Method for preparing and expanding t scm cell in vitro

Abstract

The present application relates to a method for preparing and expanding a TSCM cell in vitro. Specifically, the method comprises: 1) obtaining an isolated CD8+ T cell; 2) activating the CD8+ T cell; 3) mixing the activated CD8+ T cell, a fibrinogen solution and a thrombin solution, and culturing same under conditions suitable for cell growth, so as to obtain a TSCM cell culture; and 4) bringing the culture into contact with a dispase so as to obtain a TSCM cell. The present application also relates to a kit for preparing and expanding a TSCM cell in vitro, the use of fibrinogen and thrombin in the preparation of a kit for preparing and expanding a TSCM cell in vitro, and a TSCM cell obtained by means of the method or kit of the present application.

Description

In vitro preparation and amplification of T SCM Cellular approach Technical Field

[0001] This application relates to the fields of biomedicine and immunotherapy. Specifically, it relates to the in vitro preparation and amplification of T cells. SCM This application also relates to a method for the in vitro preparation and amplification of T cells. SCM The use of cell kits, fibrinogen and thrombin in the preparation of said kits, and T cells obtained by the methods or kits of this application. SCM cell. Background Technology

[0002] In recent years, with the development of immunological technologies, various cell types, including lymphokine-activated killer cells (LAK), cytokine-induced killer cells (CIK), dendritic cells (DC), and T lymphocytes with specific targets, such as tumor-infiltrating T cells (TIL), T cell receptor chimeric T cells (TCR-T), and chimeric antigen receptor T cells (CAR-T), have shown good efficacy in the treatment of various tumors. CAR-T therapy, in particular, has gained widespread recognition for its effectiveness in treating hematologic malignancies. However, its efficacy in solid tumors remains less than satisfactory. Immunotherapy strategies, represented by T cells, are limited by the cell's survival time within the cell. Furthermore, the self-renewal, homeostasis maintenance, and tumor-killing capabilities of immune cells weaken with differentiation, eventually leading to terminally differentiated cells and a senescent state. Most commercially available CIK and CTL cells are close to terminally differentiated cells.

[0003] Stem-like memory T cells (T cells) SCM T cells are a rare subset of memory T lymphocytes with stem cell-like self-renewal capacity and the pluripotent potential to reconstruct the entire memory and effector T cell subset. SCM T cells can specifically recognize a variety of viruses and autoantigens, and compared with other known memory cells, T cells... SCM These cells possess higher proliferative capacity, can be maintained in vivo for extended periods, and can rapidly produce large amounts of cytokines upon antigen stimulation, thus exerting stronger anti-tumor and anti-inflammatory immune functions. Given these numerous advantages, an increasing number of researchers are attempting to explore T-cell-based... SCM Cellular development of novel adoptive immunotherapy strategies. Due to T SCM The proportion of T cells in the human body is very low; therefore, it is necessary to explore how to prepare and expand T cells on a large scale in vitro. SCM Cellular approaches become particularly important and urgent.

[0004] Currently, T SCMCell culture methods primarily rely on cytokine induction, specifically by adding high doses of IL-7 and IL-15 to block GSK-30 signaling and enhance the Wnt signaling pathway, thereby maintaining the number and phenotype of stem cells in vivo and simultaneously increasing their cytotoxic efficacy. However, this method yields a relatively small number of T cells, making it difficult to meet clinical requirements. Summary of the Invention

[0005] Human fibrinogen is a glycoprotein synthesized and secreted by hepatocytes. It is one of the most abundant clotting factors in blood plasma and can be converted into fibrin by thrombin, forming a stable, porous fibrin gel upon coagulation. As an insoluble protein, fibrin can be digested and degraded by dispersants. Utilizing fibrin gel culture systems can provide cells with a 3D culture environment, better mimicking the in vivo environment and thus offering experimental conditions closer to physiological states. This is of great significance for studying cell behavior, cell-cell interactions, and cell-microenvironment interactions.

[0006] Not limited to specific theories, the technical solution of this application is at least partly based on the discovery that applying the aforementioned 3D culture system to T cell culture helps to gain a deeper understanding of cell behavior and develop more effective cancer treatment strategies.

[0007] This application utilizes a 3D fibrin gel culture system established using fibrinogen and thrombin to develop a large-scale in vitro expansion of T cells. SCM Cell culture methods. T cells prepared using this method. SCM Cells not only highly express stemness-related genes (SOX2, NANOG, and TCF7), but also possess self-renewal capabilities and longer survival times both in vivo and in vitro.

[0008] To address the inability to prepare and amplify T cells in large quantities in vitro SCM This application is made in response to the challenges posed by cells.

[0009] In a first aspect, this application provides an in vitro preparation and amplification method for T SCM A cellular method, comprising or consisting of the following steps:

[0010] 1) Obtain the separated CD8 + T cells;

[0011] 2) Activate the CD8 + T cells;

[0012] 3) Activate CD8 + T cells, fibrinogen solution, and thrombin solution were mixed and cultured under suitable cell growth conditions to obtain T cells.SCM Cell cultures;

[0013] 4) Contact the culture with the dispersing enzyme to obtain T. SCM cell.

[0014] In some embodiments, in step 1) of the method of this application, the CD8 + T cells are used to separate peripheral blood, umbilical cord blood, cancerous tissue, pleural effusion, or ascites from an animal; preferably, the animal is a mammal, and the mammal is a non-human mammal or a human, more preferably, the mammal is a human.

[0015] In some embodiments, in step 2) of the method of this application, the CD8 is incubated with CD3 monoclonal antibody, CD28 monoclonal antibody and IL-2 under conditions suitable for cell growth. + T cells activate CD8 + T cells.

[0016] In a further embodiment, the final concentration of the CD3 monoclonal antibody is 0.1-10 μg / ml, for example, but not limited to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 μg / ml, more preferably 1-5 μg / ml, and even more preferably 2.5 μg / ml.

[0017] In a further embodiment, the final concentration of the CD28 monoclonal antibody is 0.1-10 μg / ml, such as, but not limited to, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 μg / ml, more preferably 0.1-2 μg / ml, and even more preferably 1 μg / ml.

[0018] In a further embodiment, the final concentration of IL-2 is 10-1000 IU / ml, such as, but not limited to, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000 IU / ml, more preferably 50-200 IU / ml, and even more preferably 100 IU / ml.

[0019] In a further embodiment, the CD8 antibody is incubated with CD3 monoclonal antibody, CD28 monoclonal antibody, and IL-2. +T cells are stored for 24-72 hours, preferably 24-48 hours, such as but not limited to 24, 26, 28, 30, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 44, 46, 48 hours, and more preferably 36 hours.

[0020] In some implementations, in step 3) of the method of this application, the activated CD8 + T cells were mixed with a fibrinogen solution and seeded into a culture plate pre-incubated with a thrombin solution. After incubation for 30 minutes, culture medium was added, wherein the fibrinogen was human fibrinogen and the thrombin was human thrombin.

[0021] In some embodiments, the final concentration of fibrinogen is 1-20 mg / ml, such as, but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 μg / ml, more preferably 5-10 mg / ml, and even more preferably 8 mg / ml.

[0022] In some embodiments, the concentration of the thrombin is 0.01-1 U / μl, such as, but not limited to, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 U / μl, more preferably 0.05-0.2 U / μl, and even more preferably 0.1 U / μl.

[0023] In a further embodiment, in step 3) of the method of this application, the activated CD8 is cultured in a medium containing β-mercaptoethanol and human IL-2. + T cells.

[0024] In some embodiments, in step 4) of the method of this application, the culture obtained in step 3) is contacted with a dispersing enzyme after culturing for 2-3 days to obtain the T. SCM Cells; the concentration of the dispersing enzyme is 1-10 mg / ml, for example, but not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg / ml, preferably 2-5 mg / ml, more preferably 4 mg / ml.

[0025] In a second aspect, this application provides a kit for the in vitro preparation and amplification of T... SCM Cells, and contain:

[0026] Components used to activate T cells;

[0027] Fibrinogen;

[0028] thrombin; and

[0029] Optionally, a dispersant enzyme.

[0030] In some embodiments, in the kit of this application, the component for activating T cells is a combination of CD3 monoclonal antibody, CD28 monoclonal antibody and IL-2.

[0031] In a further embodiment, the final concentration of the CD3 monoclonal antibody is 0.1-10 μg / ml, more preferably 1-5 μg / ml, and even more preferably 2.5 μg / ml.

[0032] In a further embodiment, the final concentration of the CD28 monoclonal antibody is 0.1-10 μg / ml, more preferably 0.1-2 μg / ml, and even more preferably 1 μg / ml.

[0033] In a further embodiment, the final concentration of IL-2 is 10-1000 IU / ml, more preferably 50-200 IU / ml, and even more preferably 100 IU / ml.

[0034] In some embodiments, in the kit of this application, the fibrinogen is human fibrinogen and the thrombin is human thrombin.

[0035] In a further embodiment, the final concentration of fibrinogen is 1-20 mg / ml, more preferably 5-10 mg / ml, and even more preferably 8 mg / ml; the concentration of thrombin is 0.01-1 U / μl, more preferably 0.05-0.2 U / μl, and even more preferably 0.1 U / μl.

[0036] In the third aspect, this application provides fibrinogen and thrombin in the preparation of T cells for in vitro preparation and amplification. SCM Use in cell kits.

[0037] In some embodiments, the fibrinogen is human fibrinogen, and the thrombin is human thrombin.

[0038] In a further embodiment, the final concentration of fibrinogen is 1-20 mg / ml, more preferably 5-10 mg / ml, and even more preferably 8 mg / ml; the concentration of thrombin is 0.01-1 U / μl, more preferably 0.05-0.2 U / μl, and even more preferably 0.1 U / μl.

[0039] In a fourth aspect, this application provides T obtained by the method or kit of this application. SCM cell.

[0040] In some implementations, the T SCMThe cell has one or more of the following characteristics:

[0041] 1) Increased proliferation rate;

[0042] 2) Enhanced CD62L + CD45RO - CD45RA + CCR7 + CD95 + Dry phenotype;

[0043] 3) Significantly upregulated expression level of nuclear transcription factor TCF7;

[0044] 4) Significantly upregulated expression levels of stemness-related genes SOX2 and NANOG, and slightly increased expression levels of BMI-1 and C-KIT;

[0045] 5) Enhanced self-renewal capacity and long-term survival ability.

[0046] Through extensive experimental research, the applicant has discovered that T cells cultured using 3D fibrin gel possess T... SCM Phenotype (CD62L) + CD45RO - CD45RA + CCR7 + CD95 + It highly expresses stemness-related genes (SOX2, NANOG, and TCF7); possesses self-renewal capacity in vitro; and exhibits longer survival time in vivo. Based on this application, it is possible to mass-produce T cells that continuously generate immune responses and possess stronger anti-tumor immune potential. SCM cell.

[0047] In this application, when numerical values ​​are mentioned, they should be understood to include an appropriate error range, such as ±1%, ±1.5%, ±2%, ±2.5%, ±3%, ±3.5%, ±4%, ±4.5%, ±5%, ±5.5%, ±6%, ±6.5%, ±7%, ±8%, ±9%, and ±10%. This is mainly due to reasonable errors introduced by measurement methods, personnel operation, raw materials themselves, operating environment, and instruments and equipment. For example, since the accuracy of the measurement method for concentration and enzyme activity, as well as the skill level of the personnel and sampling, are related to various factors, a concentration of 100 IU / ml can refer to 100 IU / ml within the error range, and a technician can determine the specific error range based on the context. When there is no explicit limitation, the error range is ±10%, that is, "100 IU / ml" should be understood as 90-110 IU / ml.

[0048] When referring to time, it should not be understood as being accurate to the second; errors are allowed, such as ±1%, ±1.5%, ±2%, ±2.5%, ±3%, ±3.5%, ±4%, ±4.5%, ±5%, ±5.5%, ±6%, ±6.5%, ±7%, ±8%, ±9%, and ±10%. For example, due to human operation and timer errors, a 36-hour period can have an error of ±10%.

[0049] The method described in this application does not involve the diagnosis or treatment of disease. Attached Figure Description

[0050] Figure 1 shows that T cells can be cultured and proliferated in large quantities in 3D fibrin gel.

[0051] Figure 2 shows the T cells prepared by the 3D fibrin gel culture system. SCM The cells exhibit a stronger stem phenotype.

[0052] Figures 3A and 3B show the T cells prepared by the 3D fibrin gel culture system. SCM The cells have a higher expression level of the nuclear transcription factor TCF7.

[0053] Figure 4 shows the T cells prepared by the 3D fibrin gel culture system. SCM The cells have higher levels of stem cell expression.

[0054] Figures 5A to 5F show the T cells prepared using the 3D fibrin gel culture system. SCM Cells in the body have a stronger self-renewal capacity and a longer survival time. Detailed Implementation

[0055] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0056] Various cell lines, animals, etc. used in the examples:

[0057] Human peripheral blood mononuclear cells were obtained from volunteers; immunodeficient NSG mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0058] Unless otherwise specified, all raw materials and reagents used in this invention are commercially available.

[0059] Example 1: T cells can be cultured and expanded in large quantities in 3D fibrin gel.

[0060] 1. Experimental Procedure

[0061] 1) Collect human peripheral blood mononuclear cells (PBMCs) and isolate CD8+ cells. + T cells;

[0062] 2) Coat cell culture plates with monoclonal antibodies against CD3 (2.5 μg / ml) and CD28 (1 μg / ml), and add isolated CD8 antibodies. + T cells were added with cytokine IL-2 (100 IU / ml) and cultured in an incubator at 37°C, 5% CO2 and saturated humidity for 36 hours.

[0063] 3) Collect activated CD8 + T cells will release CD8 + T cells were initially at a density of 2 × 10⁻⁶. 5 10 cells / well were seeded into 48-well plates to prepare control (Con-T) cells; 3.5 μl of thrombin solution (0.1 U / μl, Sea Run Holdings, catalog number SEA-135) was pre-added to 24-well plates; activated T cells were mixed with different concentrations of human fibrinogen solution (preferably 8 mg / ml, equivalent to 1050 Pa; RiaSTAP, catalog number NDC 63833-891-90) at an initial density of 2 × 10⁶ cells / well. 5 Immediately seed each T cell into a 24-well plate and incubate at 37°C with 5% CO2 for 30 minutes to allow fibrinogen to completely convert into fibrin gel and encapsulate T cells to form a 3D network structure. Add 1 ml of culture medium containing β-mercaptoethanol (1000×, Thermo, final concentration 55 μM) and human IL-2 (100 IU / ml) to each well and continue expansion culture to prepare 3D-T cells. Depending on the growth of T cells, passage or change the medium for Con-T cells and 3D-T cells respectively.

[0064] 4) After culturing for 3 days, the 3D fibrin gel formed by fibrinogen and thrombin was digested with dispersing enzyme (4 mg / ml), centrifuged at 500g for 5 min, and Con-T cells and 3D-T cells were harvested separately. The total number of Con-T cells and 3D-T cells was counted under a microscope using a cell counting chamber.

[0065] 2. Experimental Results

[0066] The proliferation rate of T cells in the 3D fibrin gel culture system (3D-T) was significantly higher than that in the rigid plate (Con-T): the proliferation rate of T cells in the rigid plate (Con-T) increased from 5 × 10⁻⁶ cells / year. 6 The number of amplifications increased to 5.06 × 10⁻⁶. 7 The number of cells increased to 1.36 × 10⁻⁶ in 3D fibrin gel (3D-T). 8 (Figure 1).

[0067] Example 2: T cells prepared using a 3D fibrin gel culture system SCMThe cells exhibit a stronger stem phenotype.

[0068] 1. Experimental Procedure

[0069] 3D-T and Con-T cells were collected and stained with Live / Dead dye at room temperature for 10 min, followed by staining with phenotype-associated antibodies (CD45RA, CD45RO, CCR7, CD95, CD62L) at 4°C for 30 min. Data were collected using an Invitrogen Attune NxT flow cytometer and analyzed using FlowJo software.

[0070] 2. Experimental Results

[0071] Flow cytometry analysis revealed that only 0.17% of the T cells in rigid plate-cultured Con-T cells were CD45RA. + CCR7 + CD45RO - CD95 + CD62L + (T SCM (Cell phenotype); while approximately 62% of 3D-T cells are CD45RA. + CCR7 + CD45RO - CD95 + CD62L + (Figure 2). In summary, T cells prepared using 3D fibrin gel have a higher proportion of T cells. SCM Cell phenotype.

[0072] Example 3: T cells prepared using a 3D fibrin gel culture system SCM The cells had higher levels of the nuclear transcription factor TCF7.

[0073] 1. Experimental Procedure

[0074] (1) qPCR: 3D-T cells prepared by the 3D fibrin gel culture system and Con-T cells cultured on rigid plates were collected. Total RNA was extracted after Trizol lysis and reverse transcribed into cDNA. The expression level of TCF7 gene was detected by qPCR.

[0075] (2) Western blotting: 3D-T cells and Con-T cells were collected, lysed in RIPA lysis buffer, and sonicated. Protein concentration was determined using a BCA kit (Beyotime). The samples were then electrophoresed on an SDS-PAGE gel and transferred to nitrocellulose. The nitrocellulose membranes with transferred proteins were blocked with 5% bovine serum albumin (BSA) and incubated overnight with TCF1 antibody, followed by chemiluminescence enhancement with a secondary antibody conjugated with horseradish peroxidase and development.

[0076] 2. Experimental Results

[0077] (1) qPCR results showed that the expression level of the stemness-related gene TCF7 in 3D-T cells prepared by the 3D fibrin gel culture system was significantly upregulated compared with that of Con-T cells cultured on rigid plates (Figure 3A).

[0078] (2) Further verification at the protein level showed that the protein expression level of TCF1 in 3D-T cells was significantly upregulated compared with Con-T cells (Figure 3B).

[0079] Example 4: T cells prepared using a 3D fibrin gel culture system SCM Cells have higher levels of stem cell expression.

[0080] 1. Experimental Procedure

[0081] qPCR: 3D-T cells and Con-T cells were collected, lysed with Trizol, and RNA was extracted and reverse transcribed into cDNA. qPCR was then performed using the corresponding primers.

[0082] 2. Experimental Results

[0083] qPCR results showed that, compared with Con-T cells, the expression levels of SOX2 and NANOG were significantly upregulated in 3D-T cells, while the expression levels of BMI-1 and C-KIT were slightly increased, and stem genes such as Oct3 / 4 and NESTIN were undetectable.

[0084] Example 5: T cells prepared using a 3D fibrin gel culture system SCM Cells in the body have a stronger self-renewal capacity and a longer survival time.

[0085] 1. Experimental Procedure

[0086] 1×10 7 Personally derived 3D-T cells or Con-T cells were adopted into NSG mice via tail vein transfer. On days 10, 20, and 30, the number of surviving T cells and stem phenotype in bone marrow, spleen, and peripheral blood were detected using human CD8 antibody.

[0087] 2. Experimental Results

[0088] Flow cytometry analysis showed that, compared with the Con-T group mice, the 3D-T group mice had higher levels of human CD8 in their bone marrow, spleen, and peripheral blood. + The number of T cells was significantly higher, and they exhibited a greater stem phenotype (Figures 5A to 5F).

[0089] In summary, the solution proposed in this application has the following effects:

[0090] 1. This application utilizes a 3D fibrin gel culture system formed from fibrinogen and thrombin to enable the large-scale preparation and amplification of T... SCM Cells, and have T SCM It has the characteristics of high cell expansion efficiency, low degree of differentiation, and ability to self-renew.

[0091] 2. T prepared using the method and kit of this application SCM These cells exhibit high levels of stem gene expression and cytokine release, and maintain long-term persistence and function within the tumor microenvironment.

[0092] 3. The method and kit of this application can generate a large number of T cells with younger phenotypes and stronger killing ability. SCM Cell populations, which may break through the limitations of existing adoptive immunization, solve problems such as T cell transplantation, short survival time in vivo, and inability to continuously generate immune responses, and have significant clinical application prospects.

Claims

1. An in vitro preparation and amplification method for T SCM Cellular methods, including: 1) obtaining an isolated CD8 + T cell; 2) activating the CD8 + T cells; 3) the activated CD8 + T cells, the fibrinogen solution and the thrombin solution are mixed and incubated under conditions suitable for cell growth, obtaining a T SCM cell culture; Preferably, the method further comprises: 4) contacting the culture with dispersin, obtaining T SCM cells.

2. The method according to claim 1, wherein: In step 1), the CD8 + T cells are isolated from the peripheral blood, umbilical cord blood, cancer tissue, pleural fluid or ascites of an animal, Preferably, the animal is a mammal, the mammal is a non-human mammal or a human, more preferably the mammal is a human.

3. The method according to claim 1, wherein: In step 2), the CD8 + T cells are activated by incubating them with CD3 monoclonal antibodies, CD28 monoclonal antibodies and IL-2 under conditions suitable for cell growth + T cells, Preferably, the final concentration of the CD3 monoclonal antibody is 0.1-10 pg / ml, more preferably 1-5 pg / ml, further preferably 2.5 pg / ml ± 10%; Preferably, the final concentration of the CD28 monoclonal antibody is 0.1-10 pg / ml, more preferably 0.1-2 pg / ml, further preferably 1 pg / ml ± 10%; Preferably, the final concentration of the IL-2 is 10-1000 IU / ml, more preferably 50-200 IU / ml, further preferably 100 IU / ml ± 10%; Preferably, the CD8+ T cells are incubated with CD3 / CD28 monoclonal antibodies and IL-2 + T cells 24-72 hours, more preferably 24-48 hours, further preferably 36 hours ± 10%.

4. The method according to claim 1, wherein: In step 3) the activated CD8 + T cells were mixed with fibrinogen solution, seeded into culture plates pre-incubated with a thrombin solution, and after 30 minutes of incubation the culture medium was added, wherein The fibrinogen is human fibrinogen, the thrombin is human thrombin; Preferably, the final concentration of the fibrin is 1-20 mg / ml, more preferably 5-10 mg / ml, further preferably 8 mg / ml ± 10%; Preferably, the final concentration of the thrombin is 0.01-1 U / pl, more preferably 0.05-0.2 U / pl, further preferably 0.1 U / pl ± 10%; Preferably, in step 3), said activated CD8 + T cells are cultured with a culture medium containing beta-mercaptoethanol and human IL-2. T cells are cultured with a culture medium containing beta-mercaptoethanol and human IL-2.

5. The method according to claim 1, wherein: In step 4), the culture is contacted with a dispersion enzyme after 2-3 days of culturing, the final concentration of the dispersion enzyme is 1-10 mg / ml, preferably 2-5 mg / ml, more preferably 4 mg / ml ± 10%.

6. A kit for the in vitro preparation and amplification of T cells. SCM Cells, and contain: a component for activating T cells; fibrinogen; thrombin; and optionally, a dispersion enzyme.

7. The kit according to claim 6, wherein: Preferably, the component for activating T cells is a combination of a CD3 monoclonal antibody, a CD28 monoclonal antibody and IL-2, Preferably, the final concentration of the CD3 monoclonal antibody is 0.1-10 pg / ml, more preferably 1-5 pg / ml, further preferably 2.5 pg / ml ± 10%; Preferably, the final concentration of the CD28 monoclonal antibody is 0.1-10 pg / ml, more preferably 0.1-2 pg / ml, further preferably 1 pg / ml ± 10%; Preferably, the final concentration of the IL-2 is 10-1000 IU / ml, more preferably 50-200 IU / ml, further preferably 100 IU / ml ± 10%; Preferably, the fibrinogen is human fibrinogen, the thrombin is human thrombin; Preferably, the final concentration of the fibrinogen is 1-20 mg / ml, more preferably 5-10 mg / ml, further preferably 8 mg / ml ± 10%; Preferably, the final concentration of the thrombin is 0.01-1 U / pl, more preferably 0.05-0.2 U / pl, further preferably 0.1 U / pl ± 10%; Preferably, the final concentration of the dispersion enzyme is 1-10 mg / ml, preferably 2-5 mg / ml, more preferably 4 mg / ml ± 10%.

8. The use of fibrinogen and thrombin in the preparation of a kit for the in vitro preparation and expansion of T cells for adoptive immunotherapy. SCM cells. Preferably, the fibrinogen is human fibrinogen and the thrombin is human thrombin; Preferably, the final concentration of the fibrinogen is 1-20 mg / ml, more preferably 5-10 mg / ml, further preferably 8 mg / ml ± 10 %; Preferably, the concentration of the thrombin is 0.01-1 U / μl, more preferably 0.05-0.2 U / μl, further preferably 0.1 U / μl ± 10 %.

9. The T cells obtained by the method of any one of claims 1 to 5 or the kit of claim 6 or 7 SCM cells having one or more of the following characteristics: 1) increased proliferation rate; 2) enhanced CD62L + CD45RO - CD45RA + CCR7 + CD95 + stemness phenotype; 3) significantly up-regulated expression level of the nuclear transcription factor TCF7; 4) significantly up-regulated expression levels of the stemness-associated genes SOX2, NANOG, slightly increased expression levels of BMI-1 and C-KIT; 5) enhanced self-renewal capacity and long-term survival capacity in vivo.

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