Method for enriching Tcm cells and application thereof

Abstract

The invention provides a method for enriching Tcm cells and application thereof, and particularly, the method comprises the step of performing first co-culture on a T cell population and stem cell exosomes. The exosome secreted by the stem cells contains a plurality of active proteins, RNA, lipids, metabolites and other molecules for keeping the dryness of the cells, the stem cell exosome and the T cell population are co-cultured, and the stem cell exosome can keep the rejuvenation of the T cells in the culture process and induce the differentiation of the T cells to the Tcm cells, so that the proportion of the Tcm cells is increased, and the cell proliferation rate is increased. High cell viability and proliferation capacity are maintained, and guarantee is provided for the function exertion of the T cells and related cell products thereof.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological cells, in particular to a method for enriching Tcm cells and application thereof. BACKGROUND

[0002] Exosomes are a kind of cell-derived extracellular vesicles with a diameter of about 30-150 nm, having an intact membrane structure, containing proteins, lipids, DNA, RNA and metabolites, and the main exosome marker proteins are CD63, CD9, CD81 and TSG101. Exosomes carrying active substances can enter target cells or directly act on receptors on the target cells through membrane fusion, endocytosis and other ways, and participate in the physiological and pathological processes of target cells, which is a way of intercellular information transmission. Exosomes are widely distributed and can be detected in almost all body fluids, such as saliva, cerebrospinal fluid, blood, tears, urine and milk. Stem cell-derived exosomes can promote cell survival, proliferation and tissue repair, and participate in immune regulation. Exosomes can also be used as a detection index for screening certain diseases such as cancer, bringing hope for clinical application. Especially, stem cell-derived exosomes show great application potential in regenerative medicine. Studies have shown that mesenchymal stem cell-derived exosomes can effectively slow down skin aging, induce fibroblast proliferation and collagen synthesis, reduce inflammatory response, promote tissue repair after injury, and also can regulate myocardial cell function, which has great potential in treating aging-related cardiovascular diseases. Embryonic stem cell-derived exosomes also have pro-regenerative and anti-aging effects, can promote angiogenesis and anti-fibrosis, treat radiation-induced lung injury, improve ovarian function, restore the function of aging bone marrow mesenchymal stem cells, and alleviate the aging of tissue-resident stem cells. They are promising candidate drugs for treating aging-related diseases.

[0003] T cells are a kind of lymphocytes and are the main component of the immune system, which plays a role in immune surveillance, immune regulation, memory, cytotoxicity and immune tolerance in the body. The culture process of T cells is crucial for the T cell product to play a good function. During the in vitro culture of T cells, different subpopulations will appear, mainly including T N , Tcm, T EM , etc. The phenotype, homing ability and cytotoxic function of each cell subpopulation are different. Literature reports that the cell product of Tcm subpopulation has a longer survival time in vivo and can play a better cytotoxic effect on tumors. Different culture media, culture methods, cytokines and culture environments can affect the proportion of each subpopulation during the in vitro culture of T cells. Therefore, in order to improve the functional effect of T cell product, it is still necessary to optimize the culture method of T cells and improve the content of Tcm, which is the dominant cell subpopulation. SUMMARY

[0004] The present application is based on the discovery and understanding of the inventors of the following problems:

[0005] Embryonic stem cell therapy is a promising method for forming new functional heart cells and improving angiogenesis. Embryonic stem cells have totipotent differentiation properties, can be genetically engineered using genetic engineering techniques, and then differentiated into specific functional cells for treating diseases, such as embryonic stem cell-derived CART and CAR-NK cells for tumor immunotherapy. Embryonic stem cell exosomes have many similar functions to embryonic stem cells, are easy to produce, are easy to store, have a long half-life, can pass through the blood-brain barrier, and have good safety, stability and low immunogenicity. Studies have shown that embryonic stem cell exosomes have good anti-fibrosis function and promote the repair of damaged liver and heart.

[0006] During in vitro culture, T cells gradually age and even exhaust, affecting the function of T cell products. The present application finds that by using stem cell exosomes and a certain method to treat activated T cells, the activated T cells can be effectively induced to transform into Tcm cells, the proportion of Tcm cells in the cell population can be increased, the T cells can be kept young, the viability and expansion capacity of the T cells can be improved, and the function of T cells and related cell products can be better guaranteed.

[0007] Therefore, in a first aspect of the present application, a method for enriching Tcm cells is provided. According to embodiments of the present application, the method comprises co-culturing a T cell population with stem cell exosomes. The present application surprisingly finds that co-culturing stem cell exosomes and a T cell population can induce a higher proportion of Tcm cells, which can guarantee the function of T cells and related products after being reinfused into the body.

[0008] According to embodiments of the present application, the method for enriching Tcm cells described above can further comprise at least one of the following additional technical features:

[0009] According to embodiments of the present application, the working concentration of the stem cell exosomes is 50-120 μg / mL. Exemplarily, the working concentration of the stem cell exosomes is 50, 52, 55, 57, 60, 62, 65, 67, 70, 72, 75, 77, 80, 82, 85, 87, 90, 92, 95, 97, 100, 102, 105, 107, 110, 112, 115, 117, 120 μg / mL or a range value between any two numerical values, for example, 50-110, 60-110, 70-110, 80-110, 50-100, 60-100, 70-100, 80-100, 50-90, 60-90, 70-90, 80-90, 50-80, 60-80 or 70-80.

[0010] According to a preferred embodiment of the present application, the working concentration of the stem cell exosomes is 70-90 μg / mL. Thereby, the proportion of Tcm cells in the obtained cell population is higher.

[0011] According to an embodiment of the present application, the exosomes contain TSG101, Alix and HSP70 proteins, and do not contain Calnexin protein.

[0012] According to an embodiment of the present application, the time of the first co-culture is 1-14 days of the culture of the T cell population. That is, the stem cell exosomes can be added at any time period within 1-14 days of the culture of the T cell population, and the proportion of Tcm cells in the cell population can also be effectively increased.

[0013] According to some specific embodiments of the present application, the co-culture and the culture of the T cell population are performed at 35-38 °C and 4-6% CO2.

[0014] According to an embodiment of the present application, when the co-culture is performed, the seeding density of the T cell population is 1x10e6-2x10e6 cells / ml.

[0015] According to an embodiment of the present application, when the co-culture is performed, the seeding amount of the T cell population is 2x10e6-4x10e6 cells.

[0016] According to some specific embodiments of the present application, the T cell population is derived from a PBMC cell population.

[0017] According to an embodiment of the present application, the system of the co-culture contains CD3 antibody, and the mass of the CD3 antibody is 1-10 μg / mL. Exemplarily, the mass of the CD3 antibody is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 μg, or a range value between any two numerical values, for example, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9 or 9-10.

[0018] According to embodiments of the present application, the co-cultured system comprises CD28 antibody, and the mass of the CD28 antibody is 1-10 μg. Illustratively, the mass of the CD28 antibody is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μg, or a range value between any two numerical values, for example, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10.

[0019] In the present application, CD3 antibody and CD28 antibody are used to activate T cells in PBMC. According to some specific embodiments of the present application, the activation is performed for 0.5-48 h. The activation time is related to the activation temperature. Illustratively, short-time activation can be performed at room temperature, or long-time activation can be performed at a lower temperature, for example, 20-48 h at 4 °C.

[0020] According to embodiments of the present application, the co-cultured system comprises interleukin, and the working concentration of the interleukin is 100-1000 IU / ml. Illustratively, the working concentration of the interleukin is 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 IU / ml, or a range value between any two numerical values.

[0021] According to specific embodiments of the present application, the working concentration of the interleukin is 100-300 IU / ml.

[0022] According to embodiments of the present application, the interleukin comprises at least one of the following: IL-2, IL-7, IL-15, and IL-21.

[0023] According to embodiments of the present application, the culture medium of the co-culturing and the T cell population culture is independently selected from at least one of the following: RPMI 1640, CTS OpTmizer T-Cell Expansion SFM, X-VIVO 15, and AIMV TM SFM culture medium.

[0024] According to embodiments of the present application, the stem cell exosome comprises an exosome selected from at least one of the following: totipotent stem cell, pluripotent stem cell, and multipotent stem cell.

[0025] According to embodiments of the present application, the pluripotent stem cells comprise induced pluripotent stem cells.

[0026] According to embodiments of the present application, the stem cell exosomes comprise embryonic stem cell exosomes.

[0027] According to embodiments of the present application, the stem cell exosomes are obtained by separation and extraction of a culture medium of the stem cell culture.

[0028] According to embodiments of the present application, the culture medium of the stem cell culture comprises at least one selected from the group consisting of StemRD PGroF-500, mTeSR TM Plus Basal Medium, EZ-Stem TM Cell Culture Medium, Essential 8 TM Basal Medium and StemFlex TM Medium.

[0029] According to embodiments of the present application, the confluency of the stem cells is 85% to 95% when the stem cell exosomes are subjected to the separation process. Exemplarily, the confluency of the stem cells is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% or 95% or a range between any two of the values, for example, 86% to 95%, 87% to 95%, 88% to 95%, 89% to 95%, 90% to 95%, 91% to 95%, 92% to 95%, 93% to 95%, 94% to 95%, 85% to 94%, 86% to 94%, 87% to 94%, 88% to 94%, 89% to 94%, 90% to 94%, 91% to 94%, 92% to 94%, 93% to 94%, 85% to 93%, 86% to 93%, 87% to 93%, 88% to 93%, 89% to 93%, 90% to 93%, 91% to 93%, 92% to 93%, 85% to 92%, 86% to 92%, 87% to 92%, 88% to 92%, 89% to 92%, 90% to 92%, 91% to 92%, 85% to 91%, 86% to 91%, 87% to 91%, 88% to 91%, 89% to 91%, 90% to 91%, 85% to 90%, 86% to 90%, 87% to 90%, 88% to 90% or 89% to 90%.

[0030] According to some preferred embodiments of the present application, the confluency of the stem cells is 90% to 95% when the stem cell exosomes are subjected to the separation process.

[0031] According to some embodiments of the present application, the confluence of the stem cells is 95% when the stem cell exosomes are isolated.

[0032] According to embodiments of the present application, the separation treatment is performed by:

[0033] (1) subjecting the culture solution of the stem cells to a first centrifugal treatment to obtain a first centrifugal supernatant, the first centrifugal treatment being performed at 2-6°C at a speed of 1000-3000g for 20-40min;

[0034] (2) subjecting the first centrifugal supernatant to a second centrifugal treatment to obtain a second centrifugal supernatant, the second centrifugal treatment being performed at 2-6°C at a speed of 10000-20000g for 20-40min;

[0035] (3) filtering the second centrifugal supernatant through a 0.15-0.25μm filter to obtain a filtrate. Preferably, a 0.22μm filter is used.

[0036] According to embodiments of the present application, the extraction treatment is performed by purifying the filtrate in an exosome extraction device. Those skilled in the art will understand that the exosome extraction device is not particularly limited, and any device capable of purifying and extracting exosomes is included within the scope of the present application.

[0037] According to embodiments of the present application, the stem cell culture is performed at 35-38°C under 4-6% CO2.

[0038] According to some preferred embodiments of the present application, the stem cell culture is performed at 37°C under 5% CO2.

[0039] According to embodiments of the present application, the PBMC cell population is obtained by:

[0040] (1) mixing whole blood with a separation solution and a buffer in a volume ratio of (1-3):(1-3):(1-3);

[0041] (2) subjecting the product of step (1) to a third centrifugal treatment to obtain a third centrifugal treatment product, the third centrifugal treatment being performed at 20-30°C at a speed of 600-900g for 10-40min, the third centrifugal treatment product being located between the plasma layer and the separation solution layer;

[0042] (3) sequentially diluting and subjecting the third centrifugal treatment product to a fourth centrifugal treatment to obtain a fourth centrifugal precipitate, the fourth centrifugal treatment being performed at 20-30°C at a speed of 150-350g for 5-20min.

[0043] Those skilled in the art can understand that the separation solution described above is not particularly limited, and any conventional separation solution in the art can be used, all of which are within the protection scope of the present application.

[0044] According to embodiments of the present application, the buffer solution comprises at least one of the following: PBS and physiological saline.

[0045] In the second aspect of the present application, the present application provides a cell population. According to embodiments of the present application, the cell population is obtained by the method of the first aspect. As described above, the method of the first aspect can be used to obtain a cell population with a high proportion of Tcm cells, for example, not less than 50%.

[0046] According to embodiments of the present application, the proportion of Tcm cells in the cell population is not less than 50%. Illustratively, the proportion of Tcm cells in the cell population is 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a range value between any two numerical values, preferably 60% to 70%.

[0047] According to some specific embodiments of the present application, the proportion of CD45RO + CD62L + The proportion of Tcm cells is not less than 50%. Illustratively, the proportion of CD45RO + CD62L + The proportion of Tcm cells is 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a range value between any two numerical values, preferably 60% to 70%.

[0048] In a third aspect, the present application provides a culture system for enriching Tcm cells. According to an embodiment of the present application, the culture system comprises: stem cell exosomes, the working concentration of which is 50-120 μg / mL. As described above, the present application finds that stem cell exosomes can induce T cells to differentiate into Tcm cells, and thus the Tcm cells obtained account for not less than 50% of the cell population, which provides a guarantee for the function of T cells and related products after being reinfused into the body.

[0049] According to an embodiment of the present application, the culture system further comprises: a CD3 antibody, the mass of which is 1-10 μg. Illustratively, the mass of the CD3 antibody is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 μg, or a range value between any two numerical values, for example, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9 or 9-10.

[0050] According to an embodiment of the present application, the culture system further comprises: a CD28 antibody, the mass of which is 1-10 μg. Illustratively, the mass of the CD28 antibody is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 μg, or a range value between any two numerical values, for example, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9 or 9-10.

[0051] According to an embodiment of the present application, the culture system further comprises: an interleukin, the working concentration of which is 100-1000 IU / ml. Illustratively, the working concentration of the interleukin is 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 IU / ml, or a range value between any two numerical values.

[0052] According to a specific embodiment of the present application, the working concentration of the interleukin is 100-300 IU / ml.

[0053] According to embodiments of the application, the interleukin comprises at least one of: IL-2, IL-7, IL-15, and IL-21.

[0054] In a fourth aspect of the application, a composition is provided. According to embodiments of the application, the composition comprises the cell population obtained by the method of the first aspect, or the cell population of the second aspect.

[0055] According to embodiments of the application, the composition further comprises a pharmaceutically acceptable carrier.

[0056] The pharmaceutically acceptable carrier includes any solvent, solid excipient, diluent, binding agent, disintegrant, or other liquid excipient, dispersant, flavoring agent or suspending agent, surface active agent, isotonic agent, thickening agent, emulsifying agent, preservative, solid binding agent, glidant or lubricant, and the like, suitable for the particular target dosage form. Except insofar as any conventional excipient is incompatible with the cell population of the application, for example, by producing an adverse, biological effect or interacting in a deleterious manner with any other component(s) of the pharmaceutically acceptable composition, their use is contemplated to be within the scope of this application.

[0057] For example, the cell population of the application can be incorporated into a composition suitable for parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular) administration. These compositions can be prepared in various forms. For example, liquid, semi-solid, and solid dosage forms, etc., including but not limited to liquid solutions (e.g., injectable solutions and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. A typical composition is in the form of an injectable solution or infusible solution. Preferably, the cell population can be administered by intravenous infusion or injection or intramuscular or subcutaneous injection.

[0058] It is noted that the composition includes combinations that are separated in time and / or space, as long as they can act together to achieve the purpose of the application. For example, the components contained in the composition can be administered to the subject as a whole, or administered to the subject separately. When the components contained in the composition are administered to the subject separately, each component can be administered to the subject simultaneously or sequentially.

[0059] The effective amount of the cell population described herein can vary depending on the mode of administration and the severity of the disease to be treated. The selection of a preferred effective amount can be determined by one of ordinary skill in the art (e.g., through clinical trials) based on various factors. Such factors include, but are not limited to, the pharmacokinetic parameters of the active ingredient(s) such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated; the body weight of the patient; the immune status of the patient; the route of administration; etc. For example, the exigencies of the therapeutic situation can warrant the administration of several divided doses over the course of a day, or a proportionally reduced dosage for patients with severe or uncompensated hepatic or renal impairment.

[0060] It should be noted that the term "subject" refers to a mammal being evaluated for treatment and / or being treated. Subjects include, but are not limited to, individuals having cancer, individuals having autoimmune diseases, individuals having pathogen infections, etc. In one embodiment, the subject is a human, but also includes other mammals, particularly mammals useful as laboratory models for human diseases, such as other primates, mice, rats, etc.

[0061] The advantages of the present application over the prior art include at least:

[0062] The method described herein can significantly improve the content of Tcm cells in the T cell population during in vitro culture, wherein the proportion of Tcm cells in the cell population is close to 65%, and in addition, the method also improves the T cell viability and expansion capacity, and the T cell viability is close to 90%, which provides a guarantee for the better function of T cells and related cell products in vivo.

[0063] Additional aspects and advantages of the application will be set forth in part in the description that follows, and in part will become apparent to those skilled in the art upon examination of the following and / or can be learned by practice of the application. BRIEF DESCRIPTION OF DRAWINGS

[0064] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiments. It should be understood that the following drawings only show some of the embodiments of the present application, and therefore should not be considered as limiting the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0065] Figure 1 The particle distribution of the human embryonic stem cell-derived exosomes in the embodiments of the present application is shown;

[0066] Figure 2 The electron microscope observation results of the human embryonic stem cell-derived exosomes in the embodiments of the present application are shown;

[0067] Figure 3The figure shows the detection results of Marker protein of the human embryonic stem cell-derived exosomes in the embodiment of the present application;

[0068] Figure 4 The figure shows the detection results of the viability of T cells during the culture process in the embodiment of the present application;

[0069] Figure 5 The figure shows the detection results of the expansion capacity of T cells during the culture process in the embodiment of the present application;

[0070] Figure 6 The figure shows the proportion of CD4 + and CD8 + during the in vitro culture of T cells in the embodiment of the present application;

[0071] Figure 7 The figure shows the distribution of Tcm subpopulation on the 7th day of the in vitro culture of T cells in the embodiment of the present application;

[0072] Figure 8 The figure shows the statistical results of the proportion of Tcm subpopulation on the 7th day and the 14th day of the in vitro culture of T cells in the embodiment of the present application. DETAILED DESCRIPTION

[0073] The embodiments of the present application are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below by referring to the accompanying drawings are exemplary and are intended to explain the present application, and cannot be understood as limiting the present application.

[0074] In addition, the terms "first", "second" are only for the purpose of description, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined with "first", "second" can explicitly or implicitly include at least one of the features. In the description of the present application, the meaning of "a plurality of", "a plurality of kinds" is at least two, two kinds, for example, two, two kinds, three, three kinds, etc., unless otherwise specifically limited.

[0075] In this document, the terms "comprising", "having" or "including" are open-ended expressions, i.e. including the indicated content but not excluding other aspects.

[0076] In this document, the term "optionally" generally means that the event or condition described subsequently can but does not necessarily occur, and the description includes the case where the event or condition occurs and the case where the event or condition does not occur.

[0077] The endpoints of the ranges and any values stated in this disclosure are not limited to the precise values stated. The ranges and values should be construed to be approximations that allow for significant variation. For ranges, the endpoints are included within the ranges, and the ranges are inclusive of the individual points within the ranges. For ranges, the endpoints are included within the ranges, and the ranges are inclusive of the individual points within the ranges, and the individual points are included within the ranges.

[0078] In this disclosure, the working concentration and the mass of each component in the co-culture system refer to the final concentration and the final mass of each component in the co-culture system. For example, the working concentration of the stem cell exosome in the co-culture system is 50-120 μg / mL, which means the final concentration of the stem cell exosome in the co-culture system is 50-120 μg / mL, and the mass of the CD3 antibody is 1-10 μg, which means the final mass of the CD3 antibody in the co-culture system is 1-10 μg.

[0079] To facilitate understanding of the present application, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which the present application pertains.

[0080] The solutions of the present application will be explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only used to illustrate the present application and should not be regarded as limiting the scope of the present application. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the art or according to the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0081] Example 1

[0082] 1.1 Culture of human embryonic stem cells

[0083] The following steps are not specifically described using conventional methods, and the reagents and consumables used are purchased from commercial companies. The human embryonic stem cells are obtained from the Stem Cell Bank (National Stem Cell Resource Bank). The embryonic stem cells are stem cells isolated from human embryos that have not undergone in vivo development for 14 days or less after fertilization. The specific steps of stem cell culture are as follows:

[0084] (1) Prepare the culture medium: The fillers corresponding to the culture medium are thawed in a 4°C refrigerator one day in advance, and are mixed into the culture medium according to the specified proportions, and are stored at 4°C for use within the valid period.

[0085] (2) Matrigel coating: Take a tube of melted matrigel (Vitronectin (VTN-N) Recombinant Human Protein, purchased from thermo) corresponding to the culture medium, melt at room temperature, dilute and mix according to the instructions, coat the 6-well plate, add 1-2 ml per well, shake gently and evenly. Put in the carbon dioxide incubator for 60 min.

[0086] (3) Cell culture: use a 37℃ water bath to quickly melt the above-mentioned human embryonic stem cells, transfer them into an appropriate amount of culture medium (Essential 8 TM Basal Medium, purchased from thermo), centrifuge at 300g-500g for 3-5 min, discard the supernatant, resuspend and count with a small amount of culture medium, inoculate 6-150,000 cells per well into a 6-well plate, and culture at 37℃, 5% CO2. According to the color change of the culture medium, generally replace the new culture medium every 1-2 days, 2-4 ml per well, and the confluence of the cells is about 95%. Start subculture, first wash the cells with DPBS once, digest the cells with the commonly used digestion enzyme for stem cells, centrifuge at 300g-500g for 3-5 min, collect the cells, inoculate 6-150,000 cells per well into a new 6-well plate, collect the cell supernatant before subculture, and store it at -80℃ for extracting exosomes.

[0087] 1.2 Extraction and identification of human embryonic stem cell exosomes

[0088] Reagents and consumables: high-speed refrigerated centrifuge, pipette and pipette tips, 50ml centrifuge tube rack, 50ml centrifuge tube, 1.5ml centrifuge tube, 0.22um filter membrane, 20ml syringe, 84 liquid, pure water, 1XPBS or normal saline, marker pen, sealing film, gloves, 75% alcohol, -80℃ refrigerator

[0089] Collect the embryonic stem cell culture supernatant to extract exosomes, and the specific experimental steps are as follows:

[0090] (1) Centrifuge the above-mentioned embryonic stem cell culture liquid obtained after culture at 4℃, 2000g for 30min, and take the supernatant.

[0091] (2) 4℃, 12000g-15000g, centrifuge for 30min, take the supernatant.

[0092] (3) 0.2μm filter into 50ml centrifuge tube for standby.

[0093] (4) Preparation for machine, use the full-automatic exosome extraction equipment (EXODUS) to purify, according to the instrument instruction to clean the probe, load the sample, after the end, take out the chip, PBS or physiological saline blows the chip two sides about 30 times and sucks into 1.5ml centrifuge tube, take appropriate amount of about 300ul purified sample to Beijing Enze Kangtai Biological Technology Co., Ltd. for detection, mainly including exosome protein marker determination, electron microscope observation, NTA (nanoparticle tracking analysis) detection.

[0094] Results are shown in Figure 1 , Figure 2 and Figure 3 , wherein Figure 1 shows the NTA detection results, and the particle diameter is mostly between 30-150nm. Figure 2 shows the particle size and morphology of embryonic stem cells. Figure 3 shows the detection results of exosome protein marker, CL is the positive control group, and the results show that the exosome contains TSG101, Alix, and HSP70 three marker protein molecules, and does not contain Calnexin protein molecules.

[0095] 1.3 Separation of PBMC (Peripheral Blood Mononuclear Cell)

[0096] Take out the human lymphocyte separation medium, equilibrate at room temperature for 30 minutes, according to the requirements of the instruction, take appropriate amount of lymphocyte separation medium into 50ml centrifuge tube, dilute the peripheral blood with physiological saline, then slowly transfer to the surface of the lymphocyte separation medium, after centrifugation, suck the cells on the surface layer of the separation medium, wash, obtain PBMC, the specific steps are as follows:

[0097] (1) Take fresh anticoagulated blood (EDTA or heparin anticoagulant can be used). Dilute the whole blood with equal volume of PBS or physiological saline.

[0098] (2) Add a certain volume of separation medium (Ficoll-PaqueTM PREMIUM (1.077); brand: Thermo; item number: 17544202) in the centrifuge tube, place the diluted blood sample on the surface of the separation medium, and keep the interface clear. The volume ratio of separation medium, anticoagulated whole blood, PBS or physiological saline is 1:1:1.

[0099] (3) Room temperature, horizontal rotor 700g-800g centrifugation for 20-30min. Set a slower lifting speed.

[0100] (4) After centrifugation, the bottom of the tube is red blood cells, the middle layer is separation medium, and the top layer is plasma homogenate layer. There is a thin white film between the plasma layer and the separation medium layer, which is carefully sucked into another centrifuge tube.

[0101] (5) Dilute to a certain volume with PBS or normal saline, invert to mix. Centrifuge at room temperature, 250g, for 10 min, discard the supernatant. Repeat the washing for 1-2 times.

[0102] (6) Resuspend the cells with PBS or normal saline for later use.

[0103] 1.4 T cell activation culture

[0104] Coat the CD3 and CD28 antibodies in the 6-well plates according to the instructions, then inoculate the obtained PBMC cells into the 6-well plates at the required concentration, activate the T cells, add different concentrations of embryonic stem cell exosomes in the experimental group, and do not add embryonic stem cell exosomes in the control group, subculture the cells according to the growth density, and the co-culture conditions are consistent with the PBMC single culture (control group). The T cell viability and proliferation are detected during the culture process. The specific experimental steps are as follows:

[0105] (1) Dilute the CD3 antibody (1-5 μg / mL) and CD28 antibody (1-5 μg / mL) with PBS, coat the 6-well plates, and add 1-2 ml / well. After overnight incubation at 4°C, remove the PBS buffer, and wash with normal saline for 3 times.

[0106] (2) Inoculate the PBMC into the 6-well plates coated with CD3 and CD28 antibodies, with an inoculation density of 2x10e6 / ml and an inoculation amount of 2 ml / well. The culture medium is X-VIVO 15 (purchased from Lonza), and IL2 is added to the culture medium to make the final concentration in the co-culture system 200 IU / ml. The exosomes are added in the experimental group to make the final concentration in the co-culture system 80 μg / ml, and no exosomes are added in the control group. According to the cell growth, subculture the cells every 1-2 days, with a subculture concentration of 0.5x10e6-1x10e6 / ml and a culture medium volume of 2 ml / well, so that the working concentration of the stem cell exosomes is always maintained at 80 μg / ml, and the working concentration of the CD3 and CD28 antibodies in the co-culture system is 1.0 μg / mL. Incubate the above experimental group and control group at 37°C and 5% CO2, and detect the T cell viability and proliferation during the culture process.

[0107] The results are shown in Figure 4 and Figure 5 Compared with the control group, the T cell viability of the experimental group with added embryonic stem cell exosomes is significantly improved on days 5-15 of co-culture, and the T cell proliferation ability of the experimental group with added embryonic stem cell exosomes is significantly improved on days 11-15 of co-culture.

[0108] 1.5 Flow cytometry detection of human T cell subpopulations

[0109] Flow cytometry was performed according to the manufacturer's instructions. Different groups of T cells were stained with flow cytometry antibodies according to the manufacturer's instructions. After washing with PBS, the results were analyzed according to the manufacturer's instructions.

[0110] The results are shown in Figure 6 There was no significant change in the proportion of CD4 and CD8 in the control and experimental groups. Figure 7 The results are shown in Figure 8 The statistical results of the proportion of Tcm subpopulation on day 7 and day 14 are shown. Compared with the control group, the Tcm subpopulation of the experimental group was significantly increased.

[0111] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "embodiment" or "specific embodiment" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment are included in at least one embodiment of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments. Furthermore, different embodiments described in the present specification and the features of different embodiments can be combined and combined by those skilled in the art without contradiction.

[0112] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and should not be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. A method of enriching Tcm cells, characterized in that, The method comprises co-culturing a T cell population with stem cell exosomes.

2. The method of claim 1, wherein, The working concentration of the stem cell exosomes is 50-120 μg / mL. Optionally, the working concentration of the stem cell exosomes is 70-90 μg / mL. Optionally, the stem cell exosomes contain TSG101, Alix and HSP70 proteins, and do not contain Calnexin protein.

3. The method of claim 1, wherein, The co-culturing time is 1-14 days of the T cell population culture. Optionally, the co-culturing and the T cell population culture are carried out at 35-38°C and 4-6% CO2. Optionally, the seeding density of the T cell population is 1x10e6-2x10e6 / ml when the co-culturing is carried out. Optionally, the seeding amount of the T cell population is 2x10e6-4x10e6 when the co-culturing is carried out. Optionally, the T cell population is derived from a PBMC cell population. Optionally, the co-culturing system comprises: a CD3 antibody, the mass of the CD3 antibody being 1-10 μg, and / or a CD28 antibody, the mass of the CD28 antibody being 1-10 μg, and / or an interleukin, the working concentration of the interleukin being 100-1000 IU / ml. Optionally, the interleukin comprises at least one of IL-2, IL-7, IL-15 and IL-21. Optionally, the culture medium of the co-culture and the T cell population culture, respectively, is independently selected from at least one of the following: RPMI 1640, CTS OpTmizer T-Cell Expansion SFM, X-VIVO 15 and AIM V TM SFM culture medium.

4. The method of claim 1, wherein, The stem cell exosomes comprise exosomes selected from at least one of totipotent stem cells, pluripotent stem cells and multipotent stem cells. Optionally, the pluripotent stem cells comprise embryonic stem cells and / or induced pluripotent stem cells. Preferably, the stem cell exosomes comprise embryonic stem cell exosomes.

5. The method of claim 1, wherein, The stem cell exosomes are obtained by separation treatment and extraction treatment of the culture solution of the stem cell culture. Optionally, the culture medium of the stem cell culture comprises at least one selected from the group consisting of StemRD PGroF-500, mTeSR TM Plus Basal Medium, EZ-Stem TM Cell Culture Medium, Essential 8 TM Basal Medium, and StemFlex TM Medium; Optionally, when the stem cell exosomes are subjected to the separation treatment, the confluence of the stem cells is 85-95%, preferably 90-95%, and more preferably 95%.

6. The method of claim 5, wherein, The separation treatment is carried out by the following ways: (1) the culture solution of the stem cell culture is subjected to first centrifugal treatment to obtain first centrifugal supernatant, the first centrifugal treatment being carried out at 2-6°C and 1000-3000 g for 20-40 min; (2) the first centrifugal supernatant is subjected to second centrifugal treatment to obtain second centrifugal supernatant, the second centrifugal treatment being carried out at 2-6°C and 10000-20000 g for 20-40 min; (3) the second centrifugal supernatant is filtered through a 0.15-0.25 μm filter screen to obtain filtrate; Optionally, the extraction treatment is achieved by purifying the filtrate in an exosome extraction device. Optionally, the stem cell culture is carried out at 35-38°C and 4-6% CO2.

7. The method of claim 3, wherein, The PBMC cell population is obtained by the following way: (1) whole blood is mixed with separation solution and buffer solution in a volume ratio of (1-3):(1-3):(1-3). (2) performing a third centrifugal treatment on the product of step (1) to obtain a third centrifugal treatment product, the third centrifugal treatment being performed at 20-30℃ and at a speed of 600-900g for 10-40min, the third centrifugal treatment product being located between the plasma layer and the separation liquid layer; (3) performing dilution and a fourth centrifugal treatment on the third centrifugal treatment product in sequence to obtain a fourth centrifugal sediment, the fourth centrifugal treatment being performed at 20-30℃ and at a speed of 150-350g for 5-20min; Optionally, the buffer comprises at least one of the following: PBS and physiological saline.

8. A population of cells, characterized in that, The cell population is obtained by the method of any one of claims 1-7; Optionally, the proportion of Tcm cells in the cell population is not less than 50%; Optionally, the proportion of CD45RO + CD62L + the proportion of Tcm cells is not less than 50%.

9. A culture system for enriching Tcm cells, characterized in that, comprising: stem cell exosomes, the working concentration of which is 50-120μg / mL; Optionally, the culture system further comprises a CD3 antibody, the mass of the CD3 antibody being 1-10μg, and / or a CD28 antibody, the mass of the CD28 antibody being 1-10μg / mL, and / or an interleukin, the working concentration of which is 100-1000IU / ml; Optionally, the interleukin comprises at least one of the following: IL-2, IL-7, IL-15 and IL-21; Optionally, the culture medium of the culture system comprises at least one selected from the group consisting of RPMI 1640, CTS OpTmizer T-Cell Expansion SFM, X-VIVO 15 and AIM V TM SFM medium.

10. A composition characterized in that, The composition comprises the cell population obtained by the method of any one of claims 1-7, or the cell population of claim 8.

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