Method for culturing pluripotent stem cells and use thereof

Abstract

The present application relates to a method for amplifying / culturing pluripotent stem cells, which method comprises culturing pluripotent stem cells in a pluripotent stem cell culture medium under suspension culture conditions, wherein the pluripotent stem cell culture medium contains a basic culture medium, a human platelet lysate, and an additional factor thereof. Further provided in the present application is the use of the pluripotent stem cells and the pluripotent stem cell culture medium.

Description

Methods and applications of culturing pluripotent stem cells Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a method, culture medium, composition and application for expanding / culturing pluripotent stem cells. Background Art

[0002] Pluripotent stem cells have a wide range of applications in cell therapy, disease modeling, drug development and discovery, personalized medicine, and toxicology testing. Furthermore, pluripotent stem cells have the ability to differentiate into different cell types at the single-cell level, and the various cells derived from them also play an important role in cell therapy.

[0003] Human platelet lysate (hPL) is widely used in the research, development, and production of cell therapy biopharmaceuticals. Many studies have confirmed that its use as a cell culture additive in the culture of human mesenchymal stem cells (MSCs) can better maintain cell activity, promote cell proliferation, and delay cell aging caused by passage, which is more advantageous than serum-free culture and cell culture supplemented with fetal bovine serum. Currently, there are many difficulties in culturing stem cells in suspension, such as the difficulty in maintaining cell stemness, the easy death of cells after passage, and the difficulty in expanding cells in a short period of time. At present, there is no comprehensive and detailed research on the use of human platelet lysate in suspension culture to expand / cultivate pluripotent stem cells.

[0004] With the increasing demand for pluripotent stem cells, it is urgent to optimize the methods for expanding / culturing pluripotent stem cells and build a stable pluripotent stem cell production platform.

[0005] Summary of the Invention

[0006] The present application provides a method for expanding / culturing pluripotent stem cells, comprising expanding / culturing pluripotent stem cells in a pluripotent stem cell culture medium under suspension culture conditions, wherein the pluripotent stem cell culture medium comprises a basal medium, human platelet lysate, and / or other additives. By expanding / culturing pluripotent stem cells using the above method, aggregates with rounded morphology, clear edges, and uniform size can be obtained. Furthermore, the above method can extend the number of passages of pluripotent stem cells in suspension culture; improve the viability, survival rate, proliferation activity, and division activity of pluripotent stem cells; and maintain the stemness of pluripotent cells.

[0007] On the one hand, the present application provides a method for expanding / culturing pluripotent stem cells, which comprises expanding / culturing the pluripotent stem cells under suspension culture conditions, and culturing the pluripotent stem cells in a pluripotent stem cell culture medium, wherein the pluripotent stem cell culture medium comprises a basal culture medium, human platelet lysate and / or other additives.

[0008] In certain embodiments, in the methods provided herein, the pluripotent stem cells include induced pluripotent stem cells, human embryonic stem cells, or non-human embryonic stem cells.

[0009] In certain embodiments, in the methods provided herein, the induced pluripotent stem cells include induced pluripotent stem cells derived from humans and induced pluripotent stem cells derived from cynomolgus monkeys.

[0010] In certain embodiments, in the methods provided herein, the induced pluripotent stem cells are induced pluripotent stem cells derived from humans.

[0011] In certain embodiments, in the methods provided herein, the induced pluripotent stem cells are human embryonic stem cells.

[0012] In certain embodiments, in the methods provided herein, the pluripotent stem cells form cell aggregates under the suspension culture conditions.

[0013] In certain embodiments, in the methods provided herein, the cells and / or cell aggregates in the suspension culture do not adhere to the culture container.

[0014] In certain embodiments, in the methods provided herein, the culture container is non-cell-adhesive.

[0015] In certain embodiments, in the method provided herein, the basal culture medium is selected from one or more of the following groups: IMDM, MEM, Ham's F12, mTeSR1, APEL, StemSpan TM SFEM II, DMEM and RPMI1640.

[0016] In certain embodiments, in the methods provided herein, the basal culture medium is mTeSR1.

[0017] In certain embodiments, in the methods provided herein, the source of the human platelet lysate comprises one or more selected from the following groups: platelets derived from human blood, platelets differentiated from various types of stem cells, platelets transdifferentiated by reprogramming of other types of cells, and platelets obtained by biological means.

[0018] In certain embodiments, in the methods provided herein, the concentration of the human platelet lysate is 0.1%-5% (volume percentage).

[0019] In certain embodiments, in the methods provided herein, the concentration of the human platelet lysate is 0.3% (volume percentage).

[0020] In certain embodiments, in the methods provided herein, the supplement comprises one or more selected from the group consisting of nutrients, extracts, growth factors, hormones, cytokines, and culture medium additives.

[0021] In certain embodiments, in the methods provided herein, the supplement comprises one or more selected from the group consisting of: CHIR99021, PD0325901, SB431542, Methyl Cellulose, TGF-β, IGF-1, bFGF, FGF-2, Alginate hydrogel, Microcarrier, Glucose, Glutamate, 3D hydrogel, Fetal Bovine Serum (FBS), Non-Essential Amino Acids (NEAA), L-glutamine, N2, B27, Leukemia Inhibitory Factor (LIF), β-mercaptoethanol (β-ME), Insulin-Transferrin-Selenium (ITS), KnockOut TM Serum Replacement (KOSR), Bovine Serum Albumin (BSA), Human Serum Albumin (HSA), IL-6.

[0022] In certain embodiments, in the methods provided herein, the additive is a Rock inhibitor.

[0023] In certain embodiments, in the methods provided herein, the Rock inhibitor comprises one or more selected from the group consisting of Y-27632, Y-39983, Thiazovivin, Fasudil, GSK429286A, and Rkl-1447.

[0024] In certain embodiments, in the methods provided herein, the Rock inhibitor is Y-27632.

[0025] In certain embodiments, in the methods provided herein, the concentration of the Rock inhibitor is 1-20 μM.

[0026] In certain embodiments, in the methods provided herein, the concentration of the Rock inhibitor is 10 μM.

[0027] In certain embodiments, in the methods provided herein, the supplement is mTeSR1 supplement.

[0028] In certain embodiments, in the method provided herein, the additive is a double antibody, and the double antibody is penicillin and streptomycin.

[0029] In certain embodiments, in the method provided herein, the concentration of the dual antibody is 50-200 U / ml of penicillin and 0.05-0.2 mg / ml of streptomycin.

[0030] In certain embodiments, in the method provided herein, the pluripotent stem cell culture medium comprises basal culture medium, human platelet lysate, mTeSR1 supplement and double antibody.

[0031] In certain embodiments, in the methods provided herein, the pluripotent stem cell culture medium comprises mTeSR1, human platelet lysate, mTeSR1 supplement, and dual antibody.

[0032] In certain embodiments, in the method provided herein, the pluripotent stem cell culture medium comprises basal culture medium, human platelet lysate, mTeSR1 supplement, double antibody and Rock inhibitor.

[0033] In certain embodiments, in the methods provided herein, the pluripotent stem cell culture medium comprises basal culture medium, human platelet lysate, mTeSR1 supplement, double antibody, and Y-27632.

[0034] In certain embodiments, in the methods provided herein, the pluripotent stem cell culture medium comprises mTeSR1, human platelet lysate, mTeSR1 supplement, double antibody, and Y-27632.

[0035] In certain embodiments, the methods provided herein are performed in the absence of feeder cells.

[0036] In certain embodiments, the methods provided herein are performed under serum-free conditions.

[0037] In certain embodiments, in the method provided herein, the method comprises pre-treating pluripotent stem cells with DPBS and ACCUTASE.

[0038] In certain embodiments, the method provided herein comprises: isolating pluripotent stem cells at a rate of 0.5×10 5 -4×10 5 The cells were cultured at an inoculation density of 10 cells / mL.

[0039] In certain embodiments, the method provided herein comprises forming aggregates of pluripotent stem cells under suspension culture conditions provided by a shaker.

[0040] In certain embodiments, in the method provided herein, the method comprises digesting and passage the aggregates.

[0041] In certain embodiments, in the methods provided herein, the digestion passaging uses ACCUTASE.

[0042] On the other hand, the present application also provides a pluripotent stem cell culture medium, which comprises a basal culture medium, human platelet lysate and / or other additives.

[0043] In certain embodiments, in the pluripotent stem cell culture medium provided herein, the basal culture medium is selected from one or more of the following groups: IMDM, MEM, Ham's F12, mTeSR1, APEL, StemSpan TM SFEM II, DMEM and RPMI1640.

[0044] In certain embodiments, in the pluripotent stem cell culture medium provided herein, the basal culture medium is mTeSR1.

[0045] In certain embodiments, in the pluripotent stem cell culture medium provided herein, the source of the human platelet lysate comprises one or more selected from the following groups: platelets derived from human blood, platelets differentiated from various types of stem cells, platelets transdifferentiated by reprogramming of other types of cells, and platelets obtained by biological means.

[0046] In certain embodiments, in the pluripotent stem cell culture medium provided herein, the concentration of the human platelet lysate is 0.1%-5% (volume percentage).

[0047] In certain embodiments, in the pluripotent stem cell culture medium provided herein, the concentration of the human platelet lysate is 0.3% (volume percentage).

[0048] In certain embodiments, in the pluripotent stem cell culture medium provided herein, the additive comprises one or more selected from the group consisting of nutrients, extracts, growth factors, hormones, cytokines, and culture medium additives.

[0049] In certain embodiments, in the pluripotent stem cell culture medium provided herein, the additive comprises one or more selected from the group consisting of: CHIR99021, PD0325901, SB431542, Methyl Cellulose, TGF-β, IGF-1, bFGF, FGF-2, Alginate hydrogel, Microcarrier, Glucose, Glutamate, 3D hydrogel, Fetal Bovine Serum (FBS), Non-Essential Amino Acids (NEAA), L-glutamine, N2, B27, Leukemia Inhibitory Factor (LIF), β-mercaptoethanol (β-ME), Insulin-Transferrin-Selenium (ITS), KnockOut TM Serum Replacement (KOSR), Bovine Serum Albumin (BSA), Human Serum Albumin (HSA), IL-6.

[0050] In certain embodiments, in the pluripotent stem cell culture medium provided herein, the additive is a Rock inhibitor.

[0051] In certain embodiments, in the pluripotent stem cell culture medium provided herein, the Rock inhibitor comprises one or more selected from the following group: Y-27632, Y-39983, Thiazovivin, Fasudil, GSK429286A, and Rkl-1447.

[0052] In certain embodiments, in the pluripotent stem cell culture medium provided herein, the Rock inhibitor is Y-27632.

[0053] In certain embodiments, in the pluripotent stem cell culture medium provided herein, the concentration of the Rock inhibitor is 1-20 μM.

[0054] In certain embodiments, in the pluripotent stem cell culture medium provided herein, the concentration of the Rock inhibitor is 10 μM.

[0055] In certain embodiments, in the pluripotent stem cell culture medium provided herein, the supplement is mTeSR1 supplement.

[0056] In certain embodiments, in the pluripotent stem cell culture medium provided herein, the additive is a double antibody, and the double antibody is penicillin and streptomycin.

[0057] In certain embodiments, in the pluripotent stem cell culture medium provided herein, the concentration of penicillin is 50-200 U / ml, and the concentration of streptomycin is 0.05-0.2 mg / ml.

[0058] In certain embodiments, the pluripotent stem cell culture medium provided herein comprises a basal culture medium, human platelet lysate, mTeSR1 supplement, and a double antibody.

[0059] In certain embodiments, the pluripotent stem cell culture medium provided herein comprises mTeSR1, human platelet lysate, mTeSR1 supplement, and dual antibody.

[0060] In certain embodiments, the pluripotent stem cell culture medium provided herein comprises a basal culture medium, human platelet lysate, mTeSR1 supplement, dual antibody, and Rock inhibitor.

[0061] In certain embodiments, the pluripotent stem cell culture medium provided herein comprises a basal culture medium, human platelet lysate, mTeSR1 supplement, double antibody, and Y-27632.

[0062] In certain embodiments, the pluripotent stem cell culture medium provided herein comprises mTeSR1, human platelet lysate, mTeSR1 supplement, double antibody, and Y-27632.

[0063] In another aspect, the present application provides a composition comprising pluripotent cells and the culture medium.

[0064] On the other hand, the present application also provides a culture platform for expanding / culturing pluripotent stem cells, which comprises the method and the culture medium.

[0065] On the other hand, the present application also provides a method for treating, preventing and / or alleviating a disease, which comprises administering pluripotent stem cells to a subject in need thereof, wherein the pluripotent stem cells are obtained by the method, the culture medium, the composition, and the culture platform.

[0066] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0068] FIG1 is a schematic diagram of the hPSC suspension culture results described in the present application.

[0069] FIG2A is a schematic diagram showing the effect of adding hPL to hPSC suspension culture as described in the present application on promoting long-term cell culture.

[0070] FIG2B is a statistical diagram of cell count and viability described in this application.

[0071] FIG2C is a graph showing the results of measuring cell viability using the CCK-8 method described in this application.

[0072] FIG2D is a diagram showing the cell cycle detection results described in the present application.

[0073] FIG2E is a diagram showing the identification results of the expression effect of cell surface stemness markers described in the present application.

[0074] FIG2F is a diagram showing the identification results of the three-germ layer differentiation detection effect described in the present application.

[0075] FIG2G is a diagram showing the statistical results of cell counting and viability as described in this application. DETAILED DESCRIPTION

[0076] The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0077] Definition of terms

[0078] In this application, the term "pluripotent stem cells" generally refers to cells that have the ability to self-renew and differentiate. As used herein, "undifferentiated stem cells" refer to stem cells that have not undergone differentiation. Pluripotent stem cells can be functionally defined as cells that: (1) have the ability to differentiate into different cells and, in some cases, only generate one specialized cell type, and (2) can undergo long-term self-renewal to produce one or more cells that are the same or different from the original cell type. The source and preparation method of pluripotent stem cells are not limited. For example, the pluripotent stem cells can be naturally obtained or artificially modified. For example, the pluripotent stem cells can include embryonic stem cells, induced pluripotent stem cells or non-human embryonic stem cells, etc. For example, the pluripotent stem cells can be induced pluripotent stem cells (hPSC) derived from humans. For example, the pluripotent stem cells can be induced pluripotent stem cells (cPSC) derived from cynomolgus monkeys. For example, the pluripotent stem cells can be human embryonic stem cells. For example, the pluripotent stem cells can be non-human embryonic stem cells, including but not limited to non-human primates, rodents (mice, rats), ungulates (cattle, sheep, etc.), dogs (domestic and wild dogs), cats (domestic and wild cats, such as lions, tigers, cheetahs), rabbits, hamsters, gerbils, squirrels, guinea pigs, goats, elephants, pandas (including giant pandas), pigs, raccoons, horses, zebras, marine mammals (dolphins, whales, etc.), etc. In certain embodiments, the species is an endangered species. In certain embodiments, the species is a currently extinct species.

[0079] In this application, the term "human embryonic stem cells," also referred to as "embryonic stem cells" (abbreviated as "ESCs"), generally refers to cells that possess the properties of unlimited proliferation, self-renewal, and multidirectional differentiation. Embryonic stem cells are derived from the undifferentiated inner cell mass of the blastocyst (early embryonic stage). Their source and preparation methods are not limited. Embryonic stem cells can be induced to differentiate into virtually any cell type in the body, both in vitro and in vivo. For example, these cell types can include hematopoietic stem cells, neural cells, and cardiomyocytes.

[0080] In this application, the term "induced pluripotent stem cells" can be abbreviated as "iPS" cells or "iPSCs", and generally refers to a class of pluripotent stem cells prepared from non-pluripotent cells in an artificial manner. The way to obtain induced pluripotent stem cells can be to introduce specific transcription factors to reprogram terminally differentiated somatic cells. For example, the terminally differentiated somatic cells can be fibroblasts, hematopoietic stem cells, muscle cells, neurons, epidermal cells, etc. For example, at least two reprogramming proteins are transduced into somatic cells to successfully reprogram somatic cells. For example, at least three reprogramming proteins are transduced into somatic cells to successfully reprogram somatic cells. For example, at least four reprogramming proteins are transduced into somatic cells to successfully reprogram somatic cells. Reprogramming is carried out by expression of reprogramming factors (such as by viral transduction, integration or non-integration vectors, etc.) and / or contact with them (for example, by protein transduction domains, electroporation, microinjection, cationic amphiphiles, fusions with lipid bilayers, detergent permeabilization, etc.). The reprogramming factor may be selected from the group consisting of OCT3 / 4, SOX2, NANOG, LIN28, C-MYC, and KLF4. Expression of the reprogramming factor may be induced by contacting the somatic cells with at least one agent (eg, an organic small molecule agent) that induces expression of the reprogramming factor.

[0081] Induced pluripotent stem cells can be from any species. At present, mouse and human cells have been used to generate induced pluripotent stem cells, and embryonic, fetal, neonatal and adult tissues have also been used to successfully generate induced pluripotent stem cells. Therefore, donor cells from any species can be used to easily generate induced pluripotent stem cells, including but not limited to people, non-human primates, rodents (mice, rats), ungulates (cows, sheep, etc.), dogs (domestic and wild dogs), cats (domestic and wild cats, such as lions, tigers, cheetahs), rabbits, hamsters, goats, elephants, pandas (including giant pandas), pigs, raccoons, horses, zebras, marine mammals (dolphins, whales, etc.). For example, the induced pluripotent stem cells can be induced pluripotent stem cells (hPSC) derived from humans. For example, the induced pluripotent stem cells can be induced pluripotent stem cells (cPSC) derived from cynomolgus monkeys. Induced pluripotent stem cells can use almost any somatic cell of any developmental stage as a starting point to generate. For example, cells can be from embryonic, fetal, neonatal, adolescent or adult donors. For example, somatic cells can be used to include fibroblasts.

[0082] In this application, the term "suspension culture," also referred to as "suspension culture method," generally refers to a method of culturing cells or cell aggregates while maintaining them suspended in a culture medium. Specifically, suspension culture is performed under conditions where the cells or cell aggregates do not adhere to a culture vessel, etc., and is performed under conditions where they are allowed to adhere to a culture vessel, etc. However, adherent culture or adherent culture methods are not included in the scope of suspension culture. For example, cell adhesion can refer to the formation of a strong cell-substrate bond between cells or cell aggregates and the culture vessel. For example, suspension culture refers to culture under conditions where strong cell-substrate bonds are not formed between cells or cell aggregates and the culture vessel.

[0083] The cell culture container used when carrying out suspension culture is not particularly limited, as long as it can allow " suspension culture ", and those of ordinary skill in the art can appropriately determine this type of cell culture container. Examples of this type of cell culture container include flasks, tissue culture bottles, culture dishes (plates), culture dishes, tissue culture dishes, multiple culture dishes, microplates, microporous plates, micropores, multi-porous plates, test tubes, trays, culture bags, spinner flasks, conical flasks, shaker bottles, etc. In order to allow suspension culture, these culture containers can be non-cell adhesive. The culture container of the non-cell adhesive that can be used includes a surface that is not artificially treated to improve cell adhesion (for example, using the surface treatment of the extracellular matrix such as basement membrane preparations, laminin, collagen, gelatin, etc., or coated or positively charged with polymers such as polylysine, polyornithine, etc.) and similarly treated culture dishes. As non-cell adhesive cell culture containers, a surface that has been artificially treated to reduce the adhesion to cells (for example, using super-hydrophilic treatment, protein low adsorption treatment, etc. of MPC polymers, etc.) and similarly treated culture containers can be used.

[0084] In this application, the term "aggregate" generally refers to a mass formed by cells dispersed in a culture medium, wherein the cells adhere to each other. Cell clumps, embryoids, spheres, and spheroids are also included in the cell aggregates. "Aggregates of uniform size" can refer to when culturing multiple aggregates, the size of each aggregate can remain approximately consistent, and when the size of the aggregate is evaluated by the length of the maximum diameter, the length of the maximum diameter changes little. For example, the length of the maximum diameter of the aggregate is within ± 5% of the average value, within ± 10%, within ± 15%, within ± 20%, within ± 25%, or within ± 30%.

[0085] In this application, the term "human platelet lysate (hPL)" is generally derived from human platelets, which contain a variety of cell growth factors. In this application, the human platelet lysate can include platelets from various sources, for example, the human platelet lysate can be derived from human platelets collected from blood donation. For example, the human platelet lysate can be derived from platelets separated and purified from a blood sample. For example, the human platelet lysate can be derived from platelets obtained by various cell differentiation, for example, platelets obtained by differentiation of hematopoietic stem cells, for example, platelets obtained by differentiation of induced pluripotent stem cells (iPSC). After being separated, platelets can be suspended in plasma and lysed by one or more freeze / thaw cycles to obtain platelet lysate.

[0086] In this application, the term "marker phenotype" generally refers to identifying markers or antigens on cells to determine their phenotype (e.g., differentiation state and / or cell type). For example, immunophenotyping can be used, which uses antibodies to identify antigens presented on cells. Antibodies can be monoclonal or polyclonal, and are generally selected to have smaller cross-reactions with other cell markers. These markers that determine the same cell type between species can be identified based on the same markers, and there may be differences in the structure of these markers (e.g., amino acid sequence) between species. Cell markers can include cell differentiation markers, as well as gene expression markers. Gene expression markers can include expressed genes that can indicate cell type or differentiation state.

[0087] In this application, the terms "expand" and "proliferate" are used interchangeably and generally refer to the generation of multiple individual cells by division of a starting cell. The multiple individual cells can be of the same type or of different types. The starting cells used for proliferation do not need to be the same as the cells produced by proliferation. For example, the proliferated cells can be generated from the growth and differentiation of a starting cell population.

[0088] In this application, the term "differentiation" generally refers to the process by which a non-specific or less specific cell acquires specific cell characteristics. A differentiated or differentiation-induced cell is a cell that occupies a more specific position in a cell lineage.

[0089] In this application, the term "maintain" generally refers to maintaining the original state. In this application, the maintenance refers to the maintenance of cell activity at a certain level during the culture process, during which the cells can expand and / or differentiate.

[0090] In this application, the term "composition" generally refers to a product comprising a specified amount of a specified ingredient, as well as any product produced directly or indirectly by a combination of the specified amounts of the specified ingredients. In this application, the composition may also include other inactive ingredients, for example, carriers, excipients, adjuvants, stabilizers, etc.

[0091] In this application, the term "ex vivo" generally refers to operations performed on cells, tissues and / or organs that have been removed from an organism. In some embodiments, the cells, tissues and / or organs can be returned to the organism or introduced into another organism by certain methods.

[0092] In this application, the term "in vitro" generally refers to removing or releasing a part of an organism from the organism.

[0093] In this application, the term "include" generally means to include, encompass, contain or encompass. In some cases, it also means "to be", "to be composed of..."

[0094] In this application, the term "and / or" should be understood to mean any one, two, more than one or any combination of the alternatives.

[0095] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0096] In this application, the term "feeder cells" generally refers to cells other than the cultured cells that coexist when culturing cells. For example, the feeder cells may be mouse fibroblasts, human fibroblasts, SNL cells, etc. The feeder cells may be feeder cells that have been subjected to growth inhibition treatment, which may include treatment with a growth inhibitor (e.g., mitomycin C), gamma radiation, UV irradiation, etc. In this application, "feeder cell-free" culture conditions generally refer to culturing cells under conditions in which no feeder cells are present, and the cells may be pluripotent stem cells.

[0097] In this application, the term "culture medium" is used interchangeably with "culture medium" and generally refers to any culture medium that allows cell expansion / culture. For example, the culture medium may include IMDM, MEM, Ham's F12, mTeSR1, APEL, StemSpan TMOne or more of SFEM II, DMEM, RPMI1640, mTeSR™2, TeSR™-E8™, StemSpan™-ACF, etc. For example, the culture medium may further be supplemented with other additives.

[0098] In this application, the terms "additives" and "additive factors" are used interchangeably and generally refer to components added to the culture medium other than the basal medium. Additives can promote cell expansion / cultivation and can be nutrients / extracts, growth factors, hormones, cytokines, and culture medium additives. For example, additives can be suitable buffers (e.g., HEPES), compounds (e.g., N2, B27, etc.), serum-free supplements, antibiotics (e.g., penicillin / streptomycin), or non-essential amino acids. For example, additives can be glucose, L-glutamyl, ascorbic acid and / or DAPT, bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEG), basic fibroblast growth factor (bFGF), thrombopoietin (TPO) | stem cell factor (SCF), erythropoietin (EPO), FMS-like tyrosine kinase 3 ligand (Flt3L), IL-3, IL-6, IL-9, IL-7, IL-15, Rock inhibitors (e.g., Y-27632), etc.

[0099] As used herein, the terms "treat," "treat," and "alleviate" generally refer to: (1) preventing the development of a disease, disorder, and / or condition in a patient who may be susceptible to the disease but has not yet been diagnosed with the disease; (2) inhibiting the disease, disorder, or condition, that is, arresting its development; and (3) relieving the disease, disorder, or condition, that is, causing the disease, disorder, and / or condition and / or symptoms associated with the disease, disorder, and / or condition to subside.

[0100] Detailed Description of the Invention

[0101] method

[0102] On the one hand, the present application provides a method for expanding / culturing pluripotent stem cells, which comprises expanding / culturing the pluripotent stem cells under suspension culture conditions, and culturing the pluripotent stem cells in a pluripotent stem cell culture medium; wherein the pluripotent stem cell culture medium comprises a basal culture medium, human platelet lysate and / or other additives.

[0103] In the present application, the method may comprise suspending and culturing the pluripotent stem cells, induced pluripotent stem cells (e.g., induced pluripotent stem cells derived from humans and induced pluripotent stem cells derived from cynomolgus monkeys), human embryonic stem cells or non-human embryonic stem cells in the pluripotent stem cell culture medium.

[0104] In the present application, the method may include forming cell aggregates under suspension culture conditions. The manner of forming cell aggregates is not limited.

[0105] In certain embodiments, the method may include generating cell aggregates using a stirring method. The stirring method may be: (1) using a rotating flask bioreactor and (2) a rotary culture system; the principle is to place a cell suspension in a container and keep the suspension in motion, i.e., gently stir or rotate the container. The continuous movement of the suspended cells means that the cells do not adhere to the container wall, but form cell-cell interactions. A rotating flask bioreactor (commonly referred to as a "rotator") may include a container for holding the cell suspension and a stirring element for ensuring continuous mixing of the cell suspension. A rotary cell culture bioreactor works in a similar manner to a rotating flask bioreactor, but instead of using a stirring rod / rod to keep the cell suspension moving, the culture container itself is rotated.

[0106] In certain embodiments, the method may include continuously culturing a plurality of pluripotent stem cells as aggregates of uniform size in a spinner flask containing culture medium in the absence of feeder cells and a matrix. Utilizing this suspension culture system, the pluripotent stem cells and / or aggregates can be continuously passaged and continuously expanded for at least five generations. For example, the continuous expansion can be for at least six generations, at least seven generations, at least eight generations, at least nine generations, at least ten generations, at least eleven generations, or more.

[0107] In certain embodiments, the method can be performed at a passage interval of about 2-5 days, and the aggregates can grow to a size of about 200-500 μm in diameter. For example, the size of the aggregates can be monitored by taking an aliquot of the culture and observing it under a microscope. For example, the number of days can be about 0.5 days, about 1 day, about 1.5 days, about 2 days, about 2.5 days, about 3 days, about 3.5 days, about 4 days, about 4.5 days, about 5 days, about 5.5 days, about 6 days, about 6.5 days, or about 7 days. For example, the aggregate growth diameter size can be about 150 μm, about 160 μm, about 170 μm, about 180 μm, about 190 μm, about 200 μm, about 210 μm, about 220 μm, about 230 μm, about 240 μm, about 250 μm, about 260 μm, about 270 μm, about 280 μm, about 290 μm, about 300 μm, about 310 μm, about 320 μm, about 330 μm, about 340 μm, about 360 μm, about 370 μm, about 380 μm, about 390 μm, about 400 μm, about 410 μm, about 420 μm, about 430 μm, about 440 μm, about 450 μm, about 460 μm, about 470 μm, about 480 μm, about 490 μm, about 500 μm, about 510 μm, about 520 μm, about 530 μm, about 540 μm, about 550 μm, about 560 μm, about 570 μm, about 580 μm, about 590 μm, about 600 μm, about 610 μm, about 620 μm, about 630 μm, about 640 μm, about 650 μm, about 660 μm, about 670 μm, about 680 μm, about 690 μm, about 0μm, about 350μm, about 360μm, about 370μm, about 380μm, about 390μm, about 400μm, about 410μm, about 420μm, about 430μm, about 440μm, about 450μm, about 460μm, about 470μm, about 480μm, about 490μm, about 500μm, about 510μm, about 520μm, about 530μm, about 5540μm, about 550μm.

[0108] In certain embodiments, the method may comprise dissociating the aggregates into single (or substantially single) cells using an enzyme having proteolytic and collagenolytic activity. For example, the enzyme may be ACCUTASE, TrypLE, or trypsin / EDTA.

[0109] In certain embodiments, the method may comprise reaggregating the dissociated cells to form aggregates under continuous stirring. In certain embodiments, the method may comprise reaggregating the dissociated cells to form aggregates on a shaker at a speed of 40-90 rpm. For example, the speed may be about 35 rpm, about 40 rpm, about 45 rpm, about 550 rpm, about 55 rpm, about 60 rpm, about 65 rpm, about 70 rpm, about 75 rpm, about 80 rpm, about 85 rpm, about 90 rpm, about 95 rpm, about 100 rpm, about 105 rpm, or about 110 rpm.

[0110] In certain embodiments, the method may comprise using 0.5×10 5 -4×10 5 Cell culture is performed at an inoculation density of about 0.25×10 cells / mL. 5 cell / mL, about 0.5×10 5 cell / mL, approximately 0.75×10 5 cell / mL, about 1×10 5 cell / mL, about 1.25×10 5 cell / mL, about 1.5×10 5 cell / mL, approximately 1.75×10 5 cell / mL, about 2×10 5 cell / mL, approximately 2.25×10 5 cell / mL, about 2.5×10 5 cell / mL, approximately 2.75×10 5 cell / mL, about 3×10 5 cell / mL, approximately 3.25×10 5 cell / mL, about 3.5×10 5 cell / mL, approximately 3.75×10 5 cell / mL, about 4×10 5 cell / mL, approximately 4.25×10 5 cell / mL, about 4.5×10 5 cell / mL, approximately 4.75×10 5cell / mL, about 5×10 5 cell / mL.

[0111] In certain embodiments, the method can include culturing the cells uninterruptedly for 48-120 hours at various agitation rates. For example, the uninterrupted culturing can be about 20 hours, about 30 hours, about 40 hours, about 50 hours, about 60 hours, about 70 hours, about 80 hours, about 90 hours, about 100 hours, about 110 hours, about 120 hours, about 130 hours, or about 140 hours.

[0112] In certain embodiments, the method may comprise culturing pluripotent stem cells in a medium supplemented with an additive. For example, the additive may be CHIR99021, PD0325901, SB431542, Methyl Cellulose, TGF-β, IGF-1, bFGF, FGF-2, Alginate hydrogel, Microcarrier, Glucose, Glutamate, 3D hydrogel, Fetal Bovine Serum (FBS), Non-Essential Amino Acids (NEAA), L-glutamine, N2, B27, Leukemia Inhibitory Factor (LIF), β-mercaptoethanol (β-ME), Insulin-Transferrin-Selenium (ITS), KnockOut TM One or more of: serum replacement (KOSR), bovine serum albumin (BSA), human serum albumin (HSA), and IL-6. For example, the supplement can be mTeSR1 supplement. For example, the supplement can be a bispecific antibody. For example, the supplement can be a Rock inhibitor. For example, the supplement can be Y-27632.

[0113] In the present application, the method may comprise the following steps:

[0114] (1) preparing a pluripotent stem cell culture medium;

[0115] (2) Treat pluripotent stem cells with DPBS;

[0116] (3) Treating pluripotent stem cells with ACCUTASE;

[0117] (4) culturing pluripotent stem cells in suspension to form aggregates; and

[0118] (5) Digest and passage the aggregates.

[0119] In the present application, the culture method may include regularly or irregularly supplementing and / or replacing the pluripotent stem cell culture medium.

[0120] In the present application, the culturing method may comprise culturing cells at about 37-39°C, for example, about 36.5°C, about 37°C, about 37.5°C, about 38°C, about 38.5°C, about 39°C, about 39.5°C.

[0121] In the present application, the culturing method may include culturing cells under conditions of about 3-7% CO2, for example, about 3% CO2, about 3.5% CO2, about 4% CO2, about 4.5% CO2, about 5% CO2, about 5.5% CO2, about 6% CO2, about 6.5% CO2, about 7% CO, about 7.5% CO2.

[0122] In the present application, the culture method may be performed under serum-free culture conditions. In the present application, the culture method may be performed under feeder-free culture conditions. In the present application, the culture method may be performed under feeder-containing culture conditions. In the present application, the method may be an in vitro method. In the present application, the method may be an ex vivo method. In the present application, the method may be a method for purposes other than diagnosis and treatment of diseases.

[0123] culture medium

[0124] In another aspect, a pluripotent stem cell culture medium comprises a basal medium, human platelet lysate and / or other additives.

[0125] In certain embodiments, the pluripotent stem cell culture medium may include a basal culture medium for culturing cells. The basal culture medium may be a single component or a combination of multiple culture media. The basal culture medium includes but is not limited to IMDM, MEM, Ham's F12, mTeSR1, APEL, StemSpan TM SFEM II, DMEM, RPMI1640.

[0126] In certain embodiments, the source of the human platelet lysate is not limited. For example, the human platelet lysate may comprise platelets from various sources, for example, the human platelet lysate may be derived from human platelets collected from blood donations. For example, the human platelet lysate may be derived from platelets isolated and purified from a blood sample. For example, the human platelet lysate may be derived from platelets obtained by various cell differentiations, for example, platelets obtained by differentiation of hematopoietic stem cells, for example, platelets obtained by differentiation of induced pluripotent stem cells (iPSCs). In certain embodiments, after the platelets are separated, they can be suspended in plasma and lysed through one or more freeze / thaw cycles to obtain a platelet lysate. In certain embodiments, after the platelets are separated, platelet lysates can be obtained by mechanical lysis. In certain embodiments, after the platelets are separated, platelet lysates can be obtained by using a lysis buffer. In certain embodiments, after the platelets are separated, platelet lysates can be obtained by a combination of multiple lysis methods.

[0127] In certain embodiments, the human platelet lysate may further include any culture medium used in the lysed platelet process.

[0128] In certain embodiments, the concentration of the human platelet lysate is about 0.1%-5%. For example, the concentration of the human platelet lysate is about 0.05%, about 0.15%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, about 0.7%, about 0.75%, about 0.8%, about 0.85%, about 0.99%, about 0.95%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%.

[0129] In certain embodiments, the pluripotent stem cell culture medium may be supplemented with one or more additives, including but not limited to nutrients / extracts, growth factors, hormones, cytokines, and culture medium additives.

[0130] In certain embodiments, the pluripotent stem cell culture medium may be supplemented with one or more supplements, including but not limited to serum replacement, glutamine, NEAA (non-essential amino acids), ascorbic acid, epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), keratinocyte growth factor (KGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), transforming growth factor β (TGF-β), bone morphogenetic protein (BMP4), vascular endothelial growth factor (VEGF), and vascular endothelial growth factor (VEGF). The additives include, but are not limited to, various interleukins (e.g., IL-1 to IL-18), various colony-stimulating factors (e.g., granulocyte / macrophage colony-stimulating factor (GM-CSF)), various interferons (e.g., IFN-γ), stem cell factor (SCF), thrombopoietin (TPO), erythropoietin (EPO), N2 supplement, B27 supplement, Fms-related tyrosine kinase 3 ligand (FLt3), and antibiotics. The additives may be obtained from any source and may be commercially available, natural, or recombinant.

[0131] In certain embodiments, the pluripotent stem cell culture medium may be supplemented with one or more additives, including but not limited to CHIR99021, PD0325901, SB431542, Methyl Cellulose, TGF-β, IGF-1, bFGF, FGF-2, Alginate hydrogel, Microcarrier, Glucose, Glutamate, 3D hydrogel, Fetal Bovine Serum (FBS), Non-Essential Amino Acids (NEAA), L-glutamine, N2, B27, Leukemia Inhibitory Factor (LIF), β-mercaptoethanol (β-ME), Insulin-Transferrin-Selenium (ITS), KnockOut TM Serum Replacement (KOSR), Bovine Serum Albumin (BSA), Human Serum Albumin (HSA), IL-6

[0132] In certain embodiments, the pluripotent stem cell culture medium may be supplemented with inhibitors, which may include but are not limited to GSK-3 inhibitors, MEK inhibitors, ROCK inhibitors, metalloproteinase inhibitors, and the like.

[0133] In certain embodiments, the ROCK inhibitor includes, but is not limited to, polynucleotides, polypeptides, and small molecules that can reduce ROCK expression and / or ROCK activity. In certain embodiments, the ROCK inhibitor can be Y-27632, Y-39983, Thiazovivin, Fasudil (HA-1077), GSK429286A, RKI-1447, WAY-624704, H-1152, Azaindole 1 (TC-S 7001), Hydroxyfasudil (HA-1100), Y-39983, Netarsudil (AR-13324), GSK269962A, Ripasudil (K-115) hydrochloride dihydrate, Belumosudil (KD025), AT13148, Emetine hydrochloride, ZINC00881524, or a ROCK pathway-targeted antibody.

[0134] In the present application, the ROCK inhibitor concentration may be about 1-50 μM. For example, the ROCK inhibitor concentration is about 1 μM, about 3 μM, about 5 μM, about 8 μM, about 10 μM, about 15 μM, about 20 μM, about 25 μM, about 30 μM, about 35 μM, about 40 μM, about 45 μM, or about 50 μM.

[0135] In the present application, the Y-27632 concentration may be about 1-50 μM. For example, the Y27632 concentration is about 1 μM, about 3 μM, about 5 μM, about 8 μM, about 10 μM, about 15 μM, about 20 μM, about 25 μM, about 30 μM, about 35 μM, about 40 μM, about 45 μM, or about 50 μM.

[0136] In certain embodiments, the pluripotent stem cell culture medium may be supplemented with antibiotics. For example, the antibiotics may be penicillin, streptomycin, or a combination thereof. For example, the concentration of the antibiotics may be 50-200 U / ml of penicillin and 0.05-0.2 mg / ml of streptomycin. For example, the concentration of penicillin may be about 50 U / mL, about 100 U / mL, about 150 U / mL, about 200 U / mL, about 250 U / mL, about 300 U / mL, about 350 U / mL, about 400 U / mL, about 450 U / mL, or about 500 U / mL. For example, the concentration of streptomycin can be about 0.01 mg / ml, about 0.02 mg / ml, about 0.03 mg / ml, about 0.04 mg / ml, about 0.05 mg / ml, 0.06 mg / ml, about 0.07 mg / ml, about 0.08 mg / ml, about 0.09 mg / ml, about 0.1 mg / ml, about 0.11 mg / ml, about 0.12 mg / ml, about 0.13 mg / ml, about 0.14 mg / ml, about 0. 15mg / ml, 0.16mg / ml, about 0.17mg / ml, about 0.18mg / ml, about 0.19mg / ml, about 0.2mg / ml, about 0.21mg / ml, about 0.22mg / ml, about 0.23mg / ml, about 0.24mg / ml, about 0.25mg / ml, 0.26mg / ml, about 0.27mg / ml, about 0.28mg / ml, about 0.29mg / ml, about 0.3mg / ml.

[0137] In certain embodiments, the pluripotent stem cell culture medium may be mTeSR1 complete medium, which may contain mTeSR1, mTeSR 1 supplement, double antibody P / S (100X), and human platelet lysate.

[0138] In certain embodiments, the pluripotent stem cell culture medium can be mTeSR1 complete medium supplemented with a Rock inhibitor, such as Y-27632. For example, the pluripotent stem cell culture medium comprises mTeSR1, human platelet lysate, mTeSR1 supplement, double antibody, and Y-27632.

[0139] Composition and use

[0140] In another aspect, the present application provides a composition comprising pluripotent stem cells and the culture medium.

[0141] In certain embodiments, the composition and content of the composition can be varied. In certain embodiments, the composition and content of the composition can be specified. In certain embodiments, the composition can include other inactive ingredients, for example, carriers, excipients, adjuvants, stabilizers, etc.

[0142] In another aspect, the present application provides use of the method in expanding / culturing pluripotent cells, which comprises the culture medium.

[0143] On the other hand, the present application provides a culture platform for obtaining pluripotent stem cells, which comprises the method and the culture medium.

[0144] On the other hand, the present application provides a use of the culture platform, which comprises the method and the culture medium.

[0145] On the other hand, the present application provides uses of the method, the culture medium, the composition, and the culture platform for preparing pluripotent stem cells.

[0146] In another aspect, the present application provides a method for preventing and / or treating a disease, comprising using the method, the culture medium, and the composition.

[0147] Without intending to be bound by any theory, the following examples are merely intended to illustrate the fusion protein, preparation method, and use of the present application, and are not intended to limit the scope of the present invention.

[0148] Example

[0149] Experimental Materials

[0150] Example 1 Pluripotent stem cell suspension culture

[0151] 1.1 Preparation of pluripotent stem cell culture medium

[0152] 1.1.1 Preparation of mTeSR1 complete medium

[0153] 1) Thaw the mTeSR1 supplement overnight in a 4°C refrigerator.

[0154] 2) Add the thawed supplement to mTeSR1 basal medium and add double antibody P / S (100X);

[0155] 3) Store the prepared mTeSR1 complete medium at 4°C. Aliquot the medium as much as possible before use and return it to room temperature as much as possible.

[0156] 4) hPL group culture medium: 0.3% human platelet lysate was added to mTeSR1 complete culture medium for preparation.

[0157] 1.1.2 Initiation of hPSC suspension culture

[0158] 1) Prepare the culture medium and preheat it to room temperature. Do not preheat it in a 37°C water bath.

[0159] 2) After preheating, add 10 μM Y-27632 and mix thoroughly;

[0160] 3) Remove the culture medium and add 1 mL of room temperature DPBS (without calcium and magnesium);

[0161] 4) Remove DPBS and add 1 mL of room temperature ACCUTASE;

[0162] 5) Incubate at 37°C for 3-4 minutes;

[0163] 6) Remove the ACCUTASE, add 1 mL of pluripotent stem cell culture medium (containing 10 μM Y-2763), and gently tap the plate to remove the cells. Do not pipette the cells more than 10 times.

[0164] 7) Transfer the dissociated cells to a 15 mL tube. Optional: Rinse the wells with an additional 1 mL of culture medium (containing 10 μM Y-2763) to collect any remaining cells.

[0165] 8) Count and seed the cells. See the table below for recommended seeding densities. Add the appropriate number of cells to the wells and fill the remaining volume with pluripotent stem cell culture medium (containing 10 μM Y-2763).

[0166] 9) Set the shaker speed to 80 rpm.

[0167] 1.1.3 Aggregate digestion and passage

[0168] 1) Before subculturing, take photos of the aggregates under a microscope to measure their size;

[0169] 2) Collect the cells into a 15 mL tube and let them sit for 1 minute. After the cells have settled, aspirate the supernatant medium and resuspend in DPBS. Optional: Rinse the well with an additional 1 mL of DPBS to collect any remaining cells.

[0170] 3) Centrifuge at 100g for 3 minutes and discard the supernatant;

[0171] 4) Add an appropriate volume of ACCUTASE based on the amount of cells collected and incubate in a 37°C water bath for 6 minutes;

[0172] 5) If the culture is terminated, samples are taken at this step and trypan blue counts are performed;

[0173] 6) If further culture is required, add an equal volume of culture medium and centrifuge at 300 g for 5 minutes;

[0174] 7) Discard the supernatant, add pluripotent stem cell culture medium (containing 10 μM Y-2763) to resuspend, count and then inoculate.

[0175] 1.1.4 Culture and fluid supplementation

[0176] Experimental results:

[0177] As shown in Figure 1 , the cell aggregates were round in shape, with clear edges and uniform size, which was consistent with the morphological characteristics of hPSC suspension culture.

[0178] Example 2 Adding hPL to hPSC suspension culture can promote long-term cell culture

[0179] hPSC aggregates were collected and digested every 3 days, and inoculated and passaged according to a certain inoculation volume. The culture effects of the control group and the hPL-treated group at different generations during long-term culture were observed and statistically analyzed.

[0180] Experimental results:

[0181] As shown in Figure 2A, hPSCs in the control group without hPL addition exhibited significant morphological changes at passage 5 (after five passages in the 3D culture system), with a decrease in the diameter of the cell aggregates. No cell aggregates formed by passage 6. In the hPL-treated group, hPSCs were able to passage normally and form normal-sized cell aggregates, even up to passage 10.

[0182] The experimental results show that adding hPL to hPSC suspension culture can effectively maintain the growth of hPSC.

[0183] Example 3 Cell counting and viability statistics

[0184] Collect the hPS aggregates to be passaged into a 15 mL centrifuge tube, centrifuge at 100 g for 3 minutes, discard the supernatant, add ACCUTASE and incubate at 37°C for 5-6 minutes to dissociate into single cells. Take a sample and stain with trypan blue at a 1:1 dilution for cell counting and viability statistics.

[0185] Experimental results:

[0186] As shown in Figure 2B, the number of living cells in the control group with hPL added increased by more than 60%, and the cell viability remained above 90%. The survival rate of cells cultured without hPL gradually decreased to below 50%.

[0187] The experimental results show that adding hPL to hPSC suspension culture can improve hPSC viability and promote hPSC expansion.

[0188] Example 4 Determination of cell viability by CCK-8 method

[0189] On day 1 after passage, cell aggregates from both hPL-treated and non-hPL-treated cells were collected and centrifuged at 100 g for 3 minutes. The cells were resuspended in culture medium containing 10% CCK-8 and incubated at 37°C for 4 hours. The absorbance at 450 nm was measured. After obtaining the readings, cell viability (%) was calculated as [A(hPL-treated group) - A(blank)] / [A(control group) - A(blank)] × 100

[0190] A (hPL-treated group): absorbance of wells with cells, CCK-8 solution, and hPL

[0191] A (blank): absorbance of the well with culture medium and CCK-8 solution but no cells

[0192] A (control group): absorbance of wells containing cells and CCK-8 solution but no hPL

[0193] Experimental results:

[0194] As shown in Figure 2C , the cell viability of the control group without hPL addition was 100%, and the cell viability of the hPL-treated group was approximately 110%.

[0195] The experimental results show that adding hPL to hPSC suspension culture can significantly improve the proliferation activity of hPSC cells.

[0196] Example 5 Cell cycle detection

[0197] Collect 10 6 Single cells were washed twice with 1 mL of pre-cold PBS and resuspended in 300 μL of PBS. 700 μL of pre-cold anhydrous ethanol was then added and fixed overnight at 4°C. The next day, cells were washed with PBS and incubated with 250 μL of PI staining solution (containing RNase) at room temperature in the dark for 15 minutes. Analyze using flow cytometry.

[0198] Experimental results:

[0199] As shown in Figure 2D, in the control group without hPL addition, the cells in the G2 / M phase were approximately 30%, and the cells in the G1 phase were approximately 40%. After hPL addition, the cells in the G2 / M phase were approximately 45%, and the cells in the G1 phase were approximately 25%.

[0200] The experimental results showed that when hPL was added to hPSCs for suspension culture, the hPL-treated cells showed higher ratios in the G2 and M phases and lower ratios in the G1 phase, indicating enhanced cell division activity.

[0201] Example 6 Identification of cell surface stemness marker expression effects

[0202] Cell aggregates were digested into single cells using ACCUTASE, washed once with DPBS, and resuspended in 450 μL of FACS buffer (PBS with 1% FBS and 8 μM EDTA). 100 μL of FACS buffer containing the antibody was then added to each sample and incubated on ice for 20 minutes. After incubation, cells were washed twice and resuspended in 200 μL of FACS buffer containing DAPI. Flow cytometry was performed, and flow cytometric data were analyzed using FlowJo (Tree Star).

[0203] Experimental results:

[0204] As shown in FIG2E , the double positive rates of SSEA-4 and TRA-1-60 in the 4th generation control group and the hPL-treated group were 99.9%, and the double positive rate of SSEA-4 and TRA-1-60 in the 10th generation hPL-treated group was 98.1%.

[0205] The experimental results showed that when hPL was added to hPSCs for suspension culture, the expression of cell surface stem cell markers in the hPL-treated group was the same as that in the control group, indicating that the cells were still in an undifferentiated state. Supplementation with hPL can effectively preserve the characteristics of human pluripotent stem cells.

[0206] Example 7 Three germ layer differentiation detection

[0207] The 4th generation cells of the control group without hPL addition and the 4th generation and 10th generation cells of the hPL treatment group were collected and analyzed by STEMdiff. TM The Tri-Germ Layer Differentiation Kit is used to assess the differentiation potential of cultured cells into the three germ layers (ectoderm, mesoderm, and endoderm).

[0208] Experimental results:

[0209] As shown in Figure 2F , the cells in the control group were positive for the expression of ectoderm, mesoderm, and endoderm markers, and the cells in the hPL-treated group were also positive for the expression of ectoderm, mesoderm, and endoderm markers.

[0210] The experimental results showed that when hPL was added to hPSCs for suspension culture, the hPL-treated cells retained the ability to differentiate into cells of all three germ layers at the 10th generation, and hPL supplementation can effectively preserve the characteristics of human pluripotent stem cells.

[0211] Example 8 Cell Karyotype Detection

[0212] The cells of the control group cultured to the 4th passage and the cells of the hPL-treated group cultured to the 10th passage were collected and treated with colchicine for 1.5 hours before chromosome G-banding and karyotype analysis.

[0213] Experimental results:

[0214] As shown in Figure 2G , G-banding karyotype analysis of the cells after culture showed a normal number of 46 chromosomes, with sex chromosomes consisting of XY, a male karyotype; no abnormalities were found in the chromosome structure.

[0215] The experimental results showed that when hPL was added to hPSCs for suspension culture, the hPL-treated group also showed a normal diploid karyotype after long-term culture.

Claims

1. A method for expanding / culturing pluripotent stem cells, comprising: expanding / culturing the pluripotent stem cells under suspension culture conditions, and culturing pluripotent stem cells in a pluripotent stem cell medium; in, The pluripotent stem cell culture medium comprises a basal culture medium, a human platelet lysate and / or other additives.

2. The method according to claim 1, wherein the pluripotent stem cells comprise induced pluripotent stem cells, human embryonic stem cells or non-human embryonic stem cells.

3. The method according to any one of claims 1 to 2, wherein the induced pluripotent stem cells include induced pluripotent stem cells derived from humans and induced pluripotent stem cells derived from cynomolgus monkeys.

4. The method according to any one of claims 1 to 3, wherein the induced pluripotent stem cells are human-derived induced pluripotent stem cells.

5. The method according to any one of claims 1 to 4, wherein the induced pluripotent stem cells are human embryonic stem cells.

6. The method according to any one of claims 1 to 5, wherein the pluripotent stem cells form cell aggregates under the conditions of the suspension culture.

7. The method according to any one of claims 1 to 6, wherein the cells and / or cell aggregates in the suspension culture do not adhere to the culture container.

8. The method according to any one of claims 1 to 7, wherein the culture container is non-cell-adhesive.

9. The method according to any one of claims 1 to 8, wherein the basal medium is selected from one or more of the following groups: IMDM, MEM, Ham's F12, mTeSR1, APEL, StemSpan TM SFEM II, DMEM and RPMI1640.

10. The method according to any one of claims 1 to 9, wherein the basal medium is mTeSR1.

11. The method according to any one of claims 1-10, wherein the source of the human platelet lysate comprises one or more selected from the following groups: platelets derived from human blood, platelets differentiated from various types of stem cells, platelets transdifferentiated from other types of cells through reprogramming, and platelets obtained by biological means.

12. The method according to any one of claims 1 to 11, wherein the concentration of the human platelet lysate is 0.1% to 5% (volume percentage).

13. The method according to any one of claims 1 to 12, wherein the concentration of the human platelet lysate is 0.3% (volume percentage).

14. The method according to any one of claims 1 to 13, wherein the supplement comprises one or more selected from the group consisting of nutrients, extracts, growth factors, hormones, cytokines, and culture medium additives.

15. The method according to any one of claims 1 to 14, wherein the supplement comprises one or more selected from the group consisting of CHIR99021, PD0325901, SB431542, Methyl Cellulose, TGF-β, IGF-1, bFGF, FGF-2, Alginate hydrogel, Microcarrier, Glucose, Glutamate, 3D hydrogel, Fetal Bovine Serum (FBS), Non-Essential Amino Acids (NEAA), L-glutamine, N2, B27, Leukemia Inhibitory Factor (LIF), β-mercaptoethanol (β-ME), Insulin-Transferrin-Selenium (ITS), KnockOut TM Serum Replacement (KOSR), Bovine Serum Albumin (BSA), Human Serum Albumin (HSA), IL-6.

16. The method according to any one of claims 1 to 15, wherein the additive is a Rock inhibitor.

17. The method according to any one of claims 1-16, wherein the Rock inhibitor comprises one or more selected from the group consisting of Y-27632, Y-39983, Thiazovivin, Fasudil, GSK429286A, and Rkl-1447.

18. The method according to any one of claims 1-17, wherein the Rock inhibitor is Y-27632.

19. The method according to any one of claims 1-18, wherein the concentration of the Rock inhibitor is 1-20 μM.

20. The method according to any one of claims 1-19, wherein the concentration of the Rock inhibitor is 10 μM.

21. The method according to any one of claims 1-20, wherein the supplement is mTeSR1 supplement.

22. The method according to any one of claims 1 to 21, wherein the additive is a double antibody, and the double antibody is penicillin and streptomycin.

23. The method according to any one of claims 1 to 22, wherein the concentration of penicillin is 50-200 U / ml, and the concentration of streptomycin is 0.05-0.2 mg / ml. 24 . The method according to any one of claims 1 to 23 , wherein the pluripotent stem cell culture medium comprises a basal medium, human platelet lysate, mTeSR1 supplement and dual antibody. 25 . The method according to any one of claims 1 to 24 , wherein the pluripotent stem cell culture medium comprises mTeSR1, human platelet lysate, mTeSR1 supplement and dual antibody. 26 . The method according to any one of claims 1 to 25 , wherein the pluripotent stem cell culture medium comprises basal medium, human platelet lysate, mTeSR1 supplement, dual antibody and Rock inhibitor. 27 . The method according to any one of claims 1 to 26 , wherein the pluripotent stem cell culture medium comprises basal medium, human platelet lysate, mTeSR1 supplement, double antibody and Y-27632.

28. The method according to any one of claims 1-27, wherein the pluripotent stem cell culture medium comprises mTeSR1, human platelet lysate, mTeSR1 supplement, double antibody and Y-27632.

29. The method according to any one of claims 1 to 28, wherein the method is performed under feeder cell-free conditions.

30. The method according to any one of claims 1-29, wherein the method is performed under serum-free conditions.

31. The method of any one of claims 1-30, wherein the method comprises pre-treating pluripotent stem cells with DPBS and ACCUTASE.

32. The method according to any one of claims 1 to 31, wherein the method comprises isolating pluripotent stem cells at 0.5×10 5 -4×10 5 The cells were cultured at an inoculation density of 1.5 cells / mL.

33. The method according to any one of claims 1-32, wherein the method comprises forming aggregates of pluripotent stem cells under suspension culture conditions provided by a shaker.

34. The method of any one of claims 1-33, wherein the method comprises digestion passage of the aggregates.

35. The method of any one of claims 1-34, wherein the digestion passaging uses ACCUTASE.

36. A pluripotent stem cell culture medium comprising a basal culture medium, human platelet lysate and / or other additives.

37. The pluripotent stem cell culture medium according to claim 36, wherein the basal culture medium is selected from one or more of the following groups: IMDM, MEM, Ham's F12, mTeSR1, APEL, StemSpan TM SFEM II, DMEM and RPMI1640.

38. The pluripotent stem cell culture medium according to any one of claims 36-37, wherein the basal culture medium is mTeSR1.

39. The pluripotent stem cell culture medium according to any one of claims 36-38, wherein the source of human platelet lysate comprises one or more selected from the following group: platelets derived from human blood, platelets differentiated from various types of stem cells, platelets transdifferentiated from other types of cells through reprogramming, and platelets obtained by biological means.

40. The pluripotent stem cell culture medium according to any one of claims 36 to 39, wherein the concentration of the human platelet lysate is 0.1% to 5% (volume percentage).

41. The pluripotent stem cell culture medium according to any one of claims 36 to 40, wherein the concentration of the human platelet lysate is 0.3% (volume percentage).

42. The pluripotent stem cell culture medium according to any one of claims 36 to 41, wherein the supplement comprises one or more selected from the group consisting of nutrients, extracts, growth factors, hormones, cytokines, and culture medium additives.

43. The pluripotent stem cell culture medium according to any one of claims 36 to 42, wherein the additive comprises one or more selected from the group consisting of CHIR99021, PD0325901, SB431542, Methyl Cellulose, TGF-β, IGF-1, bFGF, FGF-2, Alginate hydrogel, Microcarrier, Glucose, Glutamate, 3D hydrogel, Fetal Bovine Serum (FBS), Non-Essential Amino Acids (NEAA), L-glutamine, N2, B27, Leukemia Inhibitory Factor (LIF), β-mercaptoethanol (β-ME), Insulin-Transferrin-Selenium (ITS), KnockOut TM Serum Replacement (KOSR), Bovine Serum Albumin (BSA), Human Serum Albumin (HSA), IL-6. The pluripotent stem cell culture medium according to any one of claims 36 to 43 , wherein the additive is a Rock inhibitor.

45. The pluripotent stem cell culture medium according to any one of claims 36 to 44, wherein the Rock inhibitor comprises one or more selected from the group consisting of Y-27632, Y-39983, Thiazovivin, Fasudil, GSK429286A, and Rk1-1447.

46. ​​The pluripotent stem cell culture medium according to any one of claims 36 to 45, wherein the Rock inhibitor is Y-27632. The pluripotent stem cell culture medium according to any one of claims 36 to 46 , wherein the concentration of the Rock inhibitor is 1 to 20 μM. The pluripotent stem cell culture medium according to any one of claims 36 to 47 , wherein the concentration of the Rock inhibitor is 10 μM. The pluripotent stem cell culture medium according to any one of claims 36 to 48, wherein the supplement is mTeSR1 supplement. 50 . The pluripotent stem cell culture medium according to any one of claims 36 to 49 , wherein the additive is a double antibody, and the double antibody is penicillin and streptomycin.

51. The pluripotent stem cell culture medium according to any one of claims 36 to 50, wherein the concentration of penicillin is 50-200 U / ml, and the concentration of streptomycin is 0.05-0.2 mg / ml. 52 . The pluripotent stem cell culture medium according to any one of claims 36 to 51 , wherein the pluripotent stem cell culture medium comprises a basal medium, human platelet lysate, mTeSR1 supplement and dual antibody. The pluripotent stem cell culture medium according to any one of claims 36 to 52, wherein the pluripotent stem cell culture medium comprises mTeSR1, human platelet lysate, mTeSR1 supplement and dual antibody. The pluripotent stem cell culture medium according to any one of claims 36 to 53, wherein the pluripotent stem cell culture medium comprises a basal medium, human platelet lysate, mTeSR1 supplement, dual antibody and Rock inhibitor. The pluripotent stem cell culture medium according to any one of claims 36 to 54, wherein the pluripotent stem cell culture medium comprises a basal medium, human platelet lysate, mTeSR1 supplement, double antibody and Y-27632. 56 . The pluripotent stem cell culture medium according to any one of claims 36 to 55 , wherein the pluripotent stem cell culture medium comprises mTeSR1, human platelet lysate, mTeSR1 supplement, dual antibody and Y-27632.

57. Use of the method of any one of claims 36 to 56 in expanding / culturing pluripotent stem cells.

58. A composition comprising pluripotent stem cells and the culture medium of any one of claims 36-57.

59. A culture platform for expanding / culturing pluripotent stem cells, comprising the method of any one of claims 1-35 and / or any one of the culture media of claims 36-57.

60. The method of any one of claims 1-35, the culture medium of any one of claims 36-57, the composition of claim 58 and / or the culture platform of claim 59, for expanding / culturing pluripotent stem cells.

61. The method, culture medium, composition, culture platform of claim 60, wherein the pluripotent stem cells are used for treating, preventing and / or alleviating diseases.

62. A method for treating, preventing and / or alleviating a disease, comprising administering pluripotent stem cells to a subject in need thereof, wherein the pluripotent stem cells are obtained by using any one of the methods of claims 1-35, any one of the culture media of claims 36-57, the composition of claim 58, or the culture platform of claim 59.

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