Culture medium and culture method for stem cell-like memory T lymphocytes

Abstract

The present invention relates to the field of cell culture, and in particular to a culture medium and a culture method for stem cell-like memory T lymphocytes. By adding a GSK-3β inhibitor, an mTOR inhibitor or a PI3K inhibitor, as well as adding IL-15 and using magnetic bead sorting, the present invention can provide a large number of stem cell-like memory T lymphocytes with memory characteristics and low differentiation status, which can be used alone for tumor treatment, or as a basic cell for CAR-T treatment to obtain better anti-tumor ability and survival and proliferation ability than central memory lymphocytes or effector lymphocytes in clinical practice, which has great significance and value to the entire field of cell research and application.

Description

Technical Field

[0001] The invention relates to the field of cell culture, in particular to a culture medium and a culture method for stem cell-like memory T lymphocytes. Background Art

[0002] The differentiation of T cells follows a progressive differentiation process from naive T cells (Naive T cells, CD3+ CD45RA+ CCR7+) to stem cell-like memory T cells (TSCM, CD3+ CD45RA+ CCR7+), central memory T cells (TCM, CD3+ CD45RO+ CCR7+), effector memory T cells (TEM, CD3+ CD45RO+ CCR7-) and effector T cells (Teff, CD3+ CD45RO- CCR7-). In clinical practice, T cells need to be expanded in large quantities in vitro to meet the therapeutic dose. Because the biological characteristics of TSCM cells are close to those of naive T cells, they can reverse the tumor microenvironment and alleviate T cell exhaustion, and have both long-term memory and rapid homing functions of T cells; TSCM cells express memory-related markers, such as CD95, thus showing the characteristics of stem cells, including high proliferation and self-renewal capabilities, and enhancing the killing persistence and memory of T cells; TSCM is between the initial and central memory T cells (TCM) in terms of gene expression, and can differentiate into other T cell subsets, including TCM, TEM and Teff cells, which can promote rapid immune reconstruction in patients. As the smallest differentiated cell at the top of the memory T lymphocyte hierarchy system, TSCM has the ability to survive for a long time in the body and rebuild T cell subsets. The super strong anti-tumor ability of TSCM has become a hot topic in tumor research at home and abroad.

[0003] At present, the methods for culturing and expanding TSCM cells at home and abroad cannot meet the actual clinical needs. On the one hand, the existing TSCM cell culture methods have very low yields, and it is difficult to obtain enough cells for large-scale production; on the other hand, the proportion and number of TSCM in the body are relatively small, and with the progress of in vitro expansion, TSCM can easily become TCM, TEM or terminally differentiated effector cells (TEMRA); therefore, obtaining sufficient TSCM cells and maintaining the purity of TSCM cells is currently the biggest bottleneck for TSCM in vitro culture. Summary of the invention

[0004] In view of this, the present invention provides a special culture medium and culture method for stem cell-like memory T lymphocytes, which can obtain a large number of TSCM cells with high purity.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a culture medium for stem cell-like memory T lymphocytes, comprising a lymphocyte culture medium, plasma, interleukin-7, interleukin-15, interleukin-21 and a cell signaling pathway inhibitor;

[0007] The cell signaling pathway inhibitor is a GSK-3β inhibitor, an mTOR inhibitor or a PI3K inhibitor;

[0008] The cell signaling pathway inhibitor is one of GNE-490, PI3K / mTOR Inhibitor-2, Temsirolimus, Omipalisib, α-Hydroxyglutaric Acid Lithium, and AZD1080;

[0009] The concentration of the cell signaling pathway inhibitor in the culture medium is 1-10 μM, and can be 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, 5.5 μM, 6 μM, 6.5 μM, 7 μM, 7.5 μM, 8 μM, 8.5 μM, 9 μM or 9.5 μM.

[0010] In some specific embodiments of the present invention, the volume fraction of the plasma in the above-mentioned culture medium is 2% to 10%, and can be 3%, 4%, 5%, 6%, 7%, 8% or 9%.

[0011] In some specific embodiments of the present invention, the concentration of the interleukin-7 in the above-mentioned culture medium is 5~20 ng / mL, which can be 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL or 19 ng / mL.

[0012] In some specific embodiments of the present invention, the concentration of the interleukin-15 in the above-mentioned culture medium is 5~20 ng / mL, which can be 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL or 19 ng / mL.

[0013] In some specific embodiments of the present invention, the concentration of the interleukin-21 in the above-mentioned culture medium is 10~50 ng / mL, which can be 12 ng / mL, 14 ng / mL, 16 ng / mL, 18 ng / mL, 20 ng / mL, 22ng / mL, 24 ng / mL, 26 ng / mL, 28 ng / mL, 30 ng / mL, 32 ng / mL, 34 ng / mL, 36 ng / mL, 38 ng / mL, 40 ng / mL, 42 ng / mL, 44 ng / mL, 46 ng / mL or 48 ng / mL.

[0014] The present invention also provides a method for culturing stem cell-like memory T lymphocytes, which is based on the above culture medium.

[0015] In some specific embodiments of the present invention, the above-mentioned culture method comprises the following steps:

[0016] S1. Collect blood, separate PBMCs, and sort out CD8+ naive T cells;

[0017] S2. Cultivate the CD8+ naive T cells using anti-CD3 antibody and anti-CD28 antibody and the above culture medium to harvest stem cell-like memory T lymphocytes.

[0018] In some specific embodiments of the present invention, S2 of the above culture method is: transferring the CD8+ naive T cells into a culture container coated with anti-CD3 antibodies and anti-CD28 antibodies, culturing with the above culture medium, and harvesting stem cell-like memory T lymphocytes;

[0019] The coating concentration of the anti-CD3 antibody is 0.5 μg / mL to 1.5 μg / mL, and can be 0.6 μg / mL, 0.8 μg / mL, 1 μg / mL, 1.2 μg / mL, 1.4 μg / mL, 1.6 μg / mL, 1.8 μg / mL, 2 μg / mL, 2.2 μg / mL or 2.4 μg / mL;

[0020] The coating concentration of the anti-CD28 antibody is 0.5 μg / mL to 1.5 μg / mL, and can be 0.6 μg / mL, 0.8 μg / mL, 1 μg / mL, 1.2 μg / mL, 1.4 μg / mL, 1.6 μg / mL, 1.8 μg / mL, 2 μg / mL, 2.2 μg / mL or 2.4 μg / mL.

[0021] In some specific embodiments of the present invention, the blood in the above culture method is blood rich in hematopoietic stem cells.

[0022] In some specific embodiments of the present invention, the separation in the above-mentioned culture method comprises: collecting cord blood, mixing it with lymphocyte separation solution, centrifuging and stratifying, and aspirating to obtain PBMC.

[0023] In some specific embodiments of the present invention, the sorting in the above-mentioned culture method is: using a magnetic bead sorting method, utilizing antibody binding to remove CD45RO+ or CD56+ or CD57+ or CD244+ cells in the PBMC, and then utilizing antigen-antibody binding to screen out CD8+ cells to obtain the CD8+ naive T cells.

[0024] In some specific embodiments of the present invention, the culturing time in S2 of the above culturing method is 9 to 11 days.

[0025] In some specific embodiments of the present invention, the culture temperature in S2 of the above-mentioned culture method is 36-38°C, which can be 36.2°C, 36.4°C, 36.6°C, 36.8°C, 37°C, 37.2°C, 37.4°C, 37.6°C or 37.8°C.

[0026] In some specific embodiments of the present invention, the carbon dioxide content in the culture environment described in S2 of the above-mentioned culture method is between 4% and 6%, and can be 4.2%, 4.4%, 4.6%, 4.8%, 5%, 5.2%, 5.4%, 5.6% or 5.8%.

[0027] In some specific embodiments of the present invention, the cell density during the culture in S2 of the above-mentioned culture method is maintained at (0.5-2.5)×10 6 / mL, which can be 0.6×10 6 / mL, 0.8×10 6 / mL, 1.0×10 6 / mL, 1.2×10 6 / mL, 1.4×10 6 / mL, 1.6×10 6 / mL, 1.8×10 6 / mL, 2.0×10 6 / mL, 2.2×10 6 / mL or 2.4×10 6 Pieces / mL.

[0028] In some specific embodiments of the present invention, the above-mentioned culture method is characterized in that the stem cell-like memory T lymphocytes are T lymphocytes with a phenotype of CD45RA+ CD45RO- CD197+ CD62L+ CD95+.

[0029] The beneficial effects of the present invention are as follows: the culture medium and the culture method of the present invention can provide a large number of TSCM cells with memory characteristics and a low differentiation state. The TSCM cells obtained by using the culture medium and the culture scheme can be used alone for tumor treatment, and can also be used as basic cells for CAR-T treatment to obtain better anti-tumor ability and survival and proliferation ability than central memory lymphocytes (TCM) or effector lymphocytes (Teff) in clinical practice, which has great significance and value to the entire field of cell research and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0031] Figure 1 is a cell morphology diagram under a cell microscope of Example 1 of the present invention;

[0032] Figure 2 This is a cell morphology diagram under a cell microscope of Comparative Example 1 of the present invention;

[0033] Figure 3 This is a cell morphology diagram under a cell microscope of Comparative Example 4 of the present invention;

[0034] Figure 4 This is a D10 flow cytometry image of Example 1 of the present invention;

[0035] Figure 5 This is a flow cytometry diagram of D13 in Example 1 of the present invention;

[0036] Figure 6 It is the flow detection diagram of comparative example 1 of the present invention;

[0037] Figure 7 It is the flow detection diagram of comparative example 2 of the present invention;

[0038] Figure 8 It is the flow detection diagram of comparative example 3 of the present invention. DETAILED DESCRIPTION

[0039] The present invention discloses a special culture medium and culture method for stem cell-like memory T lymphocytes. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0040] The present invention provides a special culture medium for stem cell-like memory T lymphocytes and a culture method. The stem cell-like memory T lymphocytes express the following T lymphocyte membrane molecules: leukocyte differentiation antigen CD3, leukocyte differentiation antigen CD8, leukocyte differentiation antigen CD62L, leukocyte differentiation antigen CD45RA, and leukocyte differentiation antigen CD197; the TSCM cell ratio is ≥50%, and the expansion multiple is 15-25 times; the solvent of the special culture medium for stem cell-like memory T lymphocytes (TSCM) is lymphocyte culture medium, and the solutes are: (1) plasma, (2) interleukin-7, (3) interleukin-15, (4) interleukin-21, and (5) GSK-3β inhibitor, mTOR inhibitor or PI3K inhibitor.

[0041] The plasma may specifically be human AB plasma, human autologous plasma or human AB serum.

[0042] The volume fraction of the plasma in the dedicated culture medium is 2% to 10%.

[0043] The concentration of the GSK-3β inhibitor, mTOR inhibitor or PI3K inhibitor in the dedicated culture medium is 1-10 μM.

[0044] The GSK-3β inhibitor, mTOR inhibitor or PI3K inhibitor may be at least one of GNE-490, PI3K / mTORInhibitor-2, Temsirolimus, Omipalisib, α-Hydroxyglutaric Acid Lithium and AZD1080.

[0045] The concentration of interleukin-7 in the dedicated culture medium is 5-20 ng / mL.

[0046] The concentration of interleukin-15 in the dedicated culture medium is 5-20 ng / mL.

[0047] The concentration of interleukin-21 in the dedicated culture medium is 10-50 ng / mL.

[0048] The present invention also provides a method for culturing TSCM cells, comprising the following steps: culturing the TSCM cells with the special culture medium provided by the present invention, specifically the following steps: (1) pre-coating a culture plate with Anti-CD3 antibody and Anti-CD28 antibody; (2) collecting umbilical cord blood and obtaining mononuclear cells (PBMC) by density gradient centrifugation; (3) using a magnetic bead sorting method to sort PBMC into naive T lymphocytes (CD8+ naive T cells); (4) suspending CD8+ naive T cells in a TSCM special culture medium, culturing and inoculating them on a culture plate pre-coated with CD3 and CD28 antibodies; (5) replenishing the liquid every 2 to 4 days, supplementing the above-mentioned special culture medium according to the density, and amplifying to 9 to 11 days to obtain CD8+ T cells with a TSCM phenotype.

[0049] In the TSCM cell culture method provided by the present invention, the final concentration of the Anti-CD3 antibody coated on the culture flask is 0.5 μg / mL to 1.5 μg / mL, and the final concentration of the Anti-CD28 antibody is 0.5 μg / mL to 1.5 μg / mL.

[0050] In the TSCM cell culture method provided by the present invention, the number of cells suspended in the culture medium is (0.5-2.5)×10 6 / mL.

[0051] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0052] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present application is still operable. In addition, two or more steps or actions can be performed simultaneously.

[0053] The use of any and all examples or exemplary language such as "for example" or "including" herein is intended only to better illustrate the present application and is not intended to limit the scope of the present application. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the present application.

[0054] In addition, the numerical ranges and parameters used to define the present application are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally refers to the actual value within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0055] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.

[0056] The stem cell-like memory T cells involved in the present invention have a phenotype similar to that of naive T cells and express CD45RA+ CD45RO- CD197+ CD62L+ CD95+.

[0057] The present invention will be further described below in conjunction with the embodiments.

[0058] Example 1: In vitro induction and expansion of CD8+ TSCMs using the culture medium of the present invention

[0059] The special culture medium for stem cell-like memory T lymphocytes of the present invention is prepared, and the components are as follows:

[0060] The solvent is lymphocyte culture medium, and the solutes are: (1) plasma, (2) interleukin-7, (3) interleukin-15, (4) interleukin-21, (5) GSK-3β inhibitor, mTOR inhibitor or PI3K inhibitor.

[0061] The lymphocyte culture medium can be obtained from commercial sources, such as GT-T551 H3 culture medium from TAKARA Corporation.

[0062] The plasma may specifically be human AB plasma or human autologous plasma.

[0063] The volume fraction of the plasma in the dedicated culture medium is 2% to 10%.

[0064] The GSK-3β inhibitor, mTOR inhibitor or PI3K inhibitor can be obtained from commercial sources, such as Shanghai Taoshu Biological Technology Co., Ltd.

[0065] The GSK-3β inhibitor, mTOR inhibitor or PI3K inhibitor can be one of: GNE-490, PI3K / mTORInhibitor-2, Temsirolimus, Omipalisib, α-Hydroxyglutaric Acid Lithium, and AZD1080.

[0066] The concentration of the GSK-3β inhibitor, mTOR inhibitor or PI3K inhibitor in the dedicated culture medium is 5 μM.

[0067] The interleukin-7, interleukin-15 and interleukin-21 can be obtained from commercial sources, such as NOVUS, Nearshore Protein, etc.

[0068] The concentration of interleukin-7 in the dedicated culture medium is 5-20 ng / mL.

[0069] The concentration of interleukin-15 in the dedicated culture medium is 5-20 ng / mL.

[0070] The concentration of interleukin-21 in the dedicated culture medium is 10-50 ng / mL.

[0071] The method of obtaining human TSCM cells from umbilical cord blood using a human TSCM cell-specific culture medium comprises the following steps:

[0072] 1. The culture plate is pre-coated with Anti-CD3 antibody and Anti-CD28 antibody

[0073] Anti-CD3 antibody and Anti-CD28 antibody can be purchased from commercial companies such as ACRO or Nearshore Protein.

[0074] Add Anti-CD3 antibody (final concentration of 0.5 μg / mL~1.5 μg / mL) and Anti-CD28 antibody (final concentration of 0.5 μg / mL~1.5 μg / mL) to normal saline, mix well and transfer to a 96-well plate (about 50 μL / well), and place at 4°C for coating overnight.

[0075] 2. Collect umbilical cord blood and obtain mononuclear cells (PBMC) by density gradient centrifugation

[0076] (1) Use a heparin anticoagulant tube to collect about 30-100 mL of umbilical cord blood and slowly load the umbilical cord blood into the lymphocyte separation solution to avoid damaging the interface; the ratio of umbilical cord blood to lymphocyte separation solution is 2:1;

[0077] (2) Centrifuge at 500-800 g for 20-30 min (speed 7, speed 4). The centrifuge tube is divided into four layers from top to bottom due to different densities: the first layer is the plasma layer, the second layer is the ring-shaped milky white mononuclear cell layer (PBMC), the third layer is the transparent separation fluid layer, and the fourth layer is the red blood cell layer;

[0078] (3) Use a pipette to transfer the upper layer of plasma into a new 50 mL centrifuge tube, heat at 56°C for 30 min, and set aside.

[0079] (4) Carefully pipette the second layer of milky white mononuclear cells (PBMC) into another 50 mL centrifuge tube, add physiological saline, and centrifuge at 500-800 g for 5-10 min.

[0080] (5) Discard the supernatant, resuspend the cells in saline, count the cells, and centrifuge at 500-800 g for 5-10 min.

[0081] 3. Use magnetic bead sorting to sort PBMC into naive T lymphocytes (CD8+ naive T cells)

[0082] (1) Negative selection: Non-naive CD8+ T cells and NK cells are labeled with a mixture of biotin-labeled antibodies and anti-biotin microspheres (a mixture of human CD45RO, CD56, CD57 and CD244 monoclonal antibodies coupled to biotin), thereby removing NK cells and non-naive T cells;

[0083] (2) Positive selection: The enriched cells are labeled with CD8 magnetic beads to further positively select CD8+ naive T cells.

[0084] 4. Suspend CD8+ naive T cells in TSCM-specific culture medium and inoculate them on culture plates pre-coated with CD3 and CD28 antibodies

[0085] (1) Take the above-mentioned 96-well plates coated with Anti-CD3 antibody and Anti-CD28 antibody and discard the coating solution;

[0086] (2) Resuspend the obtained CD8+ naive T cells in TSCM special culture medium and adjust the concentration of CD8+ naive T cells to (0.5~2.5)×10 6 / mL, the volume is 100~200 μL, and after mixing, transfer into a 96-well plate;

[0087] (3) Place the 96-well plate in a 37°C, 5% carbon dioxide incubator.

[0088] 5. Replenish the culture medium every 2 to 4 days according to the density. When the cells are expanded to 9 to 11 days, a large number of CD8+T cells with TSCM phenotype can be obtained.

[0089] (1) After 2 to 4 days of culture, the cells show signs of proliferation, such as Figure 1 As shown, there are a large number of T cells in the culture medium, a large number of amplified T cells, and a large number of T cell clusters, indicating that the induced amplification effect is relatively good. Add fresh TSCM special culture medium to the 96-well plate; place the culture plate in a 37°C, 5% carbon dioxide incubator for continued culture. Note: If the volume of the 96-well plate is insufficient during rehydration, the cells can be transferred to a 24-well plate for culture;

[0090] (2) Replenish TSCM cell culture medium every 2 to 4 days to maintain the cell density at (0.5 to 2.5) × 10 6 / mL, TSCM cells with higher quantity and purity can be obtained on days 9-11;

[0091] 6. The number, viability and purity of the cultured TSCM cells are tested, including the following steps:

[0092] (1) The day of obtaining PBMC from cord blood was marked as day 0, and the cells cultured on days 10 and 13 were taken to detect the cell number and viability;

[0093] (2) Detect the purity of TSCM cells in the sample by flow cytometry ( Figure 4 , Figure 5 );

[0094] Note: Since six kinds of culture media were formed by adding six compounds (GSK-3β inhibitor, mTOR inhibitor or PI3K inhibitor) to the culture medium, the number, viability and flow cytometry phenotype of cells after culture in these six culture media were not much different. The present embodiment only provides one set of pictures (the group containing GNE-490 in the culture medium). The specific data are shown in Table 2 below.

[0095] Take 100 μL of each sample to be tested, add APC-CD3, PE-CD45RO, PE / cy7-CD62L, FITC-CD45RA, APC / cy7-CD95, PerCP-CD197 (CCR7) fluorescent labeled antibodies respectively, and detect by flow cytometry. The purity calculation formula is: the number of CD3+ cells in the gate × the number of CD45RA+ CD45RO- cells × the number of CD62L+ CD197+ cells × the number of CD95+ cells. The results are based on the experimental group containing GNE-490 in the culture medium as an example ( Figure 4 , Figure 5 ), the proportion of TSCM obtained on the 10th day of cell culture was 96.73%×85.80%×77.88%×95.54%=61.75% ( Figure 4 On the 13th day of cell culture, the proportion of TSCM was detected to be 95.50%×87.17%×35.32%×59.14%=17.39% ( Figure 5 ).

[0096] It shows that the culture time has a great influence on the purity of TSCM. After the 10th day of culture, the TSCM cells continue to be cultured and differentiate downward, and the purity decreases. Table 2 shows the expansion multiples, viability and purity of TSCM cells in comparative example 1 and comparative examples 1-4.

[0097] Comparative Example 1

[0098] The difference between this comparative example and Example 1 is that no compound (GSK-3β inhibitor, mTOR inhibitor or PI3K inhibitor) is added, and other culture media and culture methods are the same.

[0099] Culture and test results:

[0100] After 4 days of cell expansion, the number of cells and cell aggregation were significantly higher than those in Example 1 ( Figure 2 ).

[0101] On the 10th day of cell culture, flow cytometry antibodies for detecting TSCM were incubated and TSCM was detected using flow cytometry. The results were as follows: Figure 6 As shown, the obtained TSCM ratio is only 98.12%×56.59%×27.07%×1.96%=0.29%, and the CD62L+CD197+ cell ratio is greatly reduced; combined with the cell count, the number is significantly higher than that in Example 1, indicating that the GSK-3β inhibitor, mTOR inhibitor or PI3K inhibitor in the culture medium is one of the important factors for inducing differentiation of TSCM cells, which can significantly inhibit the proliferation of CD8+ T cells and block the differentiation of TSCM cells, thereby regulating the stemness of CD8+ T cells and promoting the generation of TSCM cells.

[0102] Comparative Example 2

[0103] The difference between this comparative example and Example 1 is that magnetic beads are not used to sort PBMC cells into Tn cells, but PBMC cells are directly used for culture, and other culture media and culture methods are the same.

[0104] Culture and test results:

[0105] On the 10th day of cell culture, flow cytometry antibodies for detecting TSCM were incubated and TSCM was detected using flow cytometry. The results were as follows: Figure 7 As shown, the proportion of TSCM obtained was only 90.30%×63.54%×85.43%×62.77%=30.77%, indicating that the use of magnetic beads to sort PBMC into Tn and then expand and culture is one of the important factors for inducing differentiation of TSCM cells, which can significantly improve the number, viability and purity of TSCM cells.

[0106] Comparative Example 3

[0107] The difference between this comparative example and Example 1 is that the GSK-3β inhibitor, mTOR inhibitor or PI3K inhibitor added to the culture medium is different from the 6 compounds described in the present application, but 130 other known commercial GSK-3β inhibitors, mTOR inhibitors or PI3K inhibitors are used (see Table 1), and other culture media and culture methods are the same.

[0108] Table 1: 130 GSK-3β inhibitors, mTOR inhibitors or PI3K inhibitors

[0109]

[0110] Culture and test results:

[0111] On the 10th day of cell culture, flow cytometry was used to detect TSCM by incubating the flow antibody for detecting TSCM. Since 130 kinds of GSK-3β inhibitors, mTOR inhibitors or PI3K inhibitors were added to the culture medium, the number and viability of the expanded cells were different, and it was difficult to show them one by one. Only the flow results were compared. Compared with Example 1, the proportion of CD45RA+CD46RO+ cells produced in this comparative example was higher (≥50%) and the proportion of CD45RA+CD46RO- cells was lower (≤50%), and after culturing with 130 groups of compounds added to the culture medium, it was found that the purity of TSCM cells cultured in the culture medium with a high expansion multiple (≥15 times) was lower (almost 0), and the TSCM cells had differentiated downward.

[0112] Taking Ridaforolimus as an example, the results are as follows Figure 8 As shown, the phenotype of the obtained TSCM cells is different from that in Example 1, with a higher proportion of CD45RA+CD46RO+ cells (up to 92.15% in CD3+ cells) and a lower proportion of CD45RA+CD46RO- (only 6.96% in CD3+ cells), indicating that the selection of GSK-3β inhibitors, mTOR inhibitors or PI3K inhibitors is one of the key factors in TSCM cell culture, and only by correctly screening compounds can high-purity TSCM cells be obtained.

[0113] Comparative Example 4

[0114] The difference between this comparative example and Example 1 is that IL-15 is not added to the culture medium, and other culture media and culture methods are the same.

[0115] Culture and test results:

[0116] After observing the cell proliferation for 4 days, the experimental results are as follows Figure 3 As shown; the experimental results show that the number of T cells in the culture medium is small and the cell clusters rarely appear, indicating that the induced expansion effect is relatively poor, indicating that IL-15 is one of the important factors for the expansion of TSCM cells.

[0117] On the 10th day of cell culture, flow cytometry antibodies for detecting TSCM were incubated, and TSCM was detected by flow cytometry. The purity results obtained were similar to those in Example 1. At the same time, combined with cell counting, the number was significantly lower than that in Example 1, indicating that IL-15 is an important component for inducing TSCM proliferation, and adding IL-15 to the culture medium can obtain TSCM cells with higher proliferation efficiency.

[0118] Table 2 shows the comparison of the TSCM cell expansion times, viability and purity between the examples and the comparative examples.

[0119] Table 2: Example 1, Comparative Examples 1-4: TSCM cell expansion multiples, viability and purity

[0120]

[0121] As can be seen from Table 2, the TSCM cells cultured by the TSCM culture medium and culture method used in the example are significantly superior to those of other comparative examples in terms of purity and quantity. It can be seen that the use of magnetic beads in sorting Tn cells (Comparative Example 1), the addition of GSK-3β inhibitors, mTOR inhibitors or PI3K inhibitors in the culture medium (Comparative Example 2) and their specific types (Comparative Example 3), and the addition of IL-15 (Comparative Example 4) are very important for the culture of TSCM cells.

[0122] In summary, the culture medium and culture method provided in the present application can obtain a large number of TSCM cells with high purity.

[0123] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A culture medium for stem cell-like memory T lymphocytes, characterized in that: Contains lymphocyte culture medium, plasma, interleukin-7, interleukin-15, interleukin-21, and cell signaling pathway inhibitors; The cell signaling pathway inhibitor is GNE-490; The concentration of the cell signaling pathway inhibitor in the culture medium is 1-10 μM.

2. The culture medium according to claim 1, characterized in that The volume fraction of the plasma is 2% to 10%.

3. The culture medium according to claim 1, characterized in that The concentration of interleukin-7 in the culture medium is 5-20 ng / mL.

4. The culture medium according to claim 1, characterized in that The concentration of interleukin-15 in the culture medium is 5-20 ng / mL.

5. The culture medium according to claim 1, characterized in that The concentration of interleukin-21 in the culture medium is 10-50 ng / mL.

6. A method for culturing stem cell-like memory T lymphocytes, characterized in that: Cultivated based on the culture medium according to any one of claims 1 to 5.

7. The culture method according to claim 6, characterized in that The following steps are involved: S1. Collect blood, separate PBMCs, and sort out CD8+ naive T cells; S2. Cultivate the CD8+ naive T cells using anti-CD3 antibodies and anti-CD28 antibodies and the culture medium according to any one of claims 1 to 5, and harvest stem cell-like memory T lymphocytes.

8. The culture method according to claim 7, characterized in that The sorting comprises: using a magnetic bead sorting method, utilizing antigen-antibody binding to remove cells with a phenotype of CD45RO+, CD56+, CD57+, or CD244+ in the PBMC, and then utilizing antigen-antibody binding antibodies to screen out CD8+ cells to obtain the CD8+ naive T cells.

9. The culture method according to claim 7, characterized in that: The culture period described in S2 is 9 to 11 days.

10. The culture method according to any one of claims 7 to 9, characterized in that: The stem cell-like memory T lymphocytes are T lymphocytes with a phenotype of CD45RA+CD45RO-CD197+CD62L+CD95+.

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