A culture medium for γδT cells and a culture method thereof

Abstract

The present invention discloses a culture fluid and a culture method of γδT cells, the culture fluid comprises an activation culture fluid and an expansion culture fluid with a serum-free culture medium as a base culture fluid; wherein the activation culture fluid comprises IL-21, AMD3100, Metformin, IPI-145, Bezafibrate, Rapamycin and Atorvastatin components, and the expansion culture fluid comprises L-glutamine, penicillin, streptomycin, 2-mercaptoethanol and non-essential amino acids with a working concentration of 1X. The γδT cell culture fluid adopts a serum-free culture medium, avoids the risks caused by potential pathogens and immune responses in serum, improves the safety of γδT cells and the feasibility of clinical application; the combined use of each component not only promotes the proliferation of γδT cells, but also enhances cytotoxicity and cytokine secretion capacity, and improves its anti-tumor effect.

Description

Technical Field

[0001] The present invention relates to the field of immune cell preparation, and in particular to a culture medium for γδT cells and an amplification and culture method for culturing γδT cells using the culture medium. Background Art

[0002] γδT cells are a special T cell subset that plays an important role in both innate and adaptive immunity. Unlike traditional αβT cells, γδT cells can rapidly respond to pathogens and tumor cells and exert their immune functions through non-MHC restricted recognition mechanisms.

[0003] In recent years, the potential of γδT cells in tumor immunotherapy, infectious diseases and autoimmune diseases has attracted widespread attention. However, the technical challenges of in vitro expansion of γδT cells have been one of the bottlenecks restricting their clinical application.

[0004] Currently, commonly used γδT cell expansion methods usually rely on the use of zoledronic acid (ZOL) or isopropyl acrylate (IPP) as an activator and require the addition of serum (such as human AB serum, fetal bovine serum, FBS) to the culture medium. However, the addition of serum brings many problems, including batch-to-batch differences, potential pathogen contamination risks, safety impacts on clinical applications, and high costs.

[0005] Therefore, developing a serum-free culture medium for the activation and expansion of γδT cells has important practical application value. Summary of the invention

[0006] Based on the above problems, the present invention proposes a γδT cell culture fluid prepared based on a serum-free culture medium, which can efficiently activate and amplify the growth of γδT cells under serum-free conditions, and has the advantages of high cell purity and activity, low production cost and good safety.

[0007] The technical solution 1 of the present invention is as follows:

[0008] A γδT cell culture medium, comprising an activation culture medium and an expansion culture medium; wherein:

[0009] The activation culture solution comprises a serum-free culture medium, wherein the serum-free culture medium comprises IL-21 with a final concentration of 1 to 50 ng / mL, AMD3100 with a final concentration of 1 to 15 μg / mL, Metformin with a final concentration of 0.1 to 10 mM, IPI-145 with a final concentration of 50 to 500 nM, Bezafibrate with a final concentration of 50 to 500 μM, Rapamycin with a final concentration of 1 to 10 nM, and Atorvastatin with a final concentration of 0.1 to 1 μM;

[0010] The amplification culture medium includes a serum-free culture medium, which contains L-glutamine with a final concentration of 0.5 to 20 mM, penicillin with a final concentration of 10 to 500 U / mL, streptomycin with a final concentration of 10 to 500 μg / mL, 2-mercaptoethanol with a final concentration of 10 to 100 μM, vitamin C with 50 to 500 μM, and non-essential amino acids with a working concentration of 1X.

[0011] In one embodiment, the serum-free culture medium of the activation culture fluid contains IL-21 with a final concentration of 20 ng / mL, AMD3100 with a final concentration of 5 μg / mL, Metformin with a final concentration of 1 mM, IPI-145 with a final concentration of 50 to 500 nM, Bezafibrate with a final concentration of 50 to 500 μM, Rapamycin with a final concentration of 1 to 10 nM, and Atorvastatin with a final concentration of 0.1 to 1 μM.

[0012] In one embodiment, the serum-free medium of the amplification culture medium comprises L-glutamine at a final concentration of 2 mM, penicillin at a final concentration of 100 U / mL, streptomycin at a final concentration of 100 μg / mL, 2-mercaptoethanol at a final concentration of 50 μM, and non-essential amino acids at a working concentration of 1X.

[0013] In one embodiment, in the γδT cell culture medium, the serum-free culture medium is RPMI-1640, 581 or DMEM.

[0014] The present invention also provides the use of the above-prepared γδT cell culture fluid to carry out amplification and culture of γδT cells, comprising the following steps:

[0015] On day 0, mononuclear cells were inoculated into a culture flask containing the above-mentioned activation medium and the cell density was controlled to be 1×10 6 The culture bottle was placed in an incubator at 37° C. and 5 v / v% CO 2 and cultured until the 14th day of culture;

[0016] On the third day, the amplification culture medium is added to the culture bottle, and the ratio of the volume of the amplification culture medium added to the volume of the culture medium in the culture bottle is 1:1;

[0017] On the fifth day, the cells were transferred to a culture bag, and the activation culture solution and the expansion culture solution were added to the culture bag, and the volume ratio of the activation culture solution and the expansion culture solution was 1:2, and the total volume of the activation culture solution and the expansion culture solution added was 1:1 to the volume of the culture solution in the culture bottle;

[0018] On the 7th day, the expansion culture medium as claimed in claim 1 is supplemented, and the ratio of the volume of the supplemented expansion culture medium to the volume of the culture medium in the culture bag is 1:2;

[0019] On the 9th day and the 13th day, the above-mentioned expansion culture solution is supplemented, and the ratio of the volume of the expansion culture solution added each time to the volume of the culture solution in the culture bag is 1:1;

[0020] On the 14th day, the culture was stopped, the cells were collected and transferred to a centrifuge tube, and centrifuged at 800 g for 10 min, and the supernatant was removed; the cell pellet was washed with PDS to obtain the γδT cells.

[0021] The culture medium and culture method of γδT cells provided by the present invention have the following advantages:

[0022] 1) In the activated culture medium of γδT cells, IPI-145 can regulate the activity of T cells and B cells and activate the proliferation of γδT cells; Bezafibrate can regulate cell metabolism and promote T cell proliferation; Rapamycin and Atorvastatin can reduce immune metabolism and exhaustion during cell proliferation, promote the proliferation of γδT cells, and improve the activity and stability of cells;

[0023] 2) In the activation culture medium of γδT cells, components such as IPI-145, Bezafibrate, Rapamycin and Atorvastatin are all small molecule compounds that can be absorbed and utilized by cells, thereby promoting the expansion and growth of γδT cells;

[0024] 3) In the activation culture medium of γδT cells, the combination of IL-21, AMD3100 and Metformin promoted the proliferation of γδT cells, enhanced their functional activities such as cytotoxicity and cytokine secretion, and improved their anti-tumor effect;

[0025] 4) Both the activation medium and expansion medium of γδT cells are based on serum-free culture medium, which avoids the risks of potential pathogens and immune responses in serum, and improves the safety of γδT cells and the feasibility of clinical application;

[0026] 5) During the γδT cell culture process, the addition of activation culture medium in the first 5 days can regulate cell activity, reduce cellular immune metabolism, and activate the activity and cytotoxicity of γδT cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The growth curves of γδT cell culture expansion in Examples 1 to 5;

[0028] Figure 2 This is a tumor killing curve diagram of γδT cells cultured on the 14th day in Examples 1 to 5;

[0029] Figure 3This is a flow cytometry graph of the purity of γδT cells on day 14 of culture in Example 1;

[0030] Figure 4 This is a flow cytometry graph of the purity of γδT cells on day 14 of culture in Example 2;

[0031] Figure 5 This is a flow cytometry graph of the purity of γδT cells on day 14 of culture in Example 3;

[0032] Figure 6 This is a flow cytometry graph of the purity of γδT cells on the 14th day of culture in Example 4 (Comparative Example 1);

[0033] Figure 7 This is a flow cytometric graph of the purity of γδT cells on the 14th day of culture in Example 5 (Comparative Example 2). DETAILED DESCRIPTION

[0034] The preferred embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings.

[0035] 1. Obtaining Mononuclear Cells

[0036] Fresh peripheral blood drawn from a healthy person is slowly transferred to a 50 ml centrifuge tube containing Ficoll solution. The ratio of peripheral blood to Ficoll solution is 1:2. Try not to destroy the boundary layer between the two liquids during the operation.

[0037] Transfer the centrifuge tube to the centrifuge, set the setting to 5°C and 0°C, and centrifuge at 800g for 20 minutes at room temperature after the centrifuge is balanced;

[0038] After centrifugation, transfer the mononuclear cells in the middle white film layer to another 50 ml centrifuge tube, add DPBS solution to wash, and centrifuge the washed cells at 500g for 10 minutes in the room temperature. Repeat the washing and centrifugation 2 to 3 times to collect the mononuclear cells for later use.

[0039] 2. Preparation of γδT cell culture medium

[0040] The γδT cell culture fluid provided by the present invention is based on a serum-free culture medium, and an activation culture fluid and an expansion culture fluid are prepared separately according to the needs during the culture process; the activation culture fluid is used to induce and activate mononuclear cells to differentiate into γδT cells, while the expansion culture fluid promotes the rapid expansion and growth of γδT cells.

[0041] 2.1) Preparation of activation culture medium

[0042] Based on a serum-free medium, the serum-free medium contains IL-21 at a final concentration of 1 to 50 ng / mL, AMD3100 (CXCR4 receptor antagonist) at a final concentration of 1 to 15 μg / mL, Metformin (metformin) at a final concentration of 0.1 to 10 mM, IPI-145 (Duvelisib) at a final concentration of 50 to 500 nM, Bezafibrate (bezafibrate) at a final concentration of 50 to 500 μM, Rapamycin (rapamycin) at a final concentration of 1 to 10 nM, and Atorvastatin (atorvastatin) at a final concentration of 0.1 to 1 μM;

[0043] 2.2) Preparation of amplification culture medium

[0044] The serum-free medium is based on L-glutamine at a final concentration of 0.5 to 20 mM, penicillin at a final concentration of 10 to 500 U / mL, streptomycin at a final concentration of 10 to 500 μg / mL, 2-mercaptoethanol at a final concentration of 10 to 100 μM, vitamin C at a final concentration of 50 to 500 μM, and non-essential amino acids at a working concentration of 1X.

[0045] The working concentration of the non-essential amino acids is 1X (wherein 1X means 1 times the working concentration), and the non-essential amino acids include alanine (Alanine, Ala), glutamic acid (Glutamic acid, Glu), glycine (Glycine, Gly), proline (Proline, Pro), asparagine (Asparagine, Asn), aspartic acid (Aspartic acid, Asp), and serine (Serine, Ser).

[0046] In the preparation of the above-mentioned γδT cell culture medium, the preparation of the activation culture medium also includes the following preferred preparation scheme:

[0047] The serum-free medium of the activation culture medium contains IL-21 at a final concentration of 5 to 30 ng / mL, AMD3100 at a final concentration of 3 to 10 μg / mL, Metformin at a final concentration of 0.5 to 5 mM, IPI-145 at a final concentration of 50 to 500 nM, Bezafibrate at a final concentration of 50 to 500 μM, Rapamycin at a final concentration of 1 to 10 nM, and Atorvastatin at a final concentration of 0.1 to 1 μM; or,

[0048] The serum-free medium for activation culture fluid contains IL-21 at a final concentration of 20 ng / mL, AMD3100 at a final concentration of 5 μg / mL, Metformin at a final concentration of 1 mM, IPI-145 (Duvelisib) at a final concentration of 200 nM, Bezafibrate at a final concentration of 300 μM, Rapamycin at a final concentration of 6 nM, and Atorvastatin at a final concentration of 0.7 μM.

[0049] In the preparation of the above-mentioned γδT cell culture medium, the preparation of the expansion culture medium also includes the following preferred preparation scheme:

[0050] The serum-free medium of the expansion culture medium contains L-glutamine at a final concentration of 1 to 10 mM, penicillin at a final concentration of 20 to 300 U / mL, streptomycin at a final concentration of 20 to 300 μg / mL, 2-mercaptoethanol at a final concentration of 20 to 80 μM, vitamin C at a final concentration of 50 to 500 μM, and non-essential amino acids at a working concentration of 1X; or,

[0051] The serum-free medium of the expansion culture medium contains L-glutamine at a final concentration of 2 mM, penicillin at a final concentration of 100 U / mL, streptomycin at a final concentration of 100 μg / mL, 2-mercaptoethanol at a final concentration of 50 μM, vitamin C at a final concentration of 200 μM, and non-essential amino acids at a working concentration of 1X.

[0052] In the preparation of the above-mentioned γδT cell culture solution, the serum-free culture medium is selected from any one of RPMI-1640, 581 or DMEM.

[0053] Among the components of the activation culture fluid of the present invention, IL-21, as an important cytokine, can inhibit the proliferation of B cell lymphoma cell subsets and enhance the proliferation and functional activity of γδT cells; AMD3100 can promote the migration and proliferation of γδT cells by blocking the CXCR4 / SDF-1 axis; 1P1-145 (Duvelisib) can regulate the activity of T cells and B cells, and play a regulatory role in the proliferation of γδT cells, which can help improve the expansion effect and functionality; Bezafibrate can activate peroxisome proliferator-activated receptors and has the effect of regulating cell metabolism and promoting T cell proliferation; Metformin, Rapamycin and Atorvastatin enhance the proliferation, activity and stability of γδT cells by regulating the metabolism and functional pathways of immune cells.

[0054] 3. Cultivation of γδT cells

[0055] The present invention also provides a method for culturing γδT cells, wherein the culture medium prepared as above is used in the culturing process, such as activation culture medium and expansion culture medium.

[0056] The γδT cell culture method of the present invention comprises the following steps:

[0057] On day 0, the mononuclear cells obtained in item 1 above were inoculated into a culture bottle containing the activation culture medium prepared in item 2.1) above, and the cell density was controlled to be 1×10 6 / mL; the culture bottle was placed in an incubator at 37°C and 5v / v% CO2 and cultured until the end of the 14th day;

[0058] On the third day, the expansion culture medium prepared in the above item 2.2) is added to the culture bottle, and the ratio of the volume of the additional expansion culture medium to the volume of the culture medium in the culture bottle is 1:1;

[0059] On the 5th day, the cells were transferred to a culture bag, and the activation culture solution prepared in the above item 2.1) and the expansion culture solution prepared in the above item 2.2) were added to the culture bag respectively, and the volume ratio of the activation culture solution to the expansion culture solution was 1:2, and the total volume ratio of the added activation culture solution and the expansion culture solution to the volume of the culture solution in the culture bottle was 1:1;

[0060] On the 7th day, the expansion culture medium as claimed in claim 1 is supplemented, and the ratio of the volume of the supplemented expansion culture medium to the volume of the culture medium in the culture bag is 1:2;

[0061] On the 9th day and the 13th day, the expansion culture medium prepared in the above item 2.2) is added respectively, and the ratio of the volume of the expansion culture medium added each time to the volume of the culture medium in the culture bag is 1:1;

[0062] On the 14th day, the culture was stopped, the cells were collected and transferred to a centrifuge tube, and centrifuged at 800 g for 10 min, and the supernatant was removed; the cells were washed 2 to 3 times with PDS to obtain high-purity γδT cells.

[0063] The γδT cell culture fluid provided by the present invention uses a serum-free culture medium as the basic culture medium, thereby avoiding the risks caused by potential pathogens and immune responses in the serum, and improving the safety of γδT cells and the feasibility of clinical application; the combined use of the respective components in the culture fluid not only promotes the proliferation of γδT cells, but also enhances their functional activity, including cytotoxicity and cytokine secretion capacity, and improves their anti-tumor effect; at the same time, since the present invention does not use expensive serum, the cell culture process also greatly reduces the culture cost of γδT cell expansion, making large-scale production and clinical application more economical and feasible.

[0064] The γδT cell expansion method provided by the present invention can be used to directly expand γδT cells from mononuclear cells separated from peripheral blood (PBMC), simplifies the operation process, and is suitable for large-scale cell preparation and clinical treatment needs.

[0065] The γδT cells obtained by the present invention through an efficient and safe expansion method have excellent anti-tumor activity and immunomodulatory function, providing new technical support and application prospects for cell therapy for various indications such as solid tumors, infectious diseases and autoimmune diseases.

[0066] The technical solution of the present invention is further described in detail below through specific embodiments and comparative examples.

[0067] In the following examples, the mononuclear cells used were all from the above item 1; and for the convenience of statistics, the same number of mononuclear cells were inoculated.

[0068] 1. γδT cell culture

[0069] Example 1

[0070] 1.1. Preparation of γδT cell culture medium

[0071] Prepare 100 mL of activation culture medium: based on RPMI-1640 serum-free medium, add IL-21 with a final concentration of 20 ng / mL, AMD3100 with a final concentration of 5 μg / mL, Metformin with a final concentration of 1 mM, IPI-145 with a final concentration of 200 nM, Bezafibrate with a final concentration of 300 μM, Rapamycin with a final concentration of 6 nM, and Atorvastatin with a final concentration of 0.7 μM to the serum-free medium;

[0072] Prepare 1500 mL of expansion culture medium: based on RPMI-1640 serum-free medium, add L-glutamine at a final concentration of 2 mM, penicillin at a final concentration of 100 U / mL, streptomycin at a final concentration of 100 μg / mL, 2-mercaptoethanol at a final concentration of 50 μM, vitamin C at a final concentration of 500 μM, and non-essential amino acids at a working concentration of 1X to the serum-free medium.

[0073] 1.2. Expansion and culture of γδT cells

[0074] On day 0, take 0.2×10 6 mononuclear cells were inoculated into a TC75 culture flask, and the culture flask contained 30 mL of the activation culture medium prepared in step 1.1 of this example; the culture flask was placed in an incubator at 37° C. and 5 v / v% CO2 and cultured until the end of the 14th day;

[0075] On the third day, 30 mL of the expansion culture medium prepared in step 1.1 of this example was added to the culture flask;

[0076] On the 5th day, the cells were transferred to a culture bag, and 20 mL of the activation culture medium and 40 mL of the expansion culture medium prepared in step 1.1 of this example were added to the culture bag;

[0077] On the 7th day, 120 mL of the expansion culture medium prepared in step 1.1 of this example was added;

[0078] On the 9th day, 180 mL of the expansion culture medium prepared in step 1.1 of this example was added;

[0079] On the 11th day, 360 mL of the expansion culture medium prepared in step 1.1 of this example was added;

[0080] On the 13th day, 720 mL of the expansion culture medium prepared in step 1.1 of this example was added;

[0081] On the 14th day, the culture was stopped, the cells were collected and transferred to a centrifuge tube, and centrifuged at 800 g for 10 min, and the supernatant was removed; the cell pellet was repeatedly washed twice with PDS to obtain the γδT cells.

[0082] Example 2

[0083] 2.1 Preparation of γδT cell culture medium

[0084] Prepare 100 mL of activation culture medium: based on 581 serum-free medium, add IL-21 with a final concentration of 50 ng / mL, AMD3100 with a final concentration of 15 μg / mL, Metformin with a final concentration of 0.1 mM, IPI-145 with a final concentration of 500 nM, Bezafibrate with a final concentration of 50 μM, Rapamycin with a final concentration of 10 nM, and Atorvastatin with a final concentration of 0.1 μM;

[0085] Prepare 1000 mL of expansion culture medium: based on RPMI-1640 serum-free medium, add L-glutamine at a final concentration of 20 mM, penicillin at a final concentration of 500 U / mL, streptomycin at a final concentration of 10 μg / mL, 2-mercaptoethanol at a final concentration of 100 μM, vitamin C at a final concentration of 50 μM, and non-essential amino acids at a working concentration of 1X to the serum-free medium.

[0086] 3.2. Expansion and culture of γδT cells

[0087] On day 0, take 0.2×10 6mononuclear cells were inoculated into a TC75 culture flask, and the culture flask contained 25 mL of the activation culture medium prepared in step 2.1 of this example; the culture flask was placed in an incubator at 37° C. and 5 v / v% CO2 and cultured until the end of the 14th day;

[0088] On the third day, 25 mL of the expansion culture medium prepared in step 2.1 of this example was added to the culture flask;

[0089] On the 5th day, the cells were transferred to a culture bag, and 16.67 mL of activation culture medium and 33.33 mL of expansion culture medium prepared in step 2.1 of this example were added to the culture bag;

[0090] On the 7th day, 100 mL of the expansion culture medium prepared in step 2.1 of this example was added;

[0091] On the 9th day, 150 mL of the expansion culture medium prepared in step 2.1 of this example was added;

[0092] On the 11th day, 300 mL of the expansion culture medium prepared in step 2.1 of this example was added;

[0093] On the 13th day, 600 mL of the expansion culture medium prepared in step 2.1 of this example was added;

[0094] On the 14th day, the culture was stopped, the cells were collected and transferred to a centrifuge tube, and centrifuged at 800 g for 10 min, and the supernatant was removed; the cell pellet was repeatedly washed twice with PDS to obtain the γδT cells.

[0095] Example 3

[0096] 3.1 Preparation of γδT cell culture medium

[0097] Prepare 100 mL of activation culture medium: based on DMEM serum-free medium, add IL-21 with a final concentration of 1 ng / mL, AMD3100 with a final concentration of 1 μg / mL, Metformin with a final concentration of 10 mM, IPI-145 with a final concentration of 500 μM, Bezafibrate with a final concentration of 500 μM, Rapamycin with a final concentration of 1 nM, and Atorvastatin with a final concentration of 1 μM;

[0098] Prepare 2500 mL of expansion culture medium: based on DMEM serum-free medium, add L-glutamine at a final concentration of 0.5 mM, penicillin at a final concentration of 10 U / mL, streptomycin at a final concentration of 500 μg / mL, 2-mercaptoethanol at a final concentration of 10 μM, vitamin C at a final concentration of 500 μM, and non-essential amino acids at a working concentration of 1X to the serum-free medium.

[0099] 3.2. Expansion and culture of γδT cells

[0100] On day 0, take 0.2×10 6 mononuclear cells were inoculated into a TC75 culture flask, and the culture flask contained 50 mL of the activation culture solution prepared in step 3.1 of this example; the culture flask was placed in an incubator at 37° C. and 5 v / v% CO2 and cultured until the end of the 14th day;

[0101] On the third day, 50 mL of the expansion culture medium prepared in step 3.1 of this example was added to the culture flask;

[0102] On the 5th day, the cells were transferred to a culture bag, and 33.33 mL of the activation culture medium and 66.67 mL of the expansion culture medium prepared in step 3.1 of this example were added to the culture bag;

[0103] On the 7th day, 200 mL of the expansion culture medium prepared in step 3.1 of this example was added;

[0104] On the 9th day, 300 mL of the expansion culture medium prepared in step 3.1 of this example was added;

[0105] On the 11th day, 600 mL of the expansion culture medium prepared in step 3.1 of this example was added;

[0106] On the 13th day, 1200 mL of the expansion culture medium prepared in step 3.1 of this example was added;

[0107] On the 14th day, the culture was stopped, the cells were collected and transferred to a centrifuge tube, and centrifuged at 800 g for 10 min, and the supernatant was removed; the cell pellet was repeatedly washed twice with PDS to obtain the γδT cells.

[0108] Example 4 (Comparative Example 1)

[0109] 4.1 Preparation of γδT cell culture medium

[0110] The activated culture medium of this embodiment does not contain IPI-145, Bezafibrate, Rapamycin and Atorvastatin;

[0111] Prepare 100 mL of activation culture medium: based on RPMI-1640 serum-free medium, add IL-21 with a final concentration of 20 ng / mL, AMD3100 with a final concentration of 5 μg / mL, and Metformin with a final concentration of 1 mM to the serum-free medium;

[0112] Prepare 1500 mL of expansion culture medium: based on RPMI-1640 serum-free medium, add L-glutamine at a final concentration of 2 mM, penicillin at a final concentration of 100 U / mL, streptomycin at a final concentration of 100 μg / mL, 2-mercaptoethanol at a final concentration of 50 μM, vitamin C at a final concentration of 500 μM, and non-essential amino acids at a working concentration of 1X to the serum-free medium.

[0113] 4.2. Expansion and culture of γδT cells

[0114] On day 0, take 0.2×10 6 mononuclear cells are inoculated into a culture flask containing 30 mL of the activation culture medium prepared in step 4.1 of this example; the culture flask is placed in an incubator at 37° C. and 5 v / v% CO2 and cultured until the end of the 14th day;

[0115] On the third day, 30 mL of the expansion culture medium prepared in step 4.1 of this example was added to the culture flask;

[0116] On the 5th day, the cells were transferred to a culture bag, and 20 mL of the activation culture medium and 40 mL of the expansion culture medium prepared in step 4.1 of this example were added to the culture bag;

[0117] On the 7th day, 120 mL of the expansion culture medium prepared in step 4.1 of this example was added;

[0118] On the 9th day, 180 mL of the expansion culture medium prepared in step 4.1 of this example was added;

[0119] On the 11th day, 360 mL of the expansion culture medium prepared in step 4.1 of this example was added;

[0120] On the 13th day, 720 mL of the expansion culture medium prepared in step 4.1 of this example was added;

[0121] On the 14th day, the culture was stopped, the cells were collected and transferred to a centrifuge tube, and centrifuged at 800 g for 10 min, and the supernatant was removed; the cell pellet was repeatedly washed twice with PDS to obtain the γδT cells.

[0122] Example 5 (Comparative Example 2)

[0123] 5.1. Preparation of γδT cell culture medium

[0124] In this example, 10% fetal bovine serum (FBS) was used as the basic culture medium.

[0125] Prepare 100 mL of activation culture medium: based on a culture medium containing 10% fetal bovine serum (FBS), add IL-21 at a final concentration of 20 ng / mL, AMD3100 at a final concentration of 55 μg / mL, Metformin at a final concentration of 1 mM, zoledronic acid (ZOL) at a final concentration of 50 ng / mL, and isopropyl acrylate (IPP) at a final concentration of 1 μg / mL to the serum-free culture medium;

[0126] Prepare 2500 mL of expansion culture medium: based on a culture medium with 10% fetal bovine serum (FBS), this serum-free culture medium is supplemented with L-glutamine at a final concentration of 20 mM, penicillin at a final concentration of 500 U / mL, streptomycin at a final concentration of 10 μg / mL, 2-mercaptoethanol at a final concentration of 10 μM, vitamin C at a final concentration of 500 μM, and non-essential amino acids at a working concentration of 1X.

[0127] 5.2. Expansion and culture of γδT cells

[0128] On day 0, take 0.2×10 6 mononuclear cells were inoculated into a TC75 culture flask containing 50 mL of the activation culture medium prepared in step 4.1 of this example; the culture flask was placed in an incubator at 37° C. and 5 v / v% CO2 and cultured until the end of the 14th day;

[0129] On the third day, 50 mL of the expansion culture medium prepared in step 5.1 of this example was added to the culture flask;

[0130] On the 5th day, the cells were transferred to a culture bag, and 33.33 mL of the activation culture medium and 66.67 mL of the expansion culture medium prepared in step 5.1 of this example were added to the culture bag;

[0131] On the 7th day, 200 mL of the expansion culture medium prepared in step 5.1 of this example was added;

[0132] On the 9th day, 300 mL of the expansion culture medium prepared in step 5.1 of this example was added;

[0133] On the 11th day, 600 mL of the expansion culture medium prepared in step 5.1 of this example was added;

[0134] On the 13th day, the cells were transferred to a culture bag, and 1200 mL of the expansion culture medium prepared in step 5.1 of this example was added to the culture bag;

[0135] On the 14th day, the culture was stopped, the cells were collected and transferred to a centrifuge tube, and centrifuged at 800 g for 10 min, and the supernatant was removed; the cell pellet was repeatedly washed twice with PDS to obtain the γδT cells.

[0136] 2. γδT cell detection experiment

[0137] (I) Detection of γδT cell proliferation and growth

[0138] During the γδT cell culture process in Examples 1 to 5, samples were taken on days 0, 3, 5, 7, 9, 11, 13, and 14, and the γδT cells were counted. The counting results are shown in Tables 1 and Figure 1 As shown; among them, Figure 1 The results are plotted based on the values ​​in Table 1.

[0139] Table 1 Count of γδT cell culture growth and expansion

[0140]

[0141] like Figure 1 As shown in Table 1, the test results show that on the 14th day of γδT cell culture, the γδT cells in Examples 1 to 3 grew faster, with the cell numbers being 2.898×10 8 / mL, 2.499×10 8 / mL, 2.382×10 8 / mL (the cell expansion times were 1449 times, 1249.5 times, and 1191 times, respectively), while the γδT cell cultures of Examples 4 and 5 grew relatively slowly, with the cell numbers being 1.56×10 8 / mL (cell expansion multiple is 780 times), 1.62×10 8 / mL (cell expansion multiple is 810 times).

[0142] This is because: in Examples 1 to 3, the activated culture medium contains small molecule compounds such as IPI-145, Bezafibrate, Rapamycin and Atorvastatin. Among these small molecule compounds, IPI-145 can regulate the activity of T cells and B cells and activate the proliferation of γδT cells; Bezafibrate can regulate cell metabolism and promote T cell proliferation; Rapamycin and Atorvastatin can reduce immune metabolism and exhaustion during cell proliferation, promote the proliferation of γδT cells, and can be absorbed and utilized by cells, thereby promoting the expansion and growth of γδT cells.

[0143] (II) Detection of γδT cell viability

[0144] During the γδT cell culture process in Examples 1 to 5, samples were taken on the 7th day and the 14th day, and the sampled γδT cells were stained with trypan blue and stored at 18-25°C for 4 hours. The cells were counted using a cell counting plate, and then the number and viability of the γδT cells were calculated (cell viability = number of viable cells / total number of cells × 100%). The calculation results are shown in Table 2.

[0145] Table 2 γδT cell viability

[0146]

[0147] As can be seen from Table 2, the cell viability of the γδT cells cultured in Examples 1 to 3 is relatively high, about 95.1% to 99.2% or more, while the cell viability of the γδT cells cultured in Examples 4 and 5 is relatively high, about 78.3% and 81.1%. The relatively high viability of the γδT cells cultured in Examples 1 to 3 is because Bezafibrate in the activated culture medium can regulate cell metabolism and promote T cell proliferation, while Rapamycin and Atorvastatin reduce immune metabolism and exhaustion during cell proliferation, thereby improving cell activity and stability.

[0148] (III) γδT cell tumor killing test

[0149] K562 cells were used as target cells for the in vitro killing experiment of γδT cells. The γδT cells cultured on the 14th day in Examples 1 to 5 were used as effector cells. The experiment was carried out in a 96-well plate at an effector-target ratio of E:T=20:1, 10:1, 5:1, and 1:1. 100ul of target cells and effector cells were added to each well (cell concentration was 2×10 6 / mL), set up 3 replicate wells, and set up a negative control. After culturing in an incubator at 37°C and 5% CO2 saturated humidity for 24 hours, add 10μl of CCK-8 reagent to each well and incubate for 3 hours. Then use an enzyme marker to measure the absorbance (OD) value at a wavelength of 450nm. Calculate the killing rate according to the following formula:

[0150] Killing rate (%) = {[1-(OD value of experimental group-OD value of effector cell group)] / (OD value of effector cell group)} × 100%;

[0151] Among them, each OD value in the formula is the value after subtracting the OD value of the blank control group.

[0152] The calculated γδT cell killing rates are shown in Table 3 ; Figure 2 The data are drawn based on Table 3.

[0153] Table 3 Statistical table of γδT cell killing rate

[0154]

[0155] Table 3 and Figure 2 The test results show that the amplified γδT cells of the present invention have good tumor killing activity. When the effect-target ratio is E:T=1:1, 5:1, 10:1, and 20:1, the tumor killing rate of Examples 1 to 3 is better than that of Comparative Example 1 (P<0.05). Therefore, it is shown that the combined use of IL-21, AMD3100, and Metformin in the activation culture medium of the present invention promotes the proliferation of γδT cells, enhances cytotoxicity and cytokine secretion ability, etc., and thus improves the anti-tumor effect of cells; at the same time, the addition of activation culture medium in the early stage of cell culture can also regulate cell activity, reduce cell immune metabolism, and activate the activity and killing of γδT cells.

[0156] (IV) γδT cell purity test

[0157] In Examples 1 to 5, on the 14th day of the γδT cell culture process, samples were taken for flow cytometry detection of the purity of γδT cells (LD+CD3+Vd2). The detection results were as follows: Figures 3 to 7 As shown; among them, Figure 3 This is a flow cytometry graph of the purity of γδT cells on day 14 of culture in Example 1; Figure 4 This is a flow cytometry graph of the purity of γδT cells on day 14 of culture in Example 2; Figure 5 This is a flow cytometry graph of the purity of γδT cells on day 14 of culture in Example 3; Figure 6 This is a flow cytometry graph of the purity of γδT cells on day 14 of culture in Example 4 (Comparative Example 1); Figure 7 This is a flow cytometric graph of the purity of γδT cells on day 14 of culture in Example 5 (Comparative Example 2).

[0158] Depend on Figures 3 to 7 It can be seen that on the 14th day of γδT cell culture, the purity (LD+CD3+Vd2) ratios of γδT cells in Examples 1 to 4 were 87.20%, 89.21%, 82.27%, and 77.13%, respectively; while the purity (LD+CD3+Vd2) ratio of γδT cells in Example 5 was 60.10%. Therefore, the purity (LD+CD3+Vd2) of γδT cells in Examples 1 to 4 is better than the purity (LD+CD3+Vd2) of γδT cells in Example 5. This is because, in Examples 1 to 4, the activation culture medium and the expansion culture medium of γδT cells are based on serum-free culture medium, which greatly improves the purity (LD+CD3+Vd2) of γδT cells; in subsequent clinical applications, the risks caused by potential pathogens and immune responses in serum are avoided, and the safety of γδT cells and the feasibility of clinical application are improved.

[0159] It should be understood that the above description of the preferred embodiments of the present invention is relatively detailed and cannot be regarded as limiting the scope of patent protection of the present invention. The scope of patent protection of the present invention shall be based on the attached claims.

Claims

1. A γδT cell culture medium, characterized in that: It includes activation culture medium and expansion culture medium; wherein: The activation culture solution comprises a serum-free culture medium, wherein the serum-free culture medium comprises IL-21 with a final concentration of 1 to 50 ng / mL, AMD3100 with a final concentration of 1 to 15 μg / mL, Metformin with a final concentration of 0.1 to 10 mM, IPI-145 with a final concentration of 50 to 500 nM, Bezafibrate with a final concentration of 50 to 500 μM, Rapamycin with a final concentration of 1 to 10 nM, and Atorvastatin with a final concentration of 0.1 to 1 μM; The amplification culture medium includes a serum-free culture medium, which contains L-glutamine with a final concentration of 0.5 to 20 mM, penicillin with a final concentration of 10 to 500 U / mL, streptomycin with a final concentration of 10 to 500 μg / mL, 2-mercaptoethanol with a final concentration of 10 to 100 μM, vitamin C with a final concentration of 50 to 500 μM, and non-essential amino acids with a working concentration of 1X.

2. The γδT cell culture medium according to claim 1, characterized in that The serum-free culture medium of the activation culture fluid contains IL-21 with a final concentration of 20 ng / mL, AMD3100 with a final concentration of 5 μg / mL, Metformin with a final concentration of 1 mM, IPI-145 with a final concentration of 50 to 500 nM, Bezafibrate with a final concentration of 50 to 500 μM, Rapamycin with a final concentration of 1 to 10 nM, and Atorvastatin with a final concentration of 0.1 to 1 μM.

3. The γδT cell culture medium according to claim 1, characterized in that The serum-free culture medium of the amplification culture solution contains L-glutamine at a final concentration of 2 mM, penicillin at a final concentration of 100 U / mL, streptomycin at a final concentration of 100 μg / mL, 2-mercaptoethanol at a final concentration of 50 μM, and non-essential amino acids at a working concentration of 1X.

4. The γδT cell culture medium according to any one of claims 1 to 3, characterized in that The serum-free culture medium is RPMI-1640, 581 or DMEM.

5. A method for culturing γδT cells, characterized in that: The steps include: On day 0, mononuclear cells were inoculated into a culture bottle containing the activation culture medium as claimed in claim 1, and the cell density was controlled to be 1×10 6 The culture bottle was placed in an incubator at 37° C. and 5 v / v% CO 2 and cultured until the 14th day of culture; On the third day, the expansion culture medium according to claim 1 is added to the culture bottle, and the ratio of the volume of the additional expansion culture medium to the volume of the culture medium in the culture bottle is 1:1; On the fifth day, the cells are transferred to a culture bag, and the activation culture solution and the expansion culture solution as claimed in claim 1 are added to the culture bag, and the volume ratio of the activation culture solution and the expansion culture solution is 1:2, and the ratio of the total volume of the activation culture solution and the expansion culture solution added to the volume of the culture solution in the culture bottle is 1:1; On the 7th day, the expansion culture medium as claimed in claim 1 is supplemented, and the ratio of the volume of the supplemented expansion culture medium to the volume of the culture medium in the culture bag is 1:2; On the 9th day and the 13th day, the expansion culture medium according to claim 1 is added respectively, and the ratio of the volume of the expansion culture medium added each time to the volume of the culture medium in the culture bag is 1:1; On the 14th day, the culture was stopped, the cells were collected and transferred to a centrifuge tube, centrifuged at 800 g for 10 min, and the supernatant was removed; The cell pellet was washed with PDS to obtain the γδ T cells.

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