

s5: adding the culture medium into a cell culture container, and culturing for 24 hours under a third culture condition in an incubator;

s6: suspending cells with a disposable pipette, collecting cell suspension, transferring the cell suspension into a 15mL centrifuge tube, centrifuging, removing supernatant, suspending cell sediment by using the culture medium, placing the cells in a new cell culture container, continuing culturing in a culture box, and culturing under a fourth culture condition;

s7: the cell density is adjusted by adopting a mode of half liquid exchange or liquid supplement, and the cells are harvested after 12 days of culture.

2. The method of claim 1, wherein the concentration of Gomesin in the medium is 40 μg/ml to 80 μg/ml.

3. The method of claim 2, wherein the cell culture vessel is a 6-well plate.

4. A method of culturing CAR-T cells according to claim 3, wherein the cell culture vessel is coated with a coating liquid; the coating liquid adopts one of polylysine, fibrin and gelatin.

5. The method of claim 4, wherein the step of coating the cell culture vessel comprises the steps of:

S11, diluting the retroNectin to 100 mug/mL by using PBS buffer solution to obtain retroNectin diluent;

s12, adding the retroNectin diluent solution into a 6-hole plate, and adding 2mL of the retroNectin diluent solution into each hole;

S13, then placing a 6-well plate containing the retroNectin diluent in an incubator. Standing for 8h at 35 ℃.

6. The method of claim 5, wherein the medium comprises 10% autologous plasma, alyS N-0 serum-free cell culture broth of 600U/mL IL2, and 60 μg/mL Gomesin.

7. The method of claim 6, wherein the adjusting the density of PBMCs with a medium containing 5 μΜ zoledronic acid, and then adding the medium to a cell culture vessel comprises the steps of:

PBMCs were density adjusted to 2X 10 ⁵ cells/mL medium with 5. Mu.M zoledronic acid and then added to the 6-well plate at 2mL per well.

8. The method of claim 7, wherein the first, second, third, and fourth culture conditions are the same.

9. The method for culturing CAR-T cells according to claim 1, wherein the first culturing condition is that the temperature in the incubator is 35-38 ℃, and the concentration of CO ₂ in the incubator is 4-8%; and/or

The second culture condition is that the temperature in the incubator is 35-38 ℃, and the concentration of CO ₂ in the incubator is 4-8%; and/or;

The third culture condition is that the temperature in the incubator is 35-38 ℃, and the concentration of CO ₂ in the incubator is 4-8%; and/or;

The fourth culture condition is that the temperature in the incubator is 35-38 ℃, and the concentration of CO ₂ in the incubator is 4-8%.

10. The method of claim 8, wherein the lentivirus of the Mesothelin CAR is added to one well at a ratio of MOI = 10.