Blood preservation solution suitable for culturing stem cell-like memory T cells and its application

Abstract

The present invention provides a blood preservation solution suitable for culturing stem cell-like memory T cells and its application. Each 100 mL of the blood preservation solution includes: 5-50 mg of sodium heparin, 0.1-1 g of citric acid, 0.01-0.1 g of adenine, 0.1-1 g of disodium hydrogen phosphate, 1-10 g of glucose, 0.1-10 mg of transferrin, 0.1-10 mg of hydroxypropyl-β-cyclodextrin, 0.1-10 mg of ethanolamine, 0.1-10 mg of sodium selenate, 0.01-1 mg of α-zinc sulfate, 0.1-10 mg of L-ascorbic acid, and vitamin E. 0.1~10mg, cholesterol 0.01~1mg, L-glutamine 0.1~10mmol, L-methionine 0.1~10mg / 100mL, L-lysine hydrochloride 0.1~10mg / 100mL, L-valine 0.1~10mg / 100mL, L-isoleucine 0.1~10mg / 100mL, L-phenylalanine 0.1~10mg / 100mL, L-leucine 0.1~10mg / 100mL, L-tryptophan 0.1~10mg / 100mL, L-threonine 0.1~10mg / 100mL, and the solvent is an electrolyte solution. The blood preservation solution provided by the present invention provides the necessary nutrition and environment for PBMC, can preserve PBMC for a long time, and the PBMC separated subsequently still has the activity of amplifying and culturing TSCM, and the cultured Tscm accounts for more than 30% of CD3+T cells, which provides a guarantee for the clinical use of TSCM.

Description

Technical Field

[0001] The present invention relates to the technical field of cell preservation, and in particular to a blood preservation solution suitable for culturing stem cell-like memory T cells and an application thereof. Background Art

[0002] T cells are one of the main components of lymphocytes. They have multiple biological functions, such as directly killing target cells, assisting other lymphocytes to function, responding to specific antigens or mitogens, and producing cytokines. They are the main immune cells for the body to resist pathogen infection and prevent and treat tumors.

[0003] T cells can be divided into initial T cells, memory T cells, and effector T cells according to their degree of differentiation. Memory T cells are the basis for maintaining human immune surveillance, which is crucial for tumor prevention and prevention of recurrence after tumor surgery. Stem cell-like memory T cells (TSCM) belong to a subpopulation of T cells. TSCM has the characteristics of both initial T cells and memory T cells, and is believed to have the ability to self-renew and differentiate into other effector and memory T cell subpopulations. TSCM has the characteristics of memory T cells that produce cytokines and proliferate rapidly, as well as the characteristics of stem cells with strong survival, self-renewal and multipotential differentiation capabilities. Its strong survival ability overcomes the problem of short survival time of transplanted cells in vivo, and it can produce cytokines with anti-tumor effects by itself, which is expected to become an important cell source for adoptive immunotherapy of tumors.

[0004] Blood preservation fluid is a key factor in maintaining the activity of cells in the blood. Currently, the ACD preservation fluid is widely used, which is mainly composed of acid citrate and glucose. However, blood preserved at 4°C using ACD preservation fluid, PBMCs are separated after 24-28 hours, and Tscm cells are cultured on the separated PBMCs. It is found that the cultured Tscm are difficult to culture to meet clinical needs, and their cell number and memory phenotype do not meet clinical application standards. Therefore, finding a blood preservation fluid that can effectively protect the active characteristics of TSCM is an urgent need for TSCM research and application. Summary of the invention

[0005] The present invention provides a blood preservation solution suitable for culturing stem cell-like memory T cells, a preparation method and an application thereof, which are used to cultivate TSCMs that meet clinical needs by storing PBMCs at 2-10° C. for a long time.

[0006] The present invention provides a blood preservation solution suitable for culturing stem cell-like memory T cells. Each 100 mL of the blood preservation solution comprises: 5-50 mg of sodium heparin, 0.1-1 g of citric acid, 0.01-0.1 g of adenine, 0.1-1 g of disodium hydrogen phosphate, 1-10 g of glucose, 0.1-10 mg of transferrin, 0.1-10 mg of hydroxypropyl-β-cyclodextrin, 0.1-10 mg of ethanolamine, 0.1-10 mg of sodium selenate, 0.01-1 mg of α-zinc sulfate, 0.1-10 mg of L-ascorbic acid, and vitamin E. 0.1~10mg, cholesterol 0.01~1mg, L-glutamine 0.1~10mmol, L-methionine 0.1~10mg / 100mL, L-lysine hydrochloride 0.1~10mg / 100mL, L-valine 0.1~10mg / 100mL, L-isoleucine 0.1~10mg / 100mL, L-phenylalanine 0.1~10mg / 100mL, L-leucine 0.1~10mg / 100mL, L-tryptophan 0.1~10mg / 100mL, L-threonine 0.1~10mg / 100mL, the solvent is an electrolyte solution.

[0007] In a preferred embodiment, in every 100 mL of blood preservation solution, the heparin sodium is 30 mg, the citric acid is 0.5 g, the adenine is 0.05 g, the disodium hydrogen phosphate is 0.2 g, the glucose is 3 g, the transferrin is 2 mg, the hydroxypropyl-β-cyclodextrin is 1 mg, the ethanolamine is 0.5 mg, the sodium selenate is 1 mg, the α-zinc sulfate is 0.1 mg, the L-ascorbic acid is 2 mg, the vitamin E is 2 mg, the cholesterol is 0.2 mg, the L-glutamine is 2 mmol, the L-methionine is 2 mg / 100 mL, the L-lysine hydrochloride is 2 mg / 100 mL, the L-valine is 2 mg / 100 mL, the L-isoleucine is 2 mg / 100 mL, the L-phenylalanine is 2 mg / 100 mL, the L-leucine is 2 mg / 100 mL, the L-tryptophan is 1 mg / 100 mL, and the L-threonine is 2 mg / 100 mL.

[0008] In a preferred embodiment, the electrolyte solution includes 0.526% sodium chloride, 0.502% sodium gluconate, 0.368% sodium acetate, 0.037% potassium chloride, and 0.03% magnesium chloride.

[0009] In a preferred embodiment, the pH value of the blood preservation solution for culturing the stem cell-like memory T cells is 7.0-7.5.

[0010] In a preferred embodiment, the pH value of the blood preservation solution for culturing the stem cell-like memory T cells is 7.35-7.45.

[0011] In a preferred embodiment, the temperature of the blood sample stored in the blood preservation solution is 2-10°C.

[0012] In a preferred embodiment, when the blood preservative solution preserves a blood sample, the volume ratio of the blood sample to the blood preservative solution is 100:14.

[0013] The present invention also provides a kit, which includes a blood preservation solution suitable for culturing stem cell-like memory T cells as described in any one of the above schemes, and the kit is used for any one or more of blood preservation, PBMC cell extraction, TSCM amplification, TSCM culture and exosome preparation.

[0014] The present invention provides a blood preservation solution suitable for culturing stem cell-like memory T cells, which provides necessary nutrition and environment for PBMC, can preserve PBMC for a long time, and the subsequently separated PBMC still has the activity of amplifying and culturing TSCM. The cultured Tscm accounts for more than 30% of CD3+T cells, which provides a guarantee for the clinical use of TSCM.

[0015] At the same time, the present invention also provides the use of a blood preservation fluid suitable for culturing stem cell-like memory T cells, which can be applied to blood preservation, PBMC cell extraction, TSCM amplification, TSCM culture, exosomes and other kits, and improves the efficacy of the above kits by maintaining the activity of TSCM. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 This is a growth morphology diagram of Tscm cells obtained by amplifying and culturing PBMCs separated from the blood of the embodiment of the present invention after being stored for 5 days;

[0018] Figure 2 These are the flow cytometry analysis results of Tscm cells cultured from PBMCs isolated and expanded after 5 days of storage in Example 1 and Comparative Example 2 of the present invention in CD3+T cells. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] In healthy people, TSCM accounts for about 2% to 3%, and TSCM is one of the important cell sources for adoptive immunotherapy of tumors. In the process of blood preservation, there are factors such as transportation and sample processing that may require blood samples to be preserved for a long time, and the PBMC separated from blood preserved with the existing ACD preservation solution and the cultured TSCM cannot meet clinical needs.

[0021] After research, the first aspect of the present invention provides a blood preservation solution suitable for culturing stem cell-like memory T cells cultured in TSCM, and each 100 mL of the blood preservation solution suitable for culturing stem cell-like memory T cells comprises:

[0022] Heparin sodium 5-50 mg, citric acid 0.1-1 g, adenine 0.01-0.1 g, disodium hydrogen phosphate 0.1-1 g, glucose 1-10 g, transferrin 0.1-10 mg, hydroxypropyl-β-cyclodextrin 0.1-10 mg, ethanolamine 0.1-10 mg, sodium selenate 0.1-10 mg, α-zinc sulfate 0.01-1 mg, L-ascorbic acid 0.1-10 mg, vitamin E 0.1~10mg, cholesterol 0.01~1mg, L-glutamine 0.1~10mmol, L-methionine 0.1~10mg / 100mL, L-lysine hydrochloride 0.1~10mg / 100mL, L-valine 0.1~10mg / 100mL, L-isoleucine 0.1~10mg / 100mL, L-phenylalanine 0.1~10mg / 100mL, L-leucine 0.1~10mg / 100mL, L-tryptophan 0.1~10mg / 100mL, L-threonine 0.1~10mg / 100mL, the solvent is an electrolyte solution.

[0023] The electrolyte solution is composed of 0.526% sodium chloride, 0.502% sodium gluconate, 0.368% sodium acetate, 0.037% potassium chloride and 0.03% magnesium chloride.

[0024] In blood culture medium, sodium heparin is an anticoagulant that can interfere with many links of the blood coagulation process and reduce the occurrence of blood coagulation in the collected blood. Citric acid can prevent the glucose in the preservation solution from being charred during disinfection. Glucose, hydroxypropyl-β-cyclodextrin, ethanol, sodium selenate, α-zinc sulfate, L-glutamine, vitamin E, cholesterol, methionine, lysine hydrochloride, valine, isoleucine, phenylalanine, leucine, tryptophan and threonine are nutrients that provide nutrition for the normal metabolism of each cell in the blood. The use of electrolyte solution can maintain the osmotic pressure and system balance of the example, forming environmental conditions that are conducive to cell survival and the preservation of TSCM. Transferrin can bind to iron ions and supply iron to cells, participating in life activities such as respiration, DNA synthesis, and oxidoreductase metabolism. Adenine can maintain cell functions such as cell energy metabolism and cell signal transduction, and can also promote erythrocyte ATP synthesis, prolong the storage period of erythrocytes and enhance the oxygen release function of erythrocytes. The addition of sodium dihydrogen phosphate facilitates the subsequent adjustment of the pH of the preservation solution with sodium hydroxide solution.

[0025] The content of each component can be adjusted according to factors such as the type of blood sample, the difference in the individual species from which the blood sample is derived, and the cellular components in the blood sample.

[0026] The pH value of the blood preservation solution has a great influence on the blood activity. The blood preservation solution in this embodiment adopts a weakly alkaline environment with a pH value of 7.0 to 7.5, preferably 7.35 to 7.45. This pH value range is equivalent to the pH value of human blood.

[0027] In a preferred embodiment, in 100 mL of blood preservation solution, the heparin sodium is 30 mg, the citric acid is 0.5 g, the adenine is 0.05 g, the disodium hydrogen phosphate is 0.2 g, the glucose is 3 g, the transferrin is 2 mg, the hydroxypropyl-β-cyclodextrin is 1 mg, the ethanolamine is 0.5 mg, the sodium selenate is 1 mg, the α-zinc sulfate is 0.1 mg, the L-ascorbic acid is 2 mg, the vitamin E is 2 mg, the cholesterol is 0.2 mg, the L-glutamine is 2 mmol, the L-methionine is 2 mg / 100 mL, the L-lysine hydrochloride is 2 mg / 100 mL, the L-valine is 2 mg / 100 mL, the L-isoleucine is 2 mg / 100 mL, the L-phenylalanine is 2 mg / 100 mL, the L-leucine is 2 mg / 100 mL, the L-tryptophan is 1 mg / 100 mL, and the L-threonine is 2 mg / 100 mL.

[0028] In order to better illustrate the technical effect of the technical solution of the present invention, the present invention constructed the following embodiments and comparative examples for testing.

[0029] Example 1

[0030] The blood preservation solution suitable for Tscm culture in Example 1, the reagents required for preparing 100 mL of blood preservation solution are as follows: 30 mg of sodium heparin, 0.5 g of citric acid, 0.05 g of adenine, 0.2 g of disodium hydrogen phosphate, 3 g of glucose, 2 mg of transferrin, 1 mg of hydroxypropyl-β-cyclodextrin, 0.5 mg of ethanolamine, 1 mg of sodium selenate, 0.1 mg of α-zinc sulfate, 2 mg of L-ascorbic acid, 2 mg of vitamin E, 0.2 mg of cholesterol, 2 mmol of L-glutamine, 2 mg of L-methionine, 2 mg of L-lysine hydrochloride, 2 mg of L-valine, 2 mg of L-isoleucine, 2 mg of L-phenylalanine, 2 mg of L-leucine, 1 mg of L-tryptophan, and 2 mg of L-threonine, and the solvent is an electrolyte solution.

[0031] Among them, the pH of the blood preservation solution is 7.4.

[0032] The volume ratio of blood preservation solution to blood sample is 14:100, and the blood sample storage temperature is 4°C.

[0033] The configuration of the electrolyte solution is: 0.526% sodium chloride, 0.502% sodium gluconate, 0.368% sodium acetate, 0.037% potassium chloride, and 0.03% magnesium chloride.

[0034] Example 2

[0035] The blood preservation solution suitable for Tscm culture in Example 2, the reagents required for preparing 100 mL of blood preservation solution are as follows: 30 mg of sodium heparin, 0.5 g of citric acid, 0.05 g of adenine, 0.2 g of disodium hydrogen phosphate, 3 g of glucose, 2 mg of transferrin, 1 mg of hydroxypropyl-β-cyclodextrin, 0.5 mg of ethanolamine, 1 mg of sodium selenate, 0.1 mg of α-zinc sulfate, 2 mg of L-ascorbic acid, 2 mg of vitamin E, 0.2 mg of cholesterol, 2 mmol of L-glutamine, 2 mg of L-methionine, 2 mg of L-lysine hydrochloride, 2 mg of L-valine, 2 mg of L-isoleucine, 2 mg of L-phenylalanine, 2 mg of L-leucine, 1 mg of L-tryptophan, and 2 mg of L-threonine. The solvent is the same electrolyte solution as in Example 1.

[0036] The pH of the blood storage solution is 7.0.

[0037] The volume ratio of blood preservation solution to blood sample is 14:100, and the blood sample storage temperature is 4°C.

[0038] Comparative Example 1

[0039] The blood preservation solution of Comparative Example 1, the reagents required to prepare 100 mL of blood preservation solution are as follows: 30 mg of sodium heparin, 0.5 g of citric acid, 0.2 g of disodium hydrogen phosphate, 3 g of glucose, 2 mg of L-ascorbic acid, 2 mg of vitamin E, 2 mg of L-methionine, 2 mg of L-lysine hydrochloride, 2 mg of L-valine, 2 mg of L-isoleucine, 2 mg of L-phenylalanine, 2 mg of L-leucine, 1 mg of L-tryptophan, and 2 mg of L-threonine, and the solvent is the same electrolyte solution as in Example 1.

[0040] Among them, the pH of the blood preservation solution is 7.4.

[0041] Among them, the volume ratio of blood preservation fluid to blood sample is 14:100, and the blood sample storage temperature is 4°C.

[0042] Comparative Example 2

[0043] The blood preservation solution used in Comparative Example 2 is the blood preservation solution (ACD) used by the blood bank. The volume ratio of ACD to blood sample is 14:100, and the blood sample storage temperature is 4°C.

[0044] The embodiment and the comparative example used blood from the same source for treatment, the volume ratio of the blood preservation solution to the blood sample was 14:100, the mixed solution of the blood preservation solution and the blood sample was placed in the same environment, and the following experiments were performed for detection.

[0045] Experiment 1

[0046] Peripheral blood mononuclear cells (PBMC) were extracted from the peripheral blood stored in the blood storage solution of Examples 1 and 2 and Comparative Examples 1 and 2 for 0 and 5 days using the Ficoll method (density gradient centrifugation), and the PBMC survival rate was detected. The results are shown in Table 1.

[0047] Table 1. PBMC survival rate comparison table (%)

[0048] Group Save 0 days Save for 5 days Example 1 98.59±0.59 95.31±1.12 Example 2 98.72±0.78 93.38±1.03 Comparative Example 1 98.34±1.25 82.88±2.57 Comparative Example 2 98.52±0.57 56.51±1.52

[0049] Among them, it can be clearly seen from the data in the above table that the PBMC survival rate is still above 90% when the blood preservation solution corresponding to Example 1 and Example 2 of the present invention is stored for 5 days, which is significantly higher than that of Comparative Examples 1-2. Therefore, the blood preservation solution provided by the present invention can realize long-term and efficient storage of blood samples under different pH conditions and improve the survival rate of PBMC.

[0050] Experiment 2

[0051] Based on Experiment 1, the extracted PBMCs were cultured as Tscm cells, and the morphology and purity of the cultured cells were recorded. The total cell survival rate was detected after 14 days of culture. The results are shown in Table 2 below.

[0052] Table 2. Comparison of total cell survival rate after 14 days of culture (%)

[0053] Group Save 0 days Save for 5 days Example 1 91.59±1.104 86.88±1.40 Example 2 91.82±2.17 84.79±0.99 Comparative Example 1 90.65±1.67 61.80±1.56 Comparative Example 2 91.65±2.04 22.77±1.95

[0054] Among them, it can be clearly seen from the data in Table 2 above that the total cell survival rate obtained by amplifying and culturing PBMC separated from blood samples after 5 days of ACD preservation is only 22.77%, that is, Comparative Example 2, which does not meet the clinical use requirements; while the blood preservation solution corresponding to Examples 1 and 2 of the present invention, after being preserved until the 5th day, the total cell survival rate of the extracted PBMC after 14 days of culture can still reach more than 80%, which is much higher than the result of Comparative Example 2 preserved with ACD preservation solution. However, the use of Comparative Example 1 can only reach about 60%, which is also far lower than the effect of the blood preservation solution of Examples 1 and 2 in this scheme. Therefore, the blood preservation solution provided by the present invention can achieve long-term and efficient preservation of blood samples under different pH conditions and improve the survival rate of PBMC.

[0055] The test methods of Experiment 1 and Experiment 2 are as follows: AO / PI staining is performed on the collected PBMCs or the total cells obtained by culture, and the cell counting is performed using a cell counter to calculate the cell survival rate.

[0056] like Figure 1 As shown, it can be clearly seen from the images that when the blood culture medium of Example 1 and Example 2 of the present invention is used for culture, the cell culture shows obvious aggregation, and has a significant amplification effect compared with Comparative Example 1 and Comparative Example 2.

[0057] Experiment 3

[0058] Tscm cell proportion detection

[0059] After 14 days of PBMC culture, the total cells of Example 1 and Comparative Example 2 were collected and Tscm cells (CD3 + CD45RO - CD62L + ) percentage detection. + CD45RO - CD62L + ) The detection method is as follows: The antibodies used are: APC-CD3 antibody, Brilliant Violet 421-CD45RO antibody, Alexa 700-CD62L antibody, using multiple sorting. The results are as follows Figure 2 shown.

[0060] from Figure 2 It can be concluded that by extracting PBMC for Tscm cell expansion culture, after 14 days of culture, Tscm cells (CD3 + CD45RO - CD62L + ) accounted for 33.1% of CD3+T cells, which was much higher than 14.5% of Comparative Example 2. Therefore, the blood preservation solution for stem cell-like memory T cell culture provided by the embodiment of the present invention can still achieve a good blood preservation effect at a pH of 7.0 to 7.5 and a temperature of 2 to 10°C. And the PBMC cells extracted from the blood after being preserved for 5 days in the blood preservation solution of the present invention still have the ability to expand and culture TSCM.

[0061] The present invention also provides a kit, which includes the blood preservation solution for culturing stem cell-like memory T cells as described above. The kit can be applied to blood preservation, PBMC cell extraction, TSCM amplification, TSCM culture, and preparation of exosomes through TSCM supernatant, and can effectively preserve the quality and quantity of TSCM in the blood, thereby improving the efficiency of PBMC cell extraction, TSCM amplification and culture, and exosome extraction from TSCM.

[0062] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A blood preservation solution suitable for culturing stem cell-like memory T cells, characterized in that: Each 100mL of blood preservation solution contains: Heparin sodium 5-50 mg, citric acid 0.1-1 g, adenine 0.01-0.1 g, disodium hydrogen phosphate 0.1-1 g, glucose 1-10 g, transferrin 0.1-10 mg, hydroxypropyl-β-cyclodextrin 0.1-10 mg, ethanolamine 0.1-10 mg, sodium selenate 0.1-10 mg, α-zinc sulfate 0.01-1 mg, L-ascorbic acid 0.1-10 mg, vitamin E 0.1~10mg, cholesterol 0.01~1mg, L-glutamine 0.1~10mmol, L-methionine 0.1~10mg / 100mL, L-lysine hydrochloride 0.1~10mg / 100mL, L-valine 0.1~10mg / 100mL, L-isoleucine 0.1~10mg / 100mL, L-phenylalanine 0.1~10mg / 100mL, L-leucine 0.1~ 10mg / 100mL, L-tryptophan 0.1-10mg / 100mL, L-threonine 0.1-10mg / 100mL, the solvent is an electrolyte solution, the electrolyte solution includes 0.526% sodium chloride, 0.502% sodium gluconate, 0.368% sodium acetate, 0.037% potassium chloride, 0.03% magnesium chloride, and the pH value of the blood preservation solution for culturing the stem cell-like memory T cells is 7.0-7.

5.

2. The blood preservation solution suitable for culturing stem cell-like memory T cells according to claim 1, characterized in that: In every 100 mL of blood preservation solution, the sodium heparin is 30 mg, the citric acid is 0.5 g, the adenine is 0.05 g, the disodium hydrogen phosphate is 0.2 g, the glucose is 3 g, the transferrin is 2 mg, the hydroxypropyl-β-cyclodextrin is 1 mg, the ethanolamine is 0.5 mg, the sodium selenate is 1 mg, the α-zinc sulfate is 0.1 mg, the L-ascorbic acid is 2 mg, the vitamin E is 2 mg, the cholesterol is 0.2 mg, the L-glutamine is 2 mmol, the L-methionine is 2 mg / 100 mL, the L-lysine hydrochloride is 2 mg / 100 mL, the L-valine is 2 mg / 100 mL, the L-isoleucine is 2 mg / 100 mL, the L-phenylalanine is 2 mg / 100 mL, the L-leucine is 2 mg / 100 mL, the L-tryptophan is 1 mg / 100 mL, and the L-threonine is 2 mg / 100 mL.

3. The blood preservation solution suitable for culturing stem cell-like memory T cells according to claim 1, characterized in that: The pH value of the blood preservation solution for culturing the stem cell-like memory T cells is 7.35-7.

45.

4. The blood preservation solution suitable for culturing stem cell-like memory T cells according to claim 3, characterized in that: The temperature of the blood preservation solution for preserving the blood sample is 2-10°C.

5. The blood preservation solution suitable for culturing stem cell-like memory T cells according to claim 4, characterized in that: When the blood preservative solution preserves the blood sample, the volume ratio of the blood sample to the blood preservative solution is 100:

14.

6. A kit, characterized in that: The kit comprises a blood preservation solution suitable for culturing stem cell-like memory T cells as claimed in any one of claims 1 to 5, and the kit is used for any one or more of blood preservation, PBMC cell extraction, TSCM amplification and TSCM culture.

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