Serum-free and exogen-free culture media for stem cell proliferation

Abstract

The present disclosure, in particular, provides a serum-free and xeno-free culture medium capable of supporting the long-term proliferation of mesenchymal stem cells (MSCs) and a kit comprising the same. Additionally, the present disclosure provides a method for proliferating mesenchymal stem cells (MSCs) using the disclosed culture medium and a substantially homogeneous population of MSCs produced by said method.

Description

Technology Field

[0001] The present disclosure relates generally to the field of cell culture technology, particularly to serum-free and xeno-free culture media for the proliferation of mesenchymal stem cells (MSCs) and kits comprising such media. The present disclosure also relates to a method for proliferating mesenchymal stem cells (MSCs) using the disclosed culture media or kits and to a population of MSCs produced by the disclosed method. Background Technology

[0002] Mesenchymal stem cells (MSCs) are stem cells possessing pluripotency and self-renewal capabilities. MSCs can differentiate into various cell types, including osteoblasts, chondrocytes, and adipocytes. Furthermore, MSCs are known to exhibit paracrine effects and cell adhesion interactions mediated by self-generated humoral factors. Based on these effects, MSCs provide therapeutic benefits for various diseases by not only repairing and regenerating target tissues and cells but also by regulating immune responses, such as through anti-inflammation.

[0003] MSCs can be derived from various adult or fetal tissues (e.g., bone marrow, embryonic yolk sac, placenta, umbilical cord tissue, umbilical cord blood, amniotic fluid, and adipose tissue). However, these cell sources are limited. Unlimited and reproducible MSCs can be produced from induced pluripotent stem cells (iPSCs).

[0004] In clinical applications, a homogeneous and robust cell population with a large number of cells is strongly required. Currently, many protocols for MSC proliferation have been developed to obtain a large number of MSCs.

[0005] Modern MSC proliferation systems are primarily based on basal media supplemented with fetal bovine serum (FBS). However, FBS contains bacteria, viruses, and heterologous proteins capable of transmitting infectious diseases. Furthermore, some studies suggest that MSCs can absorb proteins present in the culture medium, such as fetal bovine serum proteins (7 mg to 30 mg / 10 8 (cells), it was found that repeated infusion of MSCs into patients can induce the production of anti-small protein antibodies and cell-mediated immune responses.

[0006] Numerous serum-free media have been developed for MSC proliferation. However, these media have not been able to reliably support the long-term proliferation of MSCs. For example, MSCs with a high passage number not only lack proliferative potential but also lose stem cell characteristics such as phenotype. The problem to be solved

[0007] Therefore, there remains a need to provide improved media for the proliferation of MSCs. means of solving the problem

[0008] outline

[0009] The present disclosure provides, in particular, a serum-free and exogen-free culture medium, a kit comprising the same, a method for proliferating mesenchymal stem cells (MSCs) using said culture medium or kit, and a population of MSCs produced by the disclosed method.

[0010] One aspect of the present disclosure relates to a serum-free and exogen-free culture medium capable of supporting the long-term proliferation of mesenchymal stem cells (MSCs), comprising (a) a basic medium; (b) an ethanolamine-based compound; (c) a putrescine-based compound; and (d) a human platelet lysate (HPLT).

[0011] In certain embodiments, the ethanolamine-based compound is present in the culture medium at a concentration of 1 to 30 μM.

[0012] In certain embodiments, putrescine-based compounds are present in the culture medium at a concentration of 1 to 30 μM.

[0013] In certain embodiments, human platelet lysate is present in the culture medium at a concentration of 0.1 to 20 volume%.

[0014] In certain embodiments, the ethanolamine-based compound comprises ethanolamine, and the putrescine-based compound comprises putrescine dihydrochloride.

[0015] In certain embodiments, the culture medium further comprises transferrin.

[0016] In certain embodiments, transferrin is present in the culture medium at a concentration of 1 to 200 μg / ml.

[0017] In certain embodiments, the culture medium further comprises an insulin-based compound. In certain embodiments, the insulin-based compound is present in the culture medium at a concentration of 1 to 15 μg / ml.

[0018] In certain embodiments, the culture medium further comprises an antioxidant. In certain embodiments, the antioxidant is present in the culture medium at a concentration of 1 μg / mL to 200 μg / mL.

[0019] In certain embodiments, the culture medium further comprises glutamine or a derivative thereof. In certain embodiments, glutamine or a derivative thereof is present in the culture medium at a concentration of 0.1 to 5 volume%.

[0020] In a specific embodiment, the culture medium comprises 5 to 30 μM ethanolamine, 1 to 20 μM putrescine dihydrochloride, 0.1 to 10 volume% HPLT, 1 to 150 μg / ml transferrin, 1 to 10 μg / ml insulin, 1 to 150 μg / ml ascorbate, and 0.5 to 5 volume% glutamine in the base medium.

[0021] In certain embodiments, the culture medium also additionally includes growth factors.

[0022] In certain embodiments, the growth factor is selected from the group consisting of EGF, IGF, VEGF, PDGF, FGF2, TGFβ and any combination thereof.

[0023] In certain embodiments, the growth factor is present in the culture medium at a concentration of 1 to 20 ng / ml.

[0024] In certain embodiments, the culture medium further comprises a corticosteroid compound.

[0025] In certain embodiments, the corticosteroid compound is selected from the group consisting of hydrocortisone, corticosterone, dehydrocorticosterone, cortisone, and any combination thereof.

[0026] In certain embodiments, the corticosteroid compound is present in the culture medium at a concentration of 0.1 to 5 μM.

[0027] In certain embodiments, the culture medium further comprises human serum albumin (HSA).

[0028] In certain embodiments, human serum albumin (HSA) comprises dialyzed HSA.

[0029] In certain embodiments, human serum albumin (HSA) is present in the culture medium at a concentration of 1 to 20 mg / ml.

[0030] In certain embodiments, the culture medium further comprises a heparin-based compound.

[0031] In certain embodiments, the heparin-based compound is present in the culture medium at a concentration of 1 to 150 μg / mL.

[0032] In certain embodiments, the culture medium contains lipoic acid, sulfate, ferric salt, It further comprises one or more selected from the group consisting of selenite, pyruvate, monothioglycerol (MTG), and nicotinamide-based compounds.

[0033] In a specific embodiment, the culture medium comprises 5 to 30 μM of ethanolamine, 1 to 20 μM of putrescine dihydrochloride, 0.1 to 10 vol% of HPLT, 1 to 150 μg / ml of transferrin, 1 to 10 μg / ml of insulin, 1 to 150 μg / ml of ascorbate, 0.5 to 5 vol% of glutamine, 1 to 15 ng / ml of FGF2, 0.5 to 5 μM of hydrocortisone, 1 to 10 μM of lipoic acid, 1 to 30 μM of FeSO4, and 0.1 to 10 μM of Fe(NO3)3 in the base medium.

[0034] In a specific embodiment, the culture medium comprises, in addition to the base medium, 5 to 30 μM ethanolamine, 1 to 20 μM putrescine dihydrochloride, 0.1 to 10 volume% HPLT, 1 to 150 μg / ml transferrin, 1 to 10 μg / ml insulin, 1 to 150 μg / ml ascorbate, 0.5 to 5 volume% glutamine, 1 to 15 ng / ml FGF2, 0.5 to 5 μM hydrocortisone, 5 to 20 ng / ml Na2SeO3, 0.05 to 5 mg / ml sodium pyruvate, 1 to 10 mg / ml HSA, 10 to 150 μM MTG, 0.5 to 20 mM nicotinamide (NAM), and 10 to 100 μg / ml heparin It contains sodium.

[0035] Another aspect of the present disclosure relates to a kit comprising the culture medium of the present disclosure.

[0036] Also, another aspect of the present disclosure relates to a method for proliferating mesenchymal stem cells (MSCs), comprising the step of bringing the MSCs into contact with the culture medium of the present disclosure.

[0037] In certain embodiments, MSCs are propagated continuously over several passages, such as at least passages 4, 7, and 9.

[0038] Another aspect of the present disclosure relates to a substantially homogeneous group of MSCs produced by the method of the present disclosure. Brief explanation of the drawing

[0039] Some embodiments of the present disclosure are described herein by way of example only with reference to the accompanying drawings. It is emphasized that, with specific and detailed reference to the drawings, the details shown are illustrative and are presented for the purpose of illustratively describing embodiments of the present disclosure. In this regard, the description taken together with the drawings clearly demonstrates to those skilled in the art how embodiments of the present disclosure may be practiced. Figure 1 shows bright-field images of iMSCs cultured in different media M1, SFM-M, PPRF, and MEM-FBS according to Example 1. Figures 2A and 2B show the different effects of M1 medium and M2 medium containing different concentrations of HPLT on the proliferation of iMSCs according to Example 2, where Figure 2A shows the cell morphology of the proliferated iMSCs and Figure 2B shows the number of cells of the proliferated iMSCs. Figures 3A and 3B show the different effects of M1 medium and M2 medium on the long-term proliferation of iMSCs according to Example 3, where Figure 3A shows the cell numbers of iMSCs in passages 6, 7, and 8, and Figure 3B shows the CD90 expression of iMSCs in passages 1, 4, 5, and 7. Figures 4A and 4B show the results of a CFSE-based T cell proliferation analysis of iMSCs proliferated in M1 medium and M2 medium according to Example 4, where Figure 4A shows the cell morphology of activated T cells co-cultured without or with proliferated iMSCs, and Figure 4B shows the percentage of the proliferated T cell population relative to activated T cells co-cultured without or with proliferated iMSCs. Figure 5 shows the effects of various types of growth factors and various concentrations of hydrocortisone in a proliferation medium according to Example 5 on iMSC proliferation, where F represents FGF2, FP represents a combination of FGF2(F) and PDFG-BB(P), FPT represents a combination of FGF2(F), PDFG-BB(P) and TGFβ1(T), and w / o GF represents the case without any growth factors. Figures 6A and 6B show the effects of different media I, II, III, IV, V and M1 on the proliferation of iMSCs according to Example 6, where Figure 6A shows the cell morphology of cultured iMSCs and Figure 6B shows the number of cultured iMSCs. Figure 7 shows the various effects of M1 and M3 media containing different concentrations of HPLT according to Example 7 on the proliferation of cord blood-derived mesenchymal stem cells (ucMSC) and iMSC. Figure 8 shows the effects of different media, M1, M3, and M4, on the proliferation of iMSCs according to Example 8. Figure 9 shows the effect of different media M1, M3, and M4 according to Example 9 on CD90 expression of proliferated iMSCs. Figures 10A-10B show the results of tri-lineage differentiation and CFSE-based T cell proliferation analysis of proliferated iMSCs according to Example 10, where Figure 10A shows the morphology of adipogenic (left panel), osteogenic (middle panel), and chondrogenic (right panel) cells differentiated from iMSCs proliferated in M3, and Figure 10B shows the ratio of the proliferated T cell population to activated T cells and inactivated T cells co-cultured without or with iMSCs proliferated in M1 and M3. Figure 11 shows the effect of different media M1, M3, and M3 supplemented with different concentrations of FGF2 on the proliferation of iMSCs according to Example 11. FIGS. 12A-12B show the effect of HSA, particularly dialyzed HSA, on the proliferation of iMSCs in M3 medium according to Example 12, wherein FIG. 12A shows the cell morphology of iMSCs proliferated in M3 medium containing dialyzed HSA or non-dialyzed HSA and in M3 medium containing no HSA at all, and FIG. 12B shows the number of cells of said iMSCs. Figure 13 shows the effect of HSA and heparin sodium dialyzed in M3 containing different concentrations of HPLT according to Example 13 on the proliferation of iMSCs. Figure 14 shows the effect of different concentrations of HPLT on the proliferation of iMSCs in M5 according to Example 14. Figures 15A-15B show the effects of various media on cell morphology and population doubling time (PDT) of iMSCs during long-term proliferation according to Example 15, where Figure 15A shows the PDT curves of iMSCs from passage 3 to passage 9 during long-term proliferation using M1 or M5, and Figure 15B shows the cell morphology of iMSCs at passage 8 during long-term proliferation using M1, M4, or M5. Figure 16 shows the effects of various media M1, M4, and M5 according to Example 16 on CD90 expression of iMSCs during long-term proliferation. Figures 17A-17B show the results of trilineal differentiation of proliferated iMSCs and CFSE-based T cell proliferation analysis according to Example 17, where Figure 17A shows the morphology of adipogenic (left panel), osteogenic (middle panel), and chondrogenic (right panel) cells differentiated from iMSCs proliferated in M5 medium, and Figure 17B shows the ratio of the proliferated T cell population to activated T cells and inactivated T cells co-cultured with or without iMSCs proliferated in M1 and M5. Specific details for implementing the invention

[0040] details

[0041] It should be recognized that specific aspects, modes, embodiments, variations, and features of the present disclosure are described below in detail at various levels to aid in a substantial understanding of the art.

[0042] Throughout this specification, references to “one embodiment,” “another embodiment,” “preferred embodiment(s),” “some embodiments,” or “specific embodiment(s)” mean that specific features, structures, or characteristics described in relation to the embodiment(s) are included in at least one embodiment of this disclosure. Additionally, specific features, structures, or characteristics in one embodiment may be combined with those of one or more other embodiments in any suitable manner.

[0043] The disclosure described herein by way of example may be practiced without any elements or elements, or any limitations or restrictions, that are not specifically disclosed herein. As used herein, the term “comprising” is intended to mean that the composition and method include the mentioned elements but do not exclude other elements. “essentially consisting of” means, when used to define the composition and method, that other elements of essential importance to the composition or method are excluded. “Comprising of” means that more than trace elements of other components are excluded in the claimed composition and substantial method steps. Embodiments defined by each of these transition terms are within the scope of the disclosure. Accordingly, the method and composition may include additional steps and components (comprising), alternatively include steps and compositions of non-essential importance (essentially consisting of), or alternatively intend only the specified method steps or compositions (constituting). Furthermore, in each case herein, the terms “comprising,” “essentially consisting of,” and “constituting of” may be replaced by either of the other two terms.

[0044] Of course, it should be understood that the present disclosure is not limited to specific uses, methods, reagents, compounds, compositions, or biological systems, which may be varied in many ways. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the meaning generally understood by those skilled in the art to which this disclosure pertains. However, for convenience and completeness, specific terms and their meanings are provided below and throughout this specification.

[0046] The singular forms (in English “a,” “an,” and “the”) used in this specification and the following claims include the plural forms unless the context clearly indicates otherwise. Accordingly, for example, references to “the method” include one or more methods and / or steps of the type described herein and / or that are obvious to those skilled in the art by reading this disclosure, etc.

[0047] The terms “about” or “approximately” mean within a range of statistically meaningful values. Such ranges may be within one digit of a given value or range, typically within 20%, more typically within 10%, and even more typically within 5%. The acceptable deviations covered by the terms “about” or “approximately” vary depending on the specific system under study, and those skilled in the art will readily understand this. Whenever a range is mentioned in this application, all integers within such range are also considered embodiments of this disclosure.

[0048] As used herein, the terms “substantially” or “essentially” refer to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is about or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In some embodiments, the terms “essentially identical” or “substantially identical” refer to a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is nearly identical to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0049] As used herein, the term "pluripotent stem cell" (PSC) refers to a cell derived from the inner cell mass of an embryonic blastocyst. Pluripotent stem cells may be pluripotent and, during development, produce all derivatives of the three major germ layers: ectoderm, endoderm, and mesoderm. Pluripotent stem cells may be of human origin (e.g., human PSC or hPSC). Pluripotent stem cells may be induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC). ESCs (e.g., hESC) and iPSCs (e.g., hiPSC) are known in the art and are products that can be easily obtained or are commercially available using conventional methods, such as those described in the prior art. Suitable methods for producing iPSCs from somatic cells or pluripotent stem cells are well known to those skilled in the art. For example, iPSCs can be reliably generated from somatic cells by conventional reprogramming techniques.

[0050] As used herein, the terms “pluripotent” or “pluripotency” refer to cells having the potential to differentiate into cells of all three germ layers (ectoderm, mesoderm, and endoderm). Pluripotency can be determined at least in part by evaluating the pluripotent characteristics of the cells. Pluripotent characteristics include, but are not limited to: (i) pluripotent stem cell morphology; (ii) unlimited self-renewal potential; (iii) expression of pluripotent stem cell markers including, but not limited to, SSEA1 (mouse only), SSEA3 / 4, SSEA5, TRA1-60 / 81, TRAl-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD133 / prominin, CD140a, CD56, CD73, CD90, CD105, OCT4, NANOG, SOX2, CD30, and / or CD50; (iv) the ability to differentiate into all three somatic lineages (ectoderm, mesoderm, and endoderm); (v) the formation of a teratoma composed of the three somatic lineages; and (vi) the formation of an embryoid body composed of cells from the three somatic lineages.

[0051] As used herein, the terms “mesenchymal stem cell” or “MSC” refer to pluripotent stem cells capable of differentiating into cells such as adipocytes, osteocytes, chondrocytes, myocytes, neurons, and cardiomyocytes. MSCs include primary MSCs and induced MSCs (also called iMSCs). Examples of primary MSCs include, for instance, bone marrow-derived mesenchymal stem cells (BM-MSCs), placenta-derived mesenchymal stem cells (P-MSCs), umbilical cord-derived mesenchymal stem cells (UC-MSCs), adipose-derived mesenchymal stem cells (A-MSCs), peripheral blood-derived mesenchymal stem cells (PB-MSCs), and dental pulp-derived mesenchymal stem cells (DP-MSCs). iMSCs may be differentiated from ESCs or iPSCs. As used herein, ESCs are obtained from commercially established human embryonic stem cell lines or human embryonic stem cells that have not developed in vivo within 14 days of fertilization.

[0052] The terms “proliferation” or “proliferating” as used herein refer to increasing the number of MSCs over a culture period (at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more).

[0053] As used herein, the term "culture medium" refers to a culture medium capable of supporting the survival, growth, proliferation, maintenance, and / or differentiation of cells in an in vitro environment. A culture medium may consist of a base medium and one or more supplements.

[0054] As used herein, the term “basic medium” refers to the basic component of a culture medium (e.g., differentiation culture medium or proliferation culture medium) for the supplement(s). Generally, the basic medium accounts for about 95% to 99% by volume of the culture medium (e.g., differentiation culture medium or proliferation culture medium). The basic medium of a cell proliferation culture medium serves as a source of nutrients, hormones, and / or other factors that aid in the reproduction and / or proliferation of cells. The basic medium of a cell differentiation culture medium serves as a source of nutrients, hormones, and / or other factors that aid in the differentiation of cells.

[0055] As used herein, the term “supplement(s)” refers to the addition(s) of a culture medium (e.g., differentiation culture medium or proliferation culture medium) to a base medium. In the context of this disclosure, the amount or concentration of any addition(s) or supplement(s) within a culture medium (e.g., proliferation medium) is calculated relative to the base medium. Any percentages mentioned in the description of the amount(s) or concentration(s) of the addition(s) or supplement(s) are based on volume unless otherwise specified.

[0056] As used herein, the term "supplemented" refers to the addition of a supplement to a culture medium (e.g., proliferation culture medium) as a base medium. The supplement(s) may be added to the base medium of the culture medium before or when the culture medium is used.

[0057] As used herein, the term "serum-free" refers to the absence of human or animal serum. In culture protocols, the function of serum is to provide cultured cells with an environment similar to that present in vivo (i.e., within the organism from which the cells originated). However, the use of serum derived from animal sources (e.g., bovine serum) or human sources (human serum) is limited due to significant differences in serum composition between donor individuals (where the serum was obtained) and the risk of heterologous contaminants (in the case of using animal serum).

[0058] The term "exoticity" used here refers to the complete absence of products derived from non-human animals.

[0059] The term "Serum Replacement (SR)" as used herein refers to a defined formulation that replaces the function of serum by providing components necessary for the growth and survival of pluripotent stem cells or mesenchymal stem cells.

[0060] As used herein, the term "long-term proliferation" refers to the continuous proliferation of cells in which cells are passed through multiple passages. In the context of this disclosure, long-term proliferation may be used interchangeably with continuous proliferation.

[0061] As used herein, the terms "seeding" or "seedification" refer to the process of supplying cell culture medium to a bioreactor or other vessel. Cells may have previously grown in another bioreactor or vessel. Alternatively, cells may have been frozen and thawed immediately before being supplied to the bioreactor or vessel. This term refers to any number of cells, including a single cell.

[0062] As used herein, the terms “passage” or “passaging” refer to transferring cells within a culture vessel into two or more culture vessels, typically involving the addition of new culture medium. Passaging is typically performed when the cells reach a specific confluency in the culture medium.

[0063] The term "in vitro" as used herein generally refers to activities occurring outside of a living organism.

[0064] The term "in vivo" as used herein generally refers to activities occurring within a living organism.

[0065] 1. Culture medium

[0066] According to one aspect of the present disclosure, a serum-free and exogen-free culture medium capable of supporting the long-term proliferation of mesenchymal stem cells (MSCs) is provided, comprising (a) a basic medium; (b) an ethanolamine-based compound; (c) a putrescine-based compound; and (d) a human platelet lysate (HPLT).

[0067] According to the present disclosure, the culture medium, based on the synergistic effect of a combination of ethanolamine-based compounds, putrescine-based compounds, and HPLT, [provides] the stem cell phenotype of MSCs (CD90) during long-term proliferation. + It is possible to significantly promote the proliferation of MSCs while maintaining stability. Based on this, the culture medium according to the present disclosure can substantially support the long-term proliferation of MSCs without substantially losing the characteristics of MSCs, such as morphology, phenotype, differentiation ability, and immunomodulatory effects, and is therefore more suitable for the mass production of MSC products for clinical application.

[0068] Mesenchymal stem cells (MSC)

[0069] Any MSC may be proliferated using the proliferation medium of the present disclosure. Examples of MSCs include primary MSCs and induced MSCs (iMSCs). Examples of primary MSCs include, for example, BM-MSCs, P-MSCs, UC-MSCs, A-MSCs, PB-MSCs, and DP-MSCs. iMSCs may be derived (e.g., differentiated) from pluripotent stem cells. Pluripotent stem cells may include induced pluripotent stem cells (e.g., hiPSCs), embryonic stem cells (e.g., hESCs), naive PSCs (NPSCs), and proliferating pluripotent stem cells (EPSCs). In certain embodiments, the iMSC is an MSC derived from an ESC. In certain embodiments, the iMSC is an MSC derived from an iPSC. In certain embodiments, the iMSC is an MSC derived from an NPSC. In certain embodiments, the MSC is an MSC derived from an EPSC. Commercially available MSCs may also be used in the present disclosure.

[0070] ESCs (e.g., hESCs) and iPSCs (e.g., hiPSCs) are known in the art and are products that can be easily obtained or are commercially available using conventional methods, such as those described in the prior art. For example, induced pluripotent stem cells (iPSCs) can be reliably produced from somatic cells, including PBMCs and T cells, using the CytoTune iPS 2.0 Sendai Reprogramming Kit (ThermoFisher Scientific).

[0071] Primary MSCs can be obtained using any method known in the art. For example, MSCs can be obtained from bone marrow using standard procedures. For example, MSCs can be isolated by collecting a bone marrow aspirate or biopsy from a donor (e.g., a healthy donor). Typically, monocytes are isolated from a bone marrow aspirate by concentration gradient centrifugation, seeded into a flask containing MSC medium such as Dulbecco's modified Eagle medium (DMEM)—low glucose—supplemented with 10 mM L-glutamine and 10% fetal calf serum (FCS), and incubated at 37°C under a humidified atmosphere of 5% CO2. Non-attached cells are typically removed after 24 hours (e.g., washed with PBS-HSA solution). The culture medium is replaced every 4 days, and after 2 weeks, the culture medium is mostly confluenced. MSCs are retrieved using trypsin and replatted with passage 1 cells. Cells are kept in the culture medium for at least 8 passages, and the presence of MSC-associated surface molecules can be checked periodically.

[0072] iPSCs (e.g., hiPSCs) can be differentiated using any general method for producing iMSCs. For example, iMSCs can be produced from iPSCs using the differentiation method described in CN110592007B, the full text of which is incorporated herein by reference.

[0073] MSCs can be identified by both the presence and absence of specific cell surface markers, such as CD90. MSCs can also be identified through functional analyses in both in vitro and in vivo, particularly analyses related to the ability of stem cells to generate multipotent progeny, and analyses of responsiveness to standard WNT signaling.

[0074] Basic badge

[0075] As the name implies, the basic medium is a culture medium that can support the survival, maintenance, growth, and proliferation of cells and is a basic component of the proliferation medium. Generally, the basic medium accounts for about 95% to 99% of the volume of the proliferation medium. The basic medium used in the proliferation medium of the present disclosure may be a basic medium commonly used in the art.

[0076] Examples of basic media include DMEM / F12 (e.g., Gibco Cat. #C11330500BT), BME media (e.g., Gibco Cat. #21010046, or Sigma-Aldrich Cat. #B9638), IMDM media (e.g., Gibco Cat. #12440053; or Sigma-Aldrich Cat. #I3390), Eagle MEM media (e.g., Minimal Essential Media (MEM), developed by Harry Eagle, Sigma-Aldrich Cat. #M2414 / M2279 / M5690), α-MEM media (e.g., Gibco Cat. #12561056; or, Sigma-Aldrich Cat. #M0894), DMEM media (e.g., Gibco Cat. #21068028), RPMI 1640 media (e.g., Gibco Cat. #11875093), and Ham's F12 Examples include a medium (e.g., Gibco Cat. #11765054) or a mixture thereof. If the culture medium contains a mixture of two or more basic media, the relative proportions of these basic media may be arbitrary.

[0077] Ethanolamine-based compounds

[0078] As used herein, "ethanolamine-based compounds" refers to ethanolamine, derivatives thereof, salts thereof, or mixtures thereof.

[0079] Ethanolamine (also known as 2-aminoethanol, monoethanolamine, ETA, or MEA) is an organic compound having the chemical formula HOCH2CH2NH2 or C2H7NO. It is a compound. The molecule is dual-functional, containing both a primary amine and a primary alcohol. Ethanolamine is generally referred to as monoethanolamine or MEA to distinguish it from diethanolamine (DEA) and triethanolamine (TEA).

[0080] Examples of ethanolamine derivatives include, but are not limited to, substituted ethanolamines such as phosphatidylethanolamine. Examples of salts of ethanolamine and derivatives thereof include, but are not limited to, ethanolamine hydrochloride and ethanolamine hydrobromide, as well as hydrochloride and hydrobromide of substituted ethanolamines.

[0081] According to a specific embodiment, the ethanolamine-based compound used in the culture medium may be ethanolamine. According to a specific embodiment, the ethanolamine-based compound used in the culture medium may be ethanolamine hydrochloride.

[0082] According to the present disclosure, the concentration of the ethanolamine-based compound is not particularly limited as long as it can support the long-term proliferation of MSCs. According to some embodiments, the concentration of the ethanolamine-based compound is 1 to 30 μM, for example, 1 to 5 μM, 1 to 10 μM, 1 to 15 μM, 1 to 20 μM, 1 to 25 μM, 1 to 30 μM, 2 to 5 μM, 2 to 10 μM, 2 to 15 μM, 2 to 20 μM, 2 to 25 μM, 2 to 30 μM, 3 to 5 μM, 3 to 10 μM, 3 to 15 μM, 3 to 20 μM, 3 to 25 μM, 3 to 30 μM, 4 to 5 μM, 4 to 10 μM, 4 to 15 μM, 4 to 20 μM, 4 to 25 μM, 4 to 30 μM, 5 to 10 The range is μM, 5 to 15 μM, 5 to 20 μM, 5 to 25 μM, or 5 to 30 μM. According to some embodiments, the concentration of the ethanolamine-based compound is 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, 20 μM, 21 μM, 22 μM, 23 μM, 24 μM, 25 μM, 26 μM, 27 μM, 28 μM, 29 μM, or 30 μM. According to a preferred embodiment, the concentration of the ethanolamine-based compound is 5 to 30 μM.

[0083] Putrescine-based compounds

[0084] As used herein, "putrescine-based compounds" refers to putrescine and its derivatives, its salts, or mixtures thereof.

[0085] Putrescine is an organic compound with the chemical formula (CH2)4(NH2)2. Putrescine is commonly referred to as 1,4-butanediamine and is produced on an industrial scale through the hydrogenation reaction of succinonitrile.

[0086] Examples of putrescine derivatives include, but are not limited to, substituted putrescine such as N-acetylputrescine. Examples of putrescine salts and derivatives thereof include, but are not limited to, putrescine hydrochloride, putrescine dihydrochloride, putrescine dihydrobromide, and dihydrochloride or dihydrobromide of substituted putrescine such as N-acetylputrescine.

[0087] According to a specific embodiment, the putrescine-based compound used in the culture medium may be putrescine. According to a specific embodiment, the putrescine-based compound used in the culture medium may be putrescine dihydrochloride.

[0088] According to the present disclosure, the concentration of the putrescine-based compound is not particularly limited as long as it can support the long-term proliferation of MSCs. According to some embodiments, the concentration of the putrescine-based compound is 1 to 30 μM, for example, 1 to 5 μM, 1 to 10 μM, 1 to 15 μM, 1 to 20 μM, 1 to 25 μM, 1 to 30 μM, 2 to 5 μM, 2 to 10 μM, 2 to 15 μM, 2 to 20 μM, 2 to 25 μM, 2 to 30 μM, 3 to 5 μM, 3 to 10 μM, 3 to 15 μM, 3 to 20 μM, 3 to 25 μM, 3 to 30 μM, 4 to 5 μM, 4 to 10 μM, 4 to 15 μM, 4 to 20 μM, 4 to 25 μM, 4 to 30 μM, 5 to 10 The range is μM, 5 to 15 μM, 5 to 20 μM, 5 to 25 μM, or 5 to 30 μM. According to some embodiments, the concentration of the putrescine-based compound is 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, 20 μM, 21 μM, 22 μM, 23 μM, 24 μM, 25 μM, 26 μM, 27 μM, 28 μM, 29 μM, or 30 μM. According to a preferred embodiment, the concentration of the putrescine-based compound is 1 to 20 μM.

[0089] Human platelet lysate

[0090] Human platelet lysate (HPLT) is derived from human platelets. Human platelet lysate may be derived from platelets of a healthy human donor and is rich in growth factors. The human platelet lysate used according to the present disclosure is a commercially available product, for example, PLTGold human platelet lysate (Biological Industries, #PLTGOLD500R). However, human platelet lysates from other sources may also be used in the present disclosure. In the present disclosure, human platelet lysate can substantially improve the proliferation efficiency of cells during long-term proliferation.

[0091] According to the present disclosure, the concentration of human platelet lysate is not particularly limited as long as it can support the long-term proliferation of MSCs. According to some embodiments, the concentration of human platelet lysate is 0.1 to 20 volume%, for example, 0.1 to 1 volume%, 0.1 to 2 volume%, 0.1 to 3 volume%, 0.1 to 4 volume%, 0.1 to 5 volume%, 0.1 to 10 volume%, 0.1 to 15 volume%, 0.1 to 20 volume%, 0.5 to 1 volume%, 0.5 to 2 volume%, 0.5 to 3 volume%, 0.5 to 4 volume%, 0.5 to 5 volume%, 0.5 to 10 volume%, 0.5 to 15 volume%, 0.5 to 20 volume%, 1 to 2 volume%, 1 to 3 volume%, 1 to 4 volume%, 1 to 5 volume%, 1 to 10 volume%, 1 to 15 volume%, 1 to 20 volume%, 2 to 3 volume%, 2 to 4 volume%, 2 to 5 volume%, 2 to 10 volume%, 2 to 15 volume%, 2 to 20 volume%, 3 to 4 volume%, 3 to 5 volume%, 3 to 10 volume%, 3 to 15 volume%, 3 to 20 volume%, 4 to 5 volume%, 4 to 10 volume%, 4 to 15 volume%, or 4 to 20 volume%, 5 to 6 volume%, 5 to 7 volume%, 5 to 8 volume%, 5 to 9 volume%, 5 to 10 volume%, 5 to 15 volume%, or 5 to 20 volume%. According to some embodiments, the concentration of human platelet lysate is 0.1 vol%, 0.2 vol%, 0.3 vol%, 0.4 vol%, 0.5 vol%, 0.6 vol%, 0.7 vol%, 0.8 vol%, 0.9 vol%, 1 vol%, 2 vol%, 3 vol%, 4 vol%, 5 vol%, 6 vol%, 7 vol%, 8 vol%, 9 vol%, 10 vol%, 15 vol%, or 20 vol%. According to a preferred embodiment, the concentration of human platelet lysate is 0.1 to 10 vol%.

[0092] Additional reagents

[0093] In accordance with the requirements of the present invention, one or more additional reagents may be optionally added to the culture medium before or at the time of use. The additional reagents are, for example, transferrin, insulin-based compounds, growth factors, corticosteroid compounds, human serum albumin (HSA), heparin-based compounds, glutamine or derivatives thereof, antioxidants, lipoic acid, sulfates, iron salts, selenites , It may include pyruvate, monothioglycerol (MTG), and nicotinamide-based compounds.

[0094] Transferrin

[0095] According to some embodiments, the culture medium further comprises transferrin. According to some embodiments, the transferrin may be holo-transferrin, partially saturated transferrin, or recombinant transferrin.

[0096] According to the present disclosure, the concentration of transferrin is not particularly limited as long as it can support the long-term proliferation of MSCs. According to some embodiments, the concentration of transferrin is 1 to 200 μg / ml, e.g., 1 to 5 μg / ml, 1 to 10 μg / ml, 1 to 15 μg / ml, 1 to 20 μg / ml, 1 to 25 μg / ml, 1 to 30 μg / ml, 1 to 35 μg / ml, 1 to 40 μg / ml, 1 to 45 μg / ml, 1 to 50 μg / ml, 1 to 60 μg / ml, 1 to 70 μg / ml, 1 to 80 μg / ml, 1 to 90 μg / ml, 1 to 100 μg / ml, 1 to 110 μg / ml, 1 to 120 μg / ml, 1 to 130 μg / ml, 1 to 140 μg / ml, 1 to 150 μg / ml, 1 to 160 μg / ml, 1 to 170 μg / ml, 1 to 180 μg / ml, 1 to 190 μg / ml, 1 to 200 μg / ml, 3 to 5 μg / ml, 3 to 10 μg / ml, 3 to 15 μg / ml, 3 to 20 μg / ml, 3 to 25 μg / ml, 3 to 30 μg / ml, 3 to 35 μg / ml, 3 to 40 μg / ml, 3 to 45 μg / ml, 3 to 50 μg / ml, 3 to 60 μg / ml, 3 to 70 μg / ml, 3 to 80 μg / ml, 3 to 90 μg / ml, 3 to 100 μg / ml, 3 to 110 μg / ml, 3 to 120 μg / ml, 3 to 130 μg / ml, 3 to 140 μg / ml, 3 to 150 μg / ml, 3 to 160 μg / ml, 3 to 170 μg / ml, 3 to 180 μg / ml, 3 to 190 μg / ml, 3 to 200 μg / ml, 5 to 10 μg / ml, 5 to 15 μg / ml, 5 to 20 μg / ml, 5 to 25 μg / ml, 5 to 30 μg / ml, 5 to 35 μg / ml, 5 to 40 μg / ml, 5 to 45 μg / ml,5 to 50 μg / ml, 5 to 60 μg / ml, 5 to 70 μg / ml, 5 to 80 μg / ml, 5 to 90 μg / ml, 5 to 100 μg / ml, 5 to 110 μg / ml, 5 to 120 μg / ml, 5 to 130 μg / ml, 5 to 140 μg / ml, 5 to 150 μg / ml, 5 to 160 μg / ml, 5 to 170 μg / ml, 5 to 180 μg / ml, 5 to 190 μg / ml, 5 to 200 μg / ml, 7 to 10 μg / ml, 7 to 15 μg / ml, 7 to 20 μg / ml, 7 to 25 μg / ml, 7 to 30 μg / ml, 7 to 35 μg / ml, 7 to 40 μg / ml, 7 to 45 μg / ml, 7 to 50 μg / ml, 7 to 60 μg / ml, 7 to 70 μg / ml, 7 to 80 μg / ml, 7 to 90 μg / ml, 7 to 100 μg / ml, 7 to 110 μg / ml, 7 to 120 μg / ml, 7 to 130 μg / ml, 7 to 140 μg / ml, 7 to 150 μg / ml, 7 to 160 μg / ml, 7 to 170 μg / ml, 7 to 180 μg / ml, 7 to 190 μg / ml, 7 to 200 μg / ml. 9 to 10 μg / ml, 9 to 15 μg / ml, 9 to 20 μg / ml, 9 to 25 μg / ml, 9 to 30 μg / ml, 9 to 35 μg / ml, 9 to 40 μg / ml, 9 to 45 μg / ml, 9 to 50 μg / ml, 9 to 60 μg / ml, 9 to 70 μg / ml, 9 to 80 μg / ml, 9 to 90 μg / ml, 9 to 100 μg / ml, 9 to 110 μg / ml, 9 to 120 μg / ml, 9 to 130 μg / ml, 9 to 140 μg / ml, 9 to 150 μg / ml, 9 to 160 μg / ml, 9 to 170 μg / ml, 9 to 180 μg / ml,It is 9 to 190 μg / ml or 9 to 200 μg / ml. According to some embodiments, the concentration of transferrin is 1 μg / ml, 2 μg / ml, 3 μg / ml, 4 μg / ml, 5 μg / ml, 6 μg / ml, 7 μg / ml, 8 μg / ml, 9 μg / ml, 10 μg / ml, 15 μg / ml, 20 μg / ml, 25 μg / ml, 30 μg / ml, 35 μg / ml, 40 μg / ml, 45 μg / ml, 50 μg / ml, 60 μg / ml, 70 μg / ml, 80 μg / ml, 90 μg / ml, 100 μg / ml, 110 μg / ml, 120 μg / ml, 130 μg / ml, 140 μg / ml, 150 μg / ml, 160 μg / ml, It is 170 μg / ml, 180 μg / ml, 190 μg / ml, or 200 μg / ml. According to a preferred embodiment, the concentration of transferrin is 1 to 150 μg / ml.

[0097] Insulin-based compounds

[0098] As used herein, "insulin-based compounds" refers to insulin, analogs thereof or derivatives thereof, or mixtures thereof.

[0099] According to some embodiments, the insulin-based compound comprises insulin. According to some embodiments, the insulin-based compound may be an insulin analog or derivative such as insulin lispro, insulin aspartate, and insulin glurisin.

[0100] According to the present invention, the concentration of the insulin-based compound is not particularly limited as long as it can support the long-term proliferation of MSCs. According to some embodiments, the concentration of the insulin-based compound is 1 to 15 μg / ml, e.g., 1 to 5 μg / ml, 1 to 10 μg / ml, 1 to 15 μg / ml, 2 to 5 μg / ml, 2 to 10 μg / ml, 2 to 15 μg / ml, 3 to 5 μg / ml, 3 to 10 μg / ml, 3 to 15 μg / ml, 4 to 5 μg / ml, 4 to 10 μg / ml, 4 to 15 μg / ml, 5 to 10 μg / ml, 5 to 15 μg / ml, 6 to 10 μg / ml, 6 to 15 μg / ml, 7 to 10 μg / ml, 7 to 15 μg / ml, 8 to 10 μg / ml, 8 to 15 μg / ml, 9 to It is 10 μg / ml, 9 to 15 μg / ml, or 10 to 15 μg / ml. According to some embodiments, the concentration of the insulin-based compound is 1 μg / ml, 2 μg / ml, 3 μg / ml, 4 μg / ml, 5 μg / ml, 6 μg / ml, 7 μg / ml, 8 μg / ml, 9 μg / ml, 10 μg / ml, 11 μg / ml, 12 μg / ml, 13 μg / ml, 14 μg / ml, or 15 μg / ml. According to a preferred embodiment, the concentration of the insulin-based compound is 1 to 10 μg / ml.

[0101] Glutamine or derivatives thereof

[0102] According to some embodiments, the culture medium further comprises glutamine or a derivative thereof.

[0103] According to some embodiments, glutamine or a derivative thereof may be glutamine such as L-glutamine or substituted glutamine such as alanyl-glutamine. Alanyl-glutamine is a compound used in dietary supplements, parenteral nutrition, and cell cultures in the form of L-alanyl-L-glutamine. L-alanyl-L-glutamine is sold by Thermo Fisher Scientific under the name GlutaMAX.

[0104] According to the present disclosure, the concentration of glutamine or its derivative is not particularly limited as long as it can support the long-term proliferation of MSCs. According to some embodiments, the concentration of glutamine or a derivative thereof is 0.1 to 5 volume%, for example, 0.1 to 1 volume%, 0.1 to 2 volume%, 0.1 to 3 volume%, 0.1 to 4 volume%, 0.1 to 5 volume%, 0.2 to 1 volume%, 0.2 to 2 volume%, 0.2 to 3 volume%, 0.2 to 4 volume%, 0.2 to 5 volume%, 0.3 to 1 volume%, 0.3 to 2 volume%, 0.3 to 3 volume%, 0.3 to 4 volume%, 0.3 to 5 volume%, 0.4 to 1 volume%, 0.4 to 2 volume%, 0.4 to 3 volume%, 0.4 to 4 volume%, 0.4 to 5 volume%, 0.5 to 1 volume%, 0.5 to It is 2 volume%, 0.5 to 3 volume%, 0.5 to 4 volume%, or 0.5 to 5 volume%. According to some embodiments, the concentration of glutamine or its derivative is 0.1 volume%, 0.2 volume%, 0.3 volume%, 0.4 volume%, 0.5 volume%, 0.6 volume%, 0.7 volume%, 0.8 volume%, 0.9 volume%, 1 volume%, 2 volume%, 3 volume%, 4 volume%, or 5 volume%. According to preferred embodiments, the concentration of glutamine or its derivative is 0.5 to 5 volume%.

[0105] Antioxidants

[0106] According to some embodiments, the culture medium additionally contains an antioxidant.

[0107] As used herein, "antioxidant" refers to a compound that inhibits intracellular oxidation, which is a chemical reaction capable of generating free radicals. The antioxidant may be ascorbic acid, its analogs, its derivatives, or its salts.

[0108] Antioxidants include ascorbic acid, ascorbic salts, such as magnesium salts like L(+)-ascorbate magnesium salt, sodium salts like L(+)-ascorbate sodium salt, and analogs or derivatives, such as ascorbyl glucoside, 3-ethylascorbic acid, ascorbyl tetraisopalmitate, ascorbic acid phosphate salt, and ascorbyl palmitate.

[0109] According to some embodiments, the antioxidant is ascorbate. According to some embodiments, the antioxidant is sodium ascorbate or magnesium ascorbate.

[0110] According to the present disclosure, the concentration of the antioxidant is not particularly limited as long as it can support the long-term proliferation of MSCs. According to some embodiments, the concentration of the antioxidant is 1 to 200 μg / ml, for example, 1 to 5 μg / ml, 1 to 10 μg / ml, 1 to 15 μg / ml, 1 to 20 μg / ml, 1 to 25 μg / ml, 1 to 30 μg / ml, 1 to 35 μg / ml, 1 to 40 μg / ml, 1 to 45 μg / ml, 1 to 50 μg / ml, 1 to 60 μg / ml, 1 to 70 μg / ml, 1 to 80 μg / ml, 1 to 90 μg / ml, 1 to 100 μg / ml, 1 to 150 μg / ml, 1 to 200 μg / ml, 2 to 5 μg / ml, 2 to 10 μg / ml, 2 to 15 μg / ml, 2 to 20 μg / ml, 2 to 25 μg / ml, 2 to 30 μg / ml, 2 to 35 μg / ml, 2 to 40 μg / ml, 2 to 45 μg / ml, 2 to 50 μg / ml, 2 to 60 μg / ml, 2 to 70 μg / ml, 2 to 80 μg / ml, 2 to 90 μg / ml, 2 to 100 μg / ml, 2 to 150 μg / ml, 2 to 200 μg / ml, 3 to 5 μg / ml, 3 to 10 μg / ml, 3 to 15 μg / ml, 3 to 20 μg / ml, 3 to 25 μg / ml, 3 to 30 μg / ml, 3 to 35 μg / ml, 3 to 40 μg / ml, 3 to 45 μg / ml, 3 to 50 μg / ml, 3 to 60 μg / ml, 3 to 70 μg / ml, 3 to 80 μg / ml, 3 to 90 μg / ml, 3 to 100 μg / ml, 3 to 150 μg / ml, 3 to 200 μg / ml, 4 to 5 μg / ml, 4 to 10 μg / ml, 4 to 15 μg / ml, 4 to 20 μg / ml, 4 to 25 μg / ml, 4 to 30 μg / ml, 4 to 35 μg / ml, 4 to 40 μg / ml,4 to 45 μg / ml, 4 to 50 μg / ml, 4 to 60 μg / ml, 4 to 70 μg / ml, 4 to 80 μg / ml, 4 to 90 μg / ml, 4 to 100 μg / ml, 4 to 150 μg / ml, 4 to 200 μg / ml, 5 to 10 μg / ml, 5 to 15 μg / ml, 5 to 20 μg / ml, 5 to 25 μg / ml, 5 to 30 μg / ml, 5 to 35 μg / ml, 5 to 40 μg / ml, 5 to 45 μg / ml, 5 to 50 μg / ml, 5 to 60 μg / ml, 5 to 70 μg / ml, 5 to 80 μg / ml, 5 to 90 μg / ml, It is 5 to 100 μg / ml, 5 to 150 μg / ml, or 5 to 200 μg / ml. According to some embodiments, the concentration of antioxidant is 1 μg / ml, 2 μg / ml, 3 μg / ml, 4 μg / ml, 5 μg / ml, 6 μg / ml, 7 μg / ml, 8 μg / ml, 9 μg / ml, 10 μg / ml, 15 μg / ml, 20 μg / ml, 25 μg / ml, 30 μg / ml, or It is 200 μg / ml. According to a preferred embodiment, the concentration of the antioxidant is 1 to 150 μg / ml.

[0111] Growth factors

[0112] According to some embodiments, the culture medium additionally includes growth factors.

[0113] Growth factors are molecules that can stimulate various cellular processes during development and tissue healing, including, for example, cell proliferation, cell migration, differentiation, and multicellular morphogenesis. Examples of growth factors include, but are not limited to, bone morphogenetic factor (BMP), epidermal growth factor (EGF), endothelial growth factor (ECGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), transforming growth factor beta (TGFβ), and vascular endothelial growth factor (VEGF). In the present disclosure, the addition of one or more growth factors can clearly promote MSC proliferation.

[0114] According to some embodiments, the growth factor is selected from the group consisting of EGF, IGF, VEGF, PDGF, FGF2, TGFβ, and any combination thereof. According to a preferred embodiment, the growth factor is FGF2, PDGF, and / or TGFβ. According to a more preferred embodiment, the growth factor is FGF2 and / or PDGF. According to the most preferred embodiment, the growth factor is FGF2.

[0115] Epidermal growth factor (EGF) refers to any polypeptide or variant thereof of the epidermal growth factor (EGF) protein family that promotes cell growth and differentiation. Typically, EGF exerts activity by binding to an epidermal growth factor receptor. Accordingly, any EGF molecular variant that retains biological activity, e.g., a C-terminal cleavage molecule or an N-terminal cleavage molecule, may be used according to the present disclosure.

[0116] The EGF used in the culture medium of some embodiments of the present disclosure may be a purified, synthetic, or recombinantly expressed EGF protein. EGF can be obtained from various commercial sources.

[0117] Insulin-like growth factor (IGF) is a protein with high sequence similarity to insulin. IGF is part of a complex system that cells use to communicate with their physiological environment. Insulin-like growth factor 1 (commonly called IGF-1, sometimes IGF-I) is primarily secreted by the liver in response to stimulation by growth hormone (GH). IGF-1 is important for regulating normal physiological functions as well as various pathological conditions, including cancer. Insulin-like growth factor 2 (IGF-2, sometimes IGF-II) is considered a major growth factor required for early development, whereas IGF-1 expression is necessary to achieve maximum growth.

[0118] The IGF used in the culture medium of some embodiments of the present disclosure may be a purified, synthetic, or recombinantly expressed IGF protein. IGF can be obtained from various commercial sources.

[0119] Vascular endothelial growth factor (VEGF) is a signaling protein produced by many cells that stimulates angiogenesis. VEGF is an important signaling protein involved in both angiogenesis (the formation of new blood vessels in the embryonic circulation) and neovascularization (the growth of blood vessels in the existing blood vessels).

[0120] The VEGF used in the culture medium of some embodiments of the present disclosure may be a purified, synthetic, or recombinantly expressed VEGF protein. VEGF can be obtained from various commercial sources.

[0121] Platelet-derived growth factor (PDGF) refers to any of the four different isoforms of PDGF that activate a cellular response through two different receptors. These isoforms include A (observed as a homodimer designated as PDGF-AA and as part of a heterodimer with isoform B designated as PDGF-AB), B (observed as a homodimer designated as PDGF-BB and as part of a heterodimer with isoform A designated as PDGF-AB), C (observed as a homodimer designated as PDGF-CC), and D (observed as a homodimer designated as PDGF-DD). Accordingly, the term “PDGF” as used herein refers to the commonly known PDGF homodimers and heterodimers (e.g., PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC, and PDGF-DD).

[0122] According to some embodiments, the PDGF used in the culture medium of some embodiments of the present disclosure is PDGF-BB.

[0123] The PDGF used in the culture medium of some embodiments of the present disclosure may be a purified, synthetic, or recombinantly expressed PDGF protein. PDGF can be obtained from various commercial sources.

[0124] FGF2 is also generally known as basic fibroblast growth factor (basic FGF, bFGF, or FGF-β) and is a member of the fibroblast growth factor family. The FGF2 used in the culture medium of some embodiments of the present invention may be a purified, synthetic, or recombinantly expressed FGF2 protein.

[0125] Transforming growth factor beta (TGFβ) refers to any isoform of transforming growth factor beta (β) that functions through the same receptor signaling system in controlling proliferation, differentiation, and other functions in many cell types. TGFβ plays a role in inducing transformation and also acts as a negative autocrine growth factor.

[0126] According to some embodiments of the present disclosure, TGFβ comprises TGFβ1, TGFβ2 and / or TGFβ3.

[0127] Transforming growth factor beta-1 (TGFβ1) is a polypeptide member of the transforming growth factor beta superfamily of cytokines. The TGFβ1 used in the culture medium of some embodiments of the present disclosure may be a purified, synthetic, or recombinantly expressed TGFβ1 protein. TGFβ1 may be available from various commercial sources.

[0128] Transforming growth factor beta-2 (TGFβ2) is a polypeptide member of the transforming growth factor beta superfamily of cytokines. TGFβ2 plays an important role in embryonic development. The TGFβ2 used in the culture medium of some embodiments of the present invention may be a purified, synthetic, or recombinantly expressed TGFβ2 protein. TGFβ2 can be obtained from various commercial sources.

[0129] Transforming growth factor beta-3 (TGFβ3) is a polypeptide member of the transforming growth factor beta superfamily of cytokines. The TGFβ3 used in the culture medium of some embodiments of the present invention may be a purified, synthetic, or recombinantly expressed TGFβ3 protein. TGFβ3 may be available from various commercial sources.

[0130] According to the present disclosure, the concentration of the growth factor is not particularly limited as long as it can support the long-term proliferation of MSCs. According to some embodiments, the concentration of the growth factor is 1 to 20 ng / ml, for example, 1 to 5 ng / ml, 1 to 10 ng / ml, 1 to 15 ng / ml, 1 to 20 ng / ml, 2 to 5 ng / ml, 2 to 10 ng / ml, 2 to 15 ng / ml, 2 to 20 ng / ml, 3 to 5 ng / ml, 3 to 10 ng / ml, 3 to 15 ng / ml, 3 to 20 ng / ml, 4 to 5 ng / ml, 4 to 10 ng / ml, 4 to 15 ng / ml, 4 to 20 ng / ml, 5 to 10 ng / ml, 5 to 15 ng / ml, or 5 to 20 ng / ml. According to some embodiments, the concentration of the growth factor is 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, 10 ng / ml, 11 ng / ml, 12 ng / ml, 13 ng / ml, 14 ng / ml, 15 ng / ml, 16 ng / ml, 17 ng / ml, 18 ng / ml, 19 ng / ml, or 20 ng / ml. According to a preferred embodiment, the concentration of the growth factor is 1 to 15 ng / ml.

[0131] corticosteroid compounds

[0132] According to some embodiments, the culture medium further comprises a corticosteroid compound.

[0133] Corticosteroids (also called corticosteroids) are not only a type of steroid hormone produced in the adrenal cortex of vertebrates, but also synthetic analogs of this hormone. Glucocorticoids and mineralocorticoids, the two major types of corticosteroids, are involved in a wide range of physiological processes, including stress responses, immune responses, as well as inflammation, carbohydrate metabolism, protein breakdown, and the regulation of blood electrolyte concentrations and behavior. In the present disclosure, corticosteroid compounds can promote MSC proliferation.

[0134] According to some embodiments, the corticosteroid compound is selected from the group consisting of hydrocortisone, corticosterone, dehydrocorticosterone, cortisone, and any combination thereof. According to a preferred embodiment, the corticosteroid compound is hydrocortisone.

[0135] According to the present disclosure, the concentration of the corticosteroid compound is not particularly limited as long as it can support the long-term proliferation of MSCs. According to some embodiments, the concentration of the corticosteroid compound is 0.1 to 5 μM, for example, 0.1 to 0.5 μM, 0.1 to 1 μM, 0.1 to 1.5 μM, 0.1 to 2 μM, 0.1 to 2.5 μM, 0.1 to 3 μM, 0.1 to 3.5 μM, 0.1 to 4 μM, 0.1 to 4.5 μM, 0.1 to 5 μM, 0.2 to 0.5 μM, 0.2 to 1 μM, 0.2 to 1.5 μM, 0.2 to 2 μM, 0.2 to 2.5 μM, 0.2 to 3 μM, 0.2 to 3.5 μM, 0.2 to 4 μM, 0.2 to 4.5 μM, 0.2 to 5 μM, 0.3 to 0.5 μM, 0.3 to 1 μM, 0.3 to 1.5 μM, 0.3 to 2 μM, 0.3 to 2.5 μM, 0.3 to 3 μM, 0.3 to 3.5 μM, 0.3 to 4 μM, 0.3 to 4.5 μM, 0.3 to 5 μM, 0.4 to 0.5 μM, 0.4 to 1 μM, 0.4 to 1.5 μM, 0.4 to 2 μM, 0.4 to 2.5 μM, 0.4 to 3 μM, 0.4 to 3.5 μM, 0.4 to 4 μM, 0.4 to 4.5 μM, 0.4 to 5 μM, 0.5 to 1 μM, 0.5 to 1.5 μM, The concentration is 0.5 to 2 μM, 0.5 to 2.5 μM, 0.5 to 3 μM, 0.5 to 3.5 μM, 0.5 to 4 μM, 0.5 to 4.5 μM, or 0.5 to 5 μM. According to some embodiments, the concentration of the corticosteroid compound is 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, or 5 μM.According to a preferred embodiment, the concentration of the corticosteroid compound is 0.5 to 5 μM.

[0136] Human serum albumin (HSA)

[0137] According to some embodiments, the culture medium further comprises human serum albumin (HSA). Human serum albumin is a serum albumin found in human blood. It is the most abundant protein in human plasma and constitutes about half of the serum proteins. In the present disclosure, HSA can promote MSC proliferation.

[0138] HSA may include dialyzed HSA or non-dialyzed HSA. According to a preferred embodiment, human serum albumin (HSA) is dialyzed HSA. Compared to non-dialyzed HSA, dialyzed HSA further promotes MSC proliferation and significantly lowers the HPLT concentration required to promote MSC proliferation.

[0139] Dialysis-induced HSA can be produced from HSA by dialysis. Specifically, HSA is placed in a dialysis bag, and dialysis is performed by leaving the dialysis bag in a DPBS solution at 4°C overnight, with the ratio of HSA to DPBS being 1:50 (v:v) and the pore size of the bag being 15 KD.

[0140] According to the present disclosure, the concentration of human serum albumin is not particularly limited as long as it can support the long-term proliferation of MSCs. According to some embodiments, the concentration of human serum albumin is 1 to 20 mg / ml, e.g., 1 to 5 mg / ml, 1 to 10 mg / ml, 1 to 15 mg / ml, 1 to 20 mg / ml, 2 to 5 mg / ml, 2 to 10 mg / ml, 2 to 15 mg / ml, 2 to 20 mg / ml, 3 to 5 mg / ml, 3 to 10 mg / ml, 3 to 15 mg / ml, 3 to 20 mg / ml, 4 to 5 mg / ml, 4 to 10 mg / ml, 4 to 15 mg / ml, 4 to 20 mg / ml, 5 to 10 mg / ml, 5 to 15 mg / ml, or 5 to 20 mg / ml. According to some embodiments, the concentration of human serum albumin is 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, or 20 mg / ml. According to a preferred embodiment, the concentration of human serum albumin is 1 to 10 mg / ml.

[0141] Heparin-based compounds

[0142] According to some embodiments, the culture medium further comprises a heparin-based compound.

[0143] As used herein, "heparin-based compounds" refers to heparin, derivatives thereof, salts thereof, or mixtures thereof.

[0144] Heparin, a highly sulfated glycosaminoglycan variant primarily produced and stored in mast cells, is understood to have the highest net negative charge density of all known biological molecules. This negative charge binds to positively charged heparin binding domains present in numerous extracellular proteins. These proteins include fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), bone morphogenetic protein (BMP), and large extracellular structural molecules such as fibronectin and laminin.

[0145] Examples of heparin derivatives include, but are not limited to, substituted heparin. Examples of heparin salts or derivatives thereof include, but are not limited to, sodium heparin salt, lithium heparin salt, and substituted heparin salt.

[0146] In the present disclosure, heparin-based compounds can further promote MSC proliferation.

[0147] According to a preferred embodiment, the heparin-based compound is heparin sodium.

[0148] According to the present disclosure, the concentration of the heparin-based compound is not particularly limited as long as it can support the long-term proliferation of MSCs. According to some embodiments, the concentration of the heparin-based compound is 1 to 150 μg / ml, for example, 1 to 5 μg / ml, 1 to 10 μg / ml, 1 to 15 μg / ml, 1 to 20 μg / ml, 1 to 25 μg / ml, 1 to 30 μg / ml, 1 to 35 μg / ml, 1 to 40 μg / ml, 1 to 45 μg / ml, 1 to 50 μg / ml, 1 to 60 μg / ml, 1 to 70 μg / ml, 1 to 80 μg / ml, 1 to 90 μg / ml, 1 to 100 μg / ml, 1 to 110 μg / ml, 1 to 120 μg / ml, 1 to 130 μg / ml, 1 to 140 μg / ml, 1 to 150 μg / ml, 2 to 5 μg / ml, 2 to 10 μg / ml, 2 to 15 μg / ml, 2 to 20 μg / ml, 2 to 25 μg / ml, 2 to 30 μg / ml, 2 to 35 μg / ml, 2 to 40 μg / ml, 2 to 45 μg / ml, 2 to 50 μg / ml, 2 to 60 μg / ml, 2 to 70 μg / ml, 2 to 80 μg / ml, 2 to 90 μg / ml, 2 to 100 μg / ml, 2 to 110 μg / ml, 2 to 120 μg / ml, 2 to 130 μg / ml, 2 to 140 μg / ml, 2 to 150 μg / ml, 3 to 5 μg / ml, 3 to 10 μg / ml, 3 to 15 μg / ml, 3 to 20 μg / ml, 3 to 25 μg / ml, 3 to 30 μg / ml, 3 to 35 μg / ml, 3 to 40 μg / ml, 3 to 45 μg / ml, 3 to 50 μg / ml, 3 to 60 μg / ml, 3 to 70 μg / ml, 3 to 80 μg / ml, 3 to 90 μg / ml, 3 to 100 μg / ml, 3 to 110 μg / ml, 3 to 120 μg / ml, 3 to 130 μg / ml,3 to 140 μg / ml, 3 to 150 μg / ml, 4 to 5 μg / ml, 4 to 10 μg / ml, 4 to 15 μg / ml, 4 to 20 μg / ml, 4 to 25 μg / ml, 4 to 30 μg / ml, 4 to 35 μg / ml, 4 to 40 μg / ml, 4 to 45 μg / ml, 4 to 50 μg / ml, 4 to 60 μg / ml, 4 to 70 μg / ml, 4 to 80 μg / ml, 4 to 90 μg / ml, 4 to 100 μg / ml, 4 to 110 μg / ml, 4 to 120 μg / ml, 4 to 130 μg / ml, 4 to 140 μg / ml, 4 to 150 μg / ml, 5 to 10 μg / ml, 5 to 15 μg / ml, 5 to 20 μg / ml, 5 to 25 μg / ml, 5 to 30 μg / ml, 5 to 35 μg / ml, 5 to 40 μg / ml, 5 to 45 μg / ml, 5 to 50 μg / ml, 5 to 60 μg / ml, 5 to 70 μg / ml, 5 to 80 μg / ml, 5 to 90 μg / ml, 5 to 100 μg / ml, 5 to 110 μg / ml, 5 to 120 μg / ml, 5 to 130 μg / ml, 5 to 140 μg / ml, 5 to 150 μg / ml, 10 to 15 μg / ml, 10 to 20 μg / ml, It is 10 to 25 μg / ml, 10 to 30 μg / ml, 10 to 35 μg / ml, 10 to 40 μg / ml, 10 to 45 μg / ml, 10 to 50 μg / ml, 10 to 60 μg / ml, 10 to 70 μg / ml, 10 to 80 μg / ml, 10 to 90 μg / ml, 10 to 100 μg / ml, 10 to 110 μg / ml, 10 to 120 μg / ml, 10 to 130 μg / ml, 10 to 140 μg / ml, or 10 to 150 μg / ml. According to some embodiments, the concentration of the heparin-based compound is 1 μg / ml, 2 μg / ml, 3 μg / ml, 4 μg / ml, 5 μg / ml,It is 6 μg / ml, 7 μg / ml, 8 μg / ml, 9 μg / ml, 10 μg / ml, 15 μg / ml, 20 μg / ml, 25 μg / ml, 30 μg / ml, 35 μg / ml, 40 μg / ml, 45 μg / ml, 50 μg / ml, 60 μg / ml, 70 μg / ml, 80 μg / ml, 90 μg / ml, 100 μg / ml, 110 μg / ml, 120 μg / ml, 130 μg / ml, 140 μg / ml, or 150 μg / ml. According to a preferred embodiment, the concentration of the heparin-based compound is 10 to 100 μg / ml.

[0149] In certain embodiments, the culture medium contains lipoic acid, sulfate, iron salt, It further comprises one or more selected from the group consisting of selenite, pyruvate, monothioglycerol (MTG), and nicotinamide-based compounds.

[0150] Lipoic acid

[0151] According to some embodiments, the culture medium additionally contains lipoic acid.

[0152] According to the present disclosure, the concentration of lipoic acid is not particularly limited as long as it can support the long-term proliferation of MSCs. According to some embodiments, the concentration of lipoic acid is 1 to 20 μM, e.g., 1 to 5 μM, 1 to 10 μM, 1 to 15 μM, 1 to 20 μM, 2 to 5 μM, 2 to 10 μM, 2 to 15 μM, 2 to 20 μM, 3 to 5 μM, 3 to 10 μM, 3 to 15 μM, 3 to 20 μM, 4 to 5 μM, 4 to 10 μM, 4 to 15 μM, 4 to 20 μM, 5 to 10 μM, 5 to 15 μM, or 5 to 20 μM. According to some embodiments, the concentration of lipoic acid is 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, or 20 μM. According to a preferred embodiment, the concentration of lipoic acid is 1 to 10 μM.

[0153] sulfate

[0154] According to some embodiments, the culture medium additionally contains sulfate. Examples of sulfates include FeSO4, MgSO4, CuSO4, and ZnSO4. . According to a preferred embodiment, the sulfate is FeSO4.

[0155] According to the present disclosure, the concentration of sulfate is not particularly limited as long as it can support the long-term proliferation of MSCs. According to some embodiments, the concentration of sulfate is 1 to 50 μM, for example, 1 to 5 μM, 1 to 10 μM, 1 to 15 μM, 1 to 20 μM, 1 to 25 μM, 1 to 30 μM, 1 to 35 μM, 1 to 40 μM, 1 to 45 μM, 1 to 50 μM, 2 to 5 μM, 2 to 10 μM, 2 to 15 μM, 2 to 20 μM, 2 to 25 μM, 2 to 30 μM, 2 to 35 μM, 2 to 40 μM, 2 to 45 μM, 2 to 50 μM, 3 to 5 μM, 3 to 10 μM, 3 to 15 μM, 3 to 20 μM, 3 to 25 μM, It is 3 to 30 μM, 3 to 35 μM, 3 to 40 μM, 3 to 45 μM, 3 to 50 μM, 4 to 5 μM, 4 to 10 μM, 4 to 15 μM, 4 to 20 μM, 4 to 25 μM, 4 to 30 μM, 4 to 35 μM, 4 to 40 μM, 4 to 45 μM, 4 to 50 μM, 5 to 10 μM, 5 to 15 μM, 5 to 20 μM, 5 to 25 μM, 5 to 30 μM, 5 to 35 μM, 5 to 40 μM, 5 to 45 μM, or 5 to 50 μM. According to some embodiments, the concentration of sulfate is 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, or 50 μM. According to a preferred embodiment, the concentration of sulfate is 1 to 30 μM.

[0156] iron salt

[0157] According to some embodiments, the culture medium further comprises an iron salt. Examples of iron salts include Fe(NO3) 3, There are FeCl3 and iron citrate. According to a preferred embodiment, the iron salt is Fe(NO3)3.

[0158] According to the present disclosure, the concentration of iron salt is not particularly limited as long as it can support the long-term proliferation of MSCs. According to some embodiments, the concentration of the iron salt is 0.1 to 20 μM, for example, 0.1 to 5 μM, 0.1 to 10 μM, 0.1 to 15 μM, 0.1 to 20 μM, 0.2 to 5 μM, 0.2 to 10 μM, 0.2 to 15 μM, 0.2 to 20 μM, 0.3 to 5 μM, 0.3 to 10 μM, 0.3 to 15 μM, 0.3 to 20 μM, 0.4 to 5 μM, 0.4 to 10 μM, 0.4 to 15 μM, 0.4 to 20 μM, 0.5 to 5 μM, 0.5 to 10 μM, 0.5 to 15 μM, 5 to 20 μM, 0.6 to 5 μM, 0.6 to 10 μM, 0.6 to 15 μM, 0.6 to 20 μM, 0.7 to 5 μM, 0.7 to 10 μM, 0.7 to 15 μM, 0.7 to 20 μM, 0.8 to 5 μM, 0.8 to 10 μM, 0.8 to 15 μM, 0.8 to 20 μM, 0.9 to 5 μM, 0.9 to 10 μM, 0.9 to 15 μM, 0.9 to 20 μM, 1 to 5 μM, 1 to 10 μM, 1 to 15 μM, 1 to 20 μM, 2 to 5 μM, 2 to 10 μM, 2 to 15 μM, 2 to 20 μM, 3 to 5 μM, 3 to 10 μM, 3 to 15 μM, 3 to 20 μM, 4 to 5 μM, 4 to 10 μM, 4 to 15 μM, 4 to 20 μM, 5 to 10 μM, 5 to 15 μM, or 5 to 20 μM. According to some embodiments, the concentration of iron salt is 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.It is 9 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, or 20 μM. According to a preferred embodiment, the concentration of the iron salt is 0.1 to 10 μM.

[0159] Selenite

[0160] According to some embodiments, the culture medium further comprises selenite. Examples of selenites include Na2SeO3 and K2SeO3. According to a preferred embodiment, the selenite is Na2SeO3.

[0161] According to the present disclosure, the concentration of selenite is not particularly limited as long as it can support the long-term growth of MSCs. According to some embodiments, the concentration of selenite is 1 to 30 ng / ml, for example, 1 to 5 ng / ml, 1 to 10 ng / ml, 1 to 15 ng / ml, 1 to 20 ng / ml, 1 to 25 ng / ml, 1 to 30 ng / ml, 2 to 5 ng / ml, 2 to 10 ng / ml, 2 to 15 ng / ml, 2 to 20 ng / ml, 2 to 25 ng / ml, 2 to 30 ng / ml, 3 to 5 ng / ml, 3 to 10 ng / ml, 3 to 15 ng / ml, 3 to 20 ng / ml, 3 to 25 ng / ml, 3 to 30 ng / ml, 4 to 5 ng / ml, 4 to 10 ng / ml, 4 It is up to 15 ng / ml, 4 to 20 ng / ml, 4 to 25 ng / ml, 4 to 30 ng / ml, 5 to 10 ng / ml, 5 to 15 ng / ml, 5 to 20 ng / ml, 5 to 25 ng / ml, or 5 to 30 ng / ml. According to some embodiments, the concentration of selenate is 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, 10 ng / ml, 11 ng / ml, 12 ng / ml, 13 ng / ml, 14 ng / ml, 15 ng / ml, 16 ng / ml, 17 ng / ml, 18 ng / ml, 19 ng / ml, 20 ng / ml, 21 ng / ml, 22 ng / ml, 23 ng / ml, 24 ng / ml, 25 ng / ml, 26 ng / ml, 27 ng / ml, 28 ng / ml, 29 ng / ml, or 30 It is ng / ml. According to a preferred embodiment, the concentration of selenite is 5 to 20 ng / ml.

[0162] pyruvate

[0163] According to some embodiments, the culture medium further comprises a pyruvate. Examples of pyruvates include sodium pyruvate and potassium pyruvate. According to a preferred embodiment, the pyruvate is sodium pyruvate.

[0164] According to the present disclosure, the concentration of pyruvate is not particularly limited as long as it can support the long-term proliferation of MSCs. According to some embodiments, the concentration of pyruvate is 0.01 to 10 mg / ml, for example, 0.01 to 1 mg / ml, 0.01 to 2 mg / ml, 0.01 to 3 mg / ml, 0.01 to 4 mg / ml, 0.01 to 5 mg / ml, 0.01 to 7.5 mg / ml, 0.01 to 10 mg / ml, 0.02 to 1 mg / ml, 0.02 to 2 mg / ml, 0.02 to 3 mg / ml, 0.03 to 1 mg / ml, 0.03 to 2 mg / ml, 0.03 to 3 mg / ml, 0.03 to 4 mg / ml, 0.03 to 5 mg / ml, 0.03 to 7.5 mg / ml, 0.03 to 10 mg / ml, 0.05 to 1 mg / ml, 0.05 to 2 mg / ml, 0.05 to 3 mg / ml, 0.05 to 4 mg / ml, 0.05 to 5 mg / ml, 0.05 to 7.5 mg / ml, 0.05 to 10 mg / ml, 0.075 to 1 mg / ml, 0.075 to 2 mg / ml, 0.075 to 3 mg / ml, 0.075 to 4 mg / ml, 0.075 to 5 mg / ml, 0.075 to 7.5 mg / ml, 0.075 to 10 mg / ml, 0.1 to 1 mg / ml, 0.1 to 2 mg / ml, 0.1 to 3 mg / ml, 0.1 to 4 mg / ml, 0.1 to 5 mg / ml, 0.1 to 7.5 mg / ml, 0.1 to 10 mg / ml, 0.25 to 1 mg / ml, 0.25 to 2 mg / ml, 0.25 to 3 mg / ml, 0.25 to 4 mg / ml, 0.25 to 5 mg / ml, 0.25 to 7.5 mg / ml, 0.25 to 10 mg / ml, 0.5 to 1 mg / ml, 0.5 to 2 mg / ml, 0.5 to 3 mg / ml, 0.5 to 4 mg / ml, 0.5 to 5 mg / ml, 0.5 to 7.5 mg / ml, 0.It is 5 to 10 mg / ml, 0.75 to 1 mg / ml, 0.75 to 2 mg / ml, 0.75 to 3 mg / ml, 0.75 to 4 mg / ml, 0.75 to 5 mg / ml, 0.75 to 7.5 mg / ml, 0.75 to 10 mg / ml, 1 to 2 mg / ml, 1 to 3 mg / ml, 1 to 4 mg / ml, 1 to 5 mg / ml, 1 to 7.5 mg / ml, or 1 to 10 mg / ml. According to some embodiments, the concentration of pyruvate is 0.01 mg / ml, 0.03 mg / ml, 0.05 mg / ml, 0.07 mg / ml, 0.09 mg / ml, 0.11 mg / ml, 0.13 mg / ml, 0.15 mg / ml, 0.17 mg / ml, 0.19 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, or 10 mg / ml. According to a preferred embodiment, the concentration of pyruvate is 0.05 to 5 mg / ml.

[0165] Monothioglycerol (MTG)

[0166] According to some embodiments, the culture medium further comprises monothioglycerol (MTG).

[0167] According to the present disclosure, the concentration of MTG is not particularly limited as long as it can support the long-term proliferation of MSCs. According to some embodiments, the concentration of MTG is 1 to 200 μM, for example, 1 to 5 μM, 1 to 10 μM, 1 to 15 μM, 1 to 20 μM, 1 to 25 μM, 1 to 30 μM, 1 to 35 μM, 1 to 40 μM, 1 to 45 μM, 1 to 50 μM, 1 to 60 μM, 1 to 70 μM, 1 to 80 μM, 1 to 90 μM, 1 to 100 μM, 1 to 150 μM, 1 to 200 μM, 2 to 5 μM, 2 to 10 μM, 2 to 15 μM, 2 to 20 μM, 2 to 25 μM, 2 to 30 μM, 2 to 35 μM, 2 to 40 μM, 2 to 45 μM, 2 to 50 μM, 2 to 60 μM, 2 to 70 μM, 2 to 80 μM, 2 to 90 μM, 2 to 100 μM, 2 to 150 μM, 2 to 200 μM, 3 to 5 μM, 3 to 10 μM, 3 to 15 μM, 3 to 20 μM, 3 to 25 μM, 3 to 30 μM, 3 to 35 μM, 3 to 40 μM, 3 to 45 μM, 3 to 50 μM, 3 to 60 μM, 3 to 70 μM, 3 to 80 μM, 3 to 90 μM, 3 to 100 μM, 3 to 150 μM, 3 to 200 μM, 4 to 5 μM, 4 to 10 μM, 4 to 15 μM, 4 to 20 μM, 4 to 25 μM, 4 to 30 μM, 4 to 35 μM, 4 to 40 μM, 4 to 45 μM, 4 to 50 μM, 4 to 60 μM, 4 to 70 μM, 4 to 80 μM, 4 to 90 μM, 4 to 100 μM, 4 to 150 μM, 4 to 200 μM, 5 to 10 μM, 5 to 15 μM, 5 to 20 μM, 5 to 25 μM, 5 to 30 μM, 5 to 35 μM,5 to 40 μM, 5 to 45 μM, 5 to 50 μM, 5 to 60 μM, 5 to 70 μM, 5 to 80 μM, 5 to 90 μM, 5 to 100 μM, 5 to 150 μM, 5 to 200 μM, 10 to 15 μM, 10 to 20 μM, 10 to 25 μM, 10 to 30 μM, 10 to 35 μM, 10 to 40 μM, 10 to 45 μM, 10 to 50 μM, 10 to 60 μM, 10 to 70 μM, 10 to 80 μM, 10 to 90 μM, 10 to 100 μM, 10 to 150 μM, or 10 to 200 It is μM. According to some embodiments, the concentration of MTG is 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 110 μM, 120 μM, 130 μM, 140 μM, 150 μM, or 200 μM. According to preferred embodiments, the concentration of MTG is 10 to 150 μM.

[0168] Nicotinamide (NAM)-based compounds

[0169] According to some embodiments, the culture medium further comprises a nicotinamide-based compound.

[0170] As used herein, "nicotinamide-based compounds" refers to nicotinamide, analogs thereof, metabolites of nicotinamide or nicotinamide analogs, such as NAD, NADH, and NADPH, salts thereof, or mixtures thereof.

[0171] According to an embodiment of the present disclosure, a nicotinamide-based compound may be selected from the group consisting of nicotinamide, nicotinamide analogs, nicotinamide metabolites, nicotinamide analog metabolites, and derivatives thereof.

[0172] Nicotinamide is the amide form of niacin, and both belong to the vitamin B3 family. They are precursors of nicotinamide adenine dinucleotide (NAD), which acts as coenzymes in various cellular processes, including energy metabolism and DNA repair. Nicotinamide can be converted to nicotinamide mononucleotide (NMN) by nicotinamide phosphoribosyltransferase (NAMPT), and then to NAD by nicotinamide mononucleotide adenylyltransferase (NMNAT). + It is converted to.

[0173] As used herein, "nicotinamide analogue" refers to any molecule known to act similarly to nicotinamide. Examples of nicotinamide analogues include, but are not limited to, nicotinthioamide (a thiol analogue of nicotinamide) and nicotinic acid. Examples of nicotinamide derivatives include, but are not limited to, substituted nicotinamide-based compounds and nicotinthioamide, and N-substituted nicotinamide-based compounds and nicotinthioamide.

[0174] Examples of salts of nicotinamide, analogs thereof, metabolites thereof and derivatives thereof include, but are not limited to, nicotinamide hydrochloride, nicotinamide hydrobromide and these salts of substituted nicotinamide.

[0175] According to a preferred embodiment, the nicotinamide-based compound is nicotinamide.

[0176] According to the present disclosure, the concentration of the nicotinamide-based compound is not particularly limited as long as it can support the long-term proliferation of MSCs. According to some embodiments, the concentration of the nicotinamide-based compound is 0.1 to 30 mM, e.g., 0.1 to 5 mM, 0.1 to 10 mM, 0.1 to 15 mM, 0.1 to 20 mM, 0.1 to 25 mM, 0.1 to 30 mM, 0.3 to 5 mM, 0.3 to 10 mM, 0.3 to 15 mM, 0.3 to 20 mM, 0.3 to 25 mM, 0.3 to 30 mM, 0.5 to 5 mM, 0.5 to 10 mM, 0.5 to 15 mM, 0.5 to 20 mM, 0.5 to 25 mM, 0.5 to 30 mM, 0.75 to 5 mM, 0.75 to 10 mM, 0.75 to 15 mM, 0.75 to 20 mM, 0.75 to 25 mM, 0.75 to 30 mM, 1 to 5 mM, 1 to 10 mM, 1 to 15 mM, 1 to 20 mM, 1 to 25 mM, 1 to 30 mM, 2 to 5 mM, 2 to 10 mM, 2 to 15 mM, 2 to 20 mM, 2 to 25 mM, 2 to 30 mM, 3 to 5 mM, 3 to 10 mM, 3 to 15 mM, 3 to 20 mM, 3 to 25 mM, 3 to 30 mM, 4 to 5 mM, 4 to 10 mM, 4 to 15 mM, 4 to 20 mM, 4 to 25 mM, 4 to 30 mM, 5 to 10 mM, 5 to 15 mM, 5 to 20 mM, 5 to 25 The concentration is mM, or 5 to 30 mM. According to some embodiments, the concentration of the nicotinamide-based compound is 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.It is 9 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, or 30 mM. According to a preferred embodiment, the concentration of the nicotinamide-based compound is 0.5 to 20 μM.

[0177] In a specific embodiment, the culture medium comprises 5 to 30 μM of an ethanolamine-based compound, 1 to 20 μM of a putrescine-based compound, 0.1 to 10 volume% of HPLT, 1 to 150 μg / ml of transferrin, 1 to 10 μg / ml of an insulin-based compound, 1 to 150 μg / ml of an antioxidant, and 0.5 to 5 volume% of glutamine or a derivative thereof in the base medium.

[0178] In a specific embodiment, the culture medium comprises 5 to 30 μM of ethanolamine, 1 to 20 μM of putrescine dihydrochloride, 0.1 to 10 volume% of HPLT, 1 to 150 μg / ml of transferrin, 1 to 10 μg / ml of insulin, 1 to 150 μg / ml of ascorbate, and 0.5 to 5 volume% of glutamine in the base medium.

[0179] In a specific embodiment, the culture medium comprises 5 to 30 μM of ethanolamine, 1 to 20 μM of putrescine dihydrochloride, 0.1 to 10 vol% of HPLT, 1 to 150 μg / ml of transferrin, 1 to 10 μg / ml of insulin, 1 to 150 μg / ml of ascorbate, 0.5 to 5 vol% of glutamine, 1 to 15 ng / ml of FGF2, 0.5 to 5 μM of hydrocortisone, 1 to 10 μM of lipoic acid, 1 to 30 μM of FeSO4, and 0.1 to 10 μM of Fe(NO3)3 in the base medium.

[0180] In a specific embodiment, the culture medium comprises, in addition to the base medium, 5 to 30 μM ethanolamine, 1 to 20 μM putrescine dihydrochloride, 0.1 to 10 volume% HPLT, 1 to 150 μg / ml transferrin, 1 to 10 μg / ml insulin, 1 to 150 μg / ml ascorbate, 0.5 to 5 volume% glutamine, 1 to 15 ng / ml FGF2, 0.5 to 5 μM hydrocortisone, 5 to 20 ng / ml Na2SeO3, 0.05 to 5 mg / ml sodium pyruvate, 1 to 10 mg / ml HSA, 10 to 150 μM MTG, 0.5 to 20 mM nicotinamide (NAM), and 10 to 100 μg / ml heparin It contains sodium.

[0181] In certain embodiments, the culture medium has a high seeding density (e.g., 5×10⁶). 4 It provides about 13 to 15-fold proliferation dilutions for MSCs in cells / well. In certain embodiments, the culture medium provides a high seeding density (e.g., 5×10⁶). 4 It provides approximately 15 to 30-fold proliferation dilutions for MSCs at cells / well. In contrast, the control medium provides the same high seeding density (e.g., 5×10⁶). 4 It provides an approximately 8 to 10-fold proliferation factor for MSCs in cells / well.

[0182] In certain embodiments, the culture medium has a low seeding density (e.g., 1×10⁶). 3 About 650 to about 1×10⁶ MSCs per cell / well 3 It provides a proliferation dilution of the embryo. In certain embodiments, the culture medium has a low seeding density (e.g., 1 × 10⁶). 3 Approximately 1×10⁶ MSCs per cell / well 3 Pear to about 4.5×10 3 It provides a proliferation dilution of the embryo. In contrast, the control medium has the same low seeding density (e.g., 1×10⁶). 3 Approximately 500 to 600-fold proliferation factor for MSCs in cells / well Provides

[0183] In a specific embodiment, the culture medium has a flat PDT curve from about 20 hours to about 24 hours, stably supporting the long-term proliferation of MSCs. In contrast, the control medium provides a steep PDT curve for MSCs from about 25 hours to about 57 hours during long-term proliferation.

[0184] 2. Kit

[0185] According to one aspect of the present invention, a kit comprising the culture medium as disclosed herein is provided. The culture medium may be packaged in a suitable packaging material, such as a container. The kit may optionally include specifications of the culture medium or components in the culture medium, or instructions for using the culture medium or components therein.

[0186] The container of the kit may generally include a vial, flask, bottle, or any other container in which the ingredients can be appropriately positioned. If the kit contains multiple ingredients, the kit may also include a second, third, or other additional container in which the additional ingredients can be positioned individually. However, a combination of various ingredients may be contained in a single container.

[0187] In a specific embodiment, the culture medium is packaged in a container. In a specific embodiment, the components of the culture medium are each individually packaged in different containers.

[0188] The components of the kit may be provided as one or more liquid solutions. However, the component(s) of the kit may also be provided as dry powder(s). If the component(s) are provided as dry powder(s), the powder(s) can be reconstituted by adding a suitable solvent.

[0189] 3. Propagation Method

[0190] According to one aspect of the present disclosure, a method for proliferating mesenchymal stem cells (MSCs) is provided, comprising the step of bringing the MSCs into contact with a culture medium as disclosed herein.

[0191] In certain embodiments, the method uses about 500 MSCs / well to about 1×10⁻⁶ 5 The method further includes a step of seeding at a density of cells / well. In certain embodiments, the method contains about 5×10 MSCs. 4 dog Cells / well to about 1×10 5 dog The method further includes a step of seeding at a high density of cells / well. In certain embodiments, the method is about 500 cells / well to about 1 × 10⁶ 3 dog It additionally includes a step of seeding MSCs at a low density of cells / wells.

[0192] In a specific embodiment, the method further includes the step of replacing the growth medium with the same medium every few days during growth. In a specific embodiment, the method further includes the step of replacing the growth medium with the same medium every three days during growth.

[0193] In certain embodiments, MSCs are propagated continuously over several passages. In certain embodiments, MSCs are propagated continuously over at least two passages. In certain embodiments, MSCs are propagated continuously over at least three passages. In certain embodiments, MSCs are propagated continuously over at least four passages. In certain embodiments, MSCs are propagated continuously over at least five passages. In certain embodiments, MSCs are propagated continuously over at least six passages. In certain embodiments, MSCs are propagated continuously over at least seven passages. In certain embodiments, MSCs are propagated continuously over at least eight passages. In certain embodiments, MSCs are propagated continuously over at least nine passages.

[0194] According to the present disclosure, mesenchymal stem cells (MSCs) produced by the method disclosed herein maintain a substantially homogeneous cell population even when proliferated continuously through at least 4, 7, or 9 passages. Specifically, mesenchymal stem cells produced by the method disclosed herein can maintain MSC characteristics such as morphology, phenotype, differentiation ability, and immunomodulatory effects even when MSCs are proliferated continuously over multiple passages.

[0195] 4. Cell population

[0196] According to one aspect of the present disclosure, a substantially homogeneous group of MSCs produced by the method of the present disclosure is provided.

[0197] According to some embodiments, the substantially homogeneous MSC population is a cell population of at least passage 2 (P2), at least passage 4 (P4), at least passage 7 (P7), or at least passage 9 (P9). According to some embodiments, the substantially homogeneous MSC population is a cell population of passage 2, passage 3, passage 4, passage 5, passage 6, passage 7, passage 8, or passage 9.

[0198] According to specific embodiments, the MSC population of the present disclosure may be cryopreserved or stored for further proliferation and / or differentiation.

[0199] According to the present disclosure, substantially homogeneous MSC populations have higher CD90 during long-term proliferation + It has a cell ratio. In certain embodiments, a substantially homogeneous MSC population has at least 90% CD90 + It includes cells. In certain embodiments, a substantially homogeneous MSC population comprises at least 95% CD90 + It includes cells. In certain embodiments, a substantially homogeneous MSC population comprises at least 99% CD90 + The cells comprise. In certain embodiments, a substantially homogeneous MSC population comprises 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of CD90 + It includes cells.

[0200] Examples

[0201] The following examples illustrate some embodiments of the present invention. However, it should be understood that these examples are for illustrative purposes only and are not intended to completely limit the conditions or scope of the present invention.

[0202] Furthermore, unless otherwise described in detail, the following embodiments were performed using standard techniques well known to those skilled in the art and common practice. As previously mentioned, the following embodiments are presented for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

[0203] ingredient

[0204] All reagents and devices used throughout the embodiments of this disclosure are commercially available. Specific reagents used in the embodiments are listed below.

[0205]

[0206] Examples 1-4

[0207] These examples were performed to demonstrate that HPLT is important for supporting the long-term proliferation of iMSCs.

[0208] Example 1

[0209] Cryopreserved iMSCs (RC01005, Nuwacell Co., Ltd.) were seeded in M1 medium at a rate of 1,000 cells per well in 6-well plates for cell thawing. After 24 hours, the medium was replaced with 2 ml per well of M1, SFM-M, PPRF, or MEM-FBS medium. M1 medium contained IMDM(+NaHCO3) / F12 (50% / 50%), 2 volume% Ultroser G (serum substitute (SR)), 1 volume% Glutamax, 1-150 μg / ml (e.g., 120 μg / ml) of transferrin, 5 μg / ml of insulin, 1-150 μg / ml (e.g., 110 μg / ml) of Mg ascorbate, 20 μM of ethanolamine, and 10 μM of putrescine 2HCl. SFM-M contains IMDM / F12 (50% / 50%), 1 vol% Glutamax, 5 μg / ml insulin, 5 μg / ml transferrin, 50 μg / ml Mg ascorbate, 4 mg / ml HSA, 50 nM 2-mercaptoethanol, 0.10% chemically defined lipids, 1 μg / ml fibronectin, 100 μg / ml pluronic F-68, 50 ng / ml hydrocortisone, 15 ng / ml progesterone, 10 IU / ml heparin sodium, 2 μg / ml serotonin, 10 ng / ml EGF, 10 ng / ml FGF2, 10 ng / ml PDGF-BB, 10 ng / ml IGF-1, and 1 ng / ml of It contained IL-3 and 1 ng / ml of GM-SCF. The PPRF medium contained F12, 2 volume% of Glutamax, 25 μg / ml of insulin, 25 μg / ml of transferrin, 50 μg / ml of Mg ascorbate, 55.9 μM of putrescine 2HCl, 20.5 mM of NaHCO3, 4.9 mM of HEPES, 4 mg / ml of HSA, 0.10% of chemically defined lipids, 36 ng / ml of hydrocortisone, 5.66 ng / ml of progesterone, 2 ng / ml of FGF2, and 1 ng / ml of TGFβ1.The MEM-FBS medium contained 90% (v / v) αMEM and 10% (v / v) FBS. Subsequently, during cell culture, the medium was replaced with the same medium every 3 days. On the 10th day of culture, cells were observed and photographs were taken using a phase-contrast microscope. The cell morphology of iMSCs cultured in the different culture media above is shown in Figure 1.

[0210] As shown in Figure 1, cells cultured in M1 and MEM-FBS proliferated well, but cells cultured in other media (SFM-M and PPRF) did not proliferate. In the above examples, the inventors demonstrated that M1 and MEM-FBS media can support iMSC proliferation; however, Ultroser G, which is included as a main component in M1, contains animal-derived components, and FBS, which is included in MEM-FBS media, is also animal-derived serum, making them unsuitable choices for clinical application. Therefore, developing serum-free and exogen-free media for MSC (e.g., iMSC) proliferation will have significant clinical implications.

[0211] Example 2

[0212] Cryopreserved iMSCs (RC01005, Nuwacell Co., Ltd.) were seeded in M1 medium at a density of 50,000 cells per well in 6-well plates for cell thawing. After 24 hours, the medium was replaced with 2 ml of M1 or M2 medium per well. M1 medium had the same composition as described in Example 1 and was used as a control. M2 medium had the same composition as M1 medium except that HPLT was substituted for Ultroser G. M2 medium contained HPLT at indicated concentrations (1%, 2%, 3%, 4%, or 5% by volume). On the 4th day of culture, cells were digested with solase (RP01021, Nuwacell Co., Ltd.) for 3 minutes, collected, centrifuged, and the supernatant removed and resuspended in the corresponding medium. Then, the number of iMSC cells was counted using a cell viability analyzer (Vi-CELL™XR, BECKMAN COULTER). Before digestion, cells were observed and photographs were taken using a phase-contrast microscope. The cell morphology of the iMSCs is shown in Figure 2A, and the number of iMSCs is shown in Figure 2B.

[0213] As shown in Fig. 2A, iMSC cells cultured in M2 containing HPLT were confirmed to proliferate well and were able to maintain their original morphological characteristics (such as spindle-shaped fibroblasts), similar to those cultured in M1 medium. As shown in Fig. 2B, the number of iMSC cells cultured in M2 containing HPLT was significantly higher than that of iMSC cells cultured in M1 using Ultroser G (the former being approximately 1.5 to 3 times that of the latter), indicating that replacing Ultroser G with HPLT not only avoids the use of animal-derived components but can also significantly improve the proliferation efficiency of iMSC cells. Furthermore, Fig. 2B showed that differences in HPLT concentration can have some influence on the proliferation efficiency of iMSCs.

[0214] Example 3

[0215] Cryopreserved iMSCs (RC01005, Nuwacell Co., Ltd.) were seeded in M1 medium at a density of 50,000 cells per well in 6-well plates for cell thawing. After 24 hours, the medium was replaced with 2 ml per well of either M1 or M2 medium (3% HPLT). M1 medium had the composition as described in Example 1 and was used as a control. M2 (3% HPLT) had the same composition as described in Example 2. The iMSCs were cultured and subcultured sequentially. The number of cells in each passage from the 6th to the 8th passage was counted using a cell viability analyzer (Vi-CELL™XR, BECKMAN COULTER) according to the protocol described in Example 2 (Fig. 3A).

[0216] iMSCs from passages 1, 4, 5, and 7 were stained with CD90-FITC antibody (BD Pharminge, 555595), and CD90 by flow cytometry + The cell ratio was measured (Fig. 3B). Briefly, cells were digested with solase (RP01021, Nuwacell Co., Ltd.) for 3 minutes, resuspended in analysis buffer (0.5% BSA and 0.5 mM EDTA in DPBS), and 5 × 10⁶ cells per tube 5 inside 1×10 6 Adjusted to the density of dog cells. CD90-FITC After incubating with the antibody at 4°C for 30 minutes, the sample was washed with analysis buffer, the analysis buffer was removed, the sample was resuspended in 100 μl of analysis buffer, and CD90 was analyzed using a flow cytometer (CytoFLEX, BECKMAN COULTER). + The percentage of cells was analyzed.

[0217] As shown in Fig. 3A, iMSCs cultured in M2 had higher cell numbers than iMSCs cultured in M1 for all P6, P7, and P8 cells, indicating that replacing Ultroser G with HPLT can improve proliferation efficiency for each passage of iMSC cells, particularly for higher passages. As shown in Fig. 3B, when iMSCs were cultured using M1, CD90-expressing cells decreased from 99.81% in P1 to 88.87% in P4, 70.04% in P5, and 44.37% in P7. It decreased sharply, indicating that M1 was unable to stably maintain the stem cell phenotype of iMSCs during long-term proliferation. In particular, CD90-expressing cells decreased more rapidly with long-term iMSC proliferation. On the other hand, when iMSCs were cultured using M2, the proportion of CD90-expressing cells remained more stable from P1 to P7. Therefore, the data in Figures 3A and 3B demonstrate that replacing Ultroser G with HPLT improves the proliferation efficiency of cells at each passage and allows for more stable maintenance of the iMSC stem cell phenotype during long-term proliferation. Additionally, when iMSCs were cultured using M2, a small peak appeared partially on the left side due to long-term iMSC proliferation (Figure 3B).

[0218] Example 4

[0219] The immunosuppressive effects of iMSCs were evaluated using a CFSE-based T cell proliferation assay. To proliferate T cells, PBMCs were activated with immobilized CD3 antibody (biogerm, 05121-25-100) and CD28 antibody (biogerm, 10311-25-100) for 4 days in TPA medium (T cell proliferation assay medium, RPMI 1640 + 10%(v / v) FBS + 1%(v / v) Glutamax) supplemented with 100 IU / ml IL-2 (Jiangsu Jinsili Pharmaceutical Co., Ltd, China). After collecting the activated T cells, they were stained with CFSE (Ab145291, Abcam) and co-cultured for 5 days with or without the two types of iMSCs cultured according to Example 3. Specifically, iMSCs from passage 3 were treated with mitomycin C prior to co-culture with T cells to block cell proliferation. Subsequently, T cells were added to the treated iMSCs in a 1:1 ratio. Cell morphology on day 5 of co-culture is shown in Fig. 4A. The percentage of the divided T cell population (dark gray, CFSE-low population) was detected by a flow cytometer according to the protocol described in Example 3 (Fig. 4B).

[0220] As shown in Fig. 4A, cell division of T cells cultured without iMSCs was highly activated, resulting in a significant decrease in CFSE fluorescence intensity (peak shifted to the left), whereas the proliferation of T cells cultured with iMSCs was significantly inhibited (almost no shift of the peak) (Fig. 4B). Furthermore, as confirmed by the proportion of the divided T cell population, iMSCs cultured in M2 exhibited an immunosuppressive effect similar to that cultured in M1. Therefore, the data in Fig. 4 indicate that P3 iMSCs cultured in M2 maintained an immunosuppressive effect on T cells.

[0221] Examples 1 to 4 above show that although M1 medium containing ethanolamine and putrescine 2HCl could support the proliferation of iMSCs, it could not stably maintain the stem cell phenotype (CD90 expression) of iMSCs during long-term proliferation due to a lack of HPLT, whereas M2 medium containing all of ethanolamine, putrescine 2HCl, and HPLT could stably maintain the stem cell phenotype (CD90 expression) of iMSCs during long-term proliferation. This indicates that HPLT is essential for stably maintaining the stem cell phenotype of iMSCs during long-term proliferation.

[0222] In addition, compared to M1 medium, M2 medium was able to promote potent iMSC proliferation and provide a similar immunosuppressive effect on iMSCs without using animal-derived components.

[0223] In summary, when combining the results of Examples 1 to 4, M2 was found to support the long-term proliferation of iMSCs without losing MSC characteristics (including morphology, phenotype, differentiation potential, and immunomodulatory effects), which suggests high clinical applicability of M2.

[0224] Example 5

[0225] This embodiment is It was performed to show that FGF2 and / or hydrocortisone can further promote iMSC proliferation.

[0226] Cryopreserved iMSCs (RC01005, Nuwacell Co., Ltd.) were seeded in M1 medium at a rate of 1,000 cells per well in 6-well plates for cell thawing. After 24 hours, the medium was replaced at a rate of 2 ml per well with M1 medium or M2 medium (5% HPLT) supplemented with or without one or more growth factors (F: FGF2 (10 ng / ml, Nuwacell Co., Ltd.); P: PDFG-BB (10 ng / ml, Cat. No.: 100-14B-100, Peprotech); T: TGFβ1 (1 ng / ml, Cat. No.: 100-21, Peprotech)) in the presence or absence of hydrocortisone (1 μM, Cat. No.: 4098, Tocris). M1 medium had the composition as described in Example 1 and was used as a control. M2 (5% HPLT) had the same composition as described in Example 2. The medium was replaced with the same medium every 3 days. On the 10th day of culture, cells were digested with solase (RP01021, Nuwacell Co., Ltd.) for 3 minutes, collected, centrifuged, the supernatant was removed, and the cells were resuspended in the corresponding medium. Then, the number of iMSC cells was counted using a cell viability analyzer (Vi-CELL™XR, BECKMAN COULTER) (Fig. 5).

[0227] As shown in Figure 5, regardless of the presence of hydrocortisone, the number of iMSCs cultured in M2 (5% HPLT) supplemented with FGF2 alone, a combination of FGF2 and PDFG-BB or TGFβ1, or a combination of FGF2, PDFG-BB, and TGFβ1 was not only significantly higher than that of iMSCs cultured in M1 (the former being about 4.5 to 9 times the latter), but was also superior compared to iMSCs cultured in M2 (5% HPLT) without added growth factors (the former being about 2.3 to 3.75 times the latter), demonstrating that the addition of one or more growth factors can clearly promote the proliferation of iMSCs. Furthermore, the addition of hydrocortisone was able to promote iMSC proliferation, particularly in the presence of the presented growth factors, and the addition of hydrocortisone clearly promoted iMSC proliferation.

[0228] Examples 6-10

[0229] This example was performed to show that ethanolamine and putrescine 2HCl are important for supporting the long-term proliferation of iMSCs.

[0230] Example 6

[0231] Cryopreserved iMSCs (RC01005, Nuwacell Co., Ltd.) were seeded into 6-well plates in M1 medium at a density of 1,000 cells per well for cell thawing. After 24 hours, the medium was replaced with 2 ml per well of I, II, III, IV, V, or M1 medium. The M1 medium had the composition as described in Example 1. Medium I is basic IMDM(+NaHCO3)(50%), Ham's F12(50%), 1-150 μg / ml (e.g., 110 μg / ml) of Mg ascorbate, 20 μM ethanolamine, 23.2 nM progesterone, 10 μM putrescine 2HCl, 5 μg / ml insulin, 1-150 μg / ml (e.g., 120 μg / ml) of transferrin, 100 μM monothioglycerol (MTG), 0.1 vol% of chemically defined lipids, 0.1 vol% of trace element A (1000x), 0.1 vol% of trace element B (1000x), 1 μM hydrocortisone, 1 vol% glutamate, 10 ng / ml of FGF2, 10 ng / ml of PDGF-BB, and contained 4 mg / ml of HSA. Medium II had the same composition as Medium I except that 10 volume% of KOSR was added. Medium III had the same composition as Medium I except that 2 volume% of B27 was additionally added. Medium IV had the same composition as Medium I except that 2 volume% of B27 and 1 volume% of N2 were additionally added. Medium V contained basic IMDM(+NaHCO3) (100%), 1-150 μg / ml (e.g., 110 μg / ml) of Mg ascorbate, 5 μg / ml of insulin, 1-150 μg / ml (e.g., 120 μg / ml) of transferrin, 1 μM of hydrocortisone, 4 ng / ml of FGF2, 1-10 μM (e.g., 10 μM) of lipoic acid, 1-30 μM (e.g., 30 μM) of FeSO4, 0.1-10 μM (e.g., 10 μM) of Fe(NO3)3, and 20 vol% of BIT9500. The medium was replaced with the same medium every 3 days.The morphology of the cells on the 10th day of culture is shown in Fig. 6A, and the number of cells was counted using a cell viability analyzer (Vi-CELL™XR, BECKMAN COULTER) according to the protocol described in Example 5 (Fig. 6B).

[0232] As shown in Figure 6, among media I, II, III, IV, and V, only medium V (also called EPM) was able to support iMSC proliferation and was superior to M1. Like M1, medium V also contains the serum substitute BIT9500, which is not a suitable choice for clinical application.

[0233] Example 7

[0234] Cryopreserved ucMSCs (RC02003, Nuwacell Co., Ltd.) and iMSCs (RC01005, Nuwacell Co., Ltd.) were seeded in M1 medium at a density of 50,000 cells per well in 6-well plates for cell thawing. After 24 hours, the medium was replaced with 2 ml of M1 or M3 medium per well. M1 medium had the composition as described in Example 6 and was used as a control. M3 medium had the same composition as the EPM medium described in Example 6, except that HPLT was used instead of BIT9500. M3 medium contained HPLT at the suggested concentrations (1%, 2%, 3%, 4%, or 5% by volume). On day 4 of culture, when the cells had confluenced, the number of iMSC cells was counted using a cell viability analyzer (Vi-CELLTMXR, BECKMAN COULTER) according to the protocol described in Example 2.

[0235] As shown in Figure 7, the number of ucMSCs or iMSCs cultured in M3 containing HPLT was significantly higher than that of ucMSCs or iMSCs cultured in M1 containing Ultroser G. Therefore, HPLT was found to completely replace BIT9500 in EPM and promote the proliferation of both ucMSCs and iMSCs in a concentration-dependent manner. This demonstrates that M3 medium can support the proliferation of MSCs (iMSCs and ucMSCs) due to the presence of HPLT.

[0236] Example 8

[0237] Cryopreserved iMSCs (RC01005, Nuwacell Co., Ltd.) were seeded in M1 medium at a density of 50,000 cells per well in 6-well plates for cell thawing. After 24 hours, the medium was replaced with 2 ml per well using M1, M3 (3% HPLT), and M4 media. M1 and M3 (3% HPLT) media had the same composition as presented in Example 7 and were used as controls. Compared to M3 (3% HPLT), M4 had the same composition except for the additional addition of 10 μM putrescine 2HCl and 20 μM ethanolamine. On the 4th day of culture, when the cells had confluenced, the number of iMSC cells was counted using a cell viability analyzer (Vi-CELLTMXR, BECKMAN COULTER) according to the protocol described in Example 2.

[0238] As shown in Figure 8, the number of iMSCs cultured in M4 was not only significantly higher than that of iMSCs cultured in M1 (the former being about 1.5 times the latter), but also slightly higher than that of iMSCs cultured in M3, which indicates that M4 can support iMSC proliferation.

[0239] Example 9

[0240] Cryopreserved iMSCs (RC01005, Nuwacell Co., Ltd.) were seeded in M1 medium at a density of 50,000 cells per well in 6-well plates for cell thawing. After 24 hours, the medium was replaced with 2 ml per well of M1, M3 (3% HPLT), or M4 medium. The iMSCs were cultured and subcultured 4 times in the presented media. M1, M3 (3% HPLT), and M4 had the same composition as in Example 8. Cells from P1 or P4 were stained with CD90-FITC antibody (BD Pharminge, 555595) and CD90 was analyzed using a flow cytometer according to the protocol described in Example 3. + The percentage of cells was detected.

[0241] Surprisingly, a high percentage of CD90 + As demonstrated by the cells, M4 maintained the stem cell phenotype of iMSCs better than M3 as well as M1 (Fig. 9). Specifically, as shown in Fig. 9, CD90-expressing cells cultured with M1 decreased sharply from 98.63% at P1 to 89.67% at P4, and CD90-expressing cells cultured with M3 decreased sharply from 96.88% at P1 to 80.74% at P4, suggesting that M1 and M3 failed to stably maintain the stem cell phenotype of iMSCs during long-term proliferation. On the other hand, the proportion of CD90-expressing cells cultured with M4 remained more stable from P1 to P4. Therefore, the data in Fig. 9 indicate that the addition of ethanolamine and putrescine 2HCl can stably maintain the stem cell phenotype of iMSCs during long-term proliferation.

[0242] Example 10

[0243] Cryopreserved iMSCs (RC01005, Nuwacell Co., Ltd.) were seeded in M1 medium at a density of 50,000 cells per well in 6-well plates for cell thawing. After 24 hours, the medium was replaced with 2 ml per well of either M1 or M3 (3% HPLT) medium. M1 and M3 (3% HPLT) had the same composition as in Example 9. After culturing iMSCs (RC01005, Nuwacell Co., Ltd.) in M3 (3% HPLT) medium, they were subcultured four times, and characterization was performed as follows:

[0244] 1. Osteogenesis

[0245] P3 iMSCs cultured in M3 (3% HPLT) were seeded into 6-well plates. When the culture medium reached 90% confluence, the medium was replaced with osteogenic differentiation medium (αMEM medium (Cat No.: 11900-024, thermo)) supplemented with 20% by volume of FBS (Cat No.: SH30071.03, Hyclone), 1% Glutamax, 50 μg / ml ascorbic acid, and 5 mM β-glycerophosphate (Cat No.: G9422, sigma). The medium was replaced with the same medium every 3 days until day 21. On day 21, osteogenic differentiation cells were seeded with Alizarin Red (Cat No.: 0223, ScienCell TM )as Stained and cell morphology are shown in Fig. 10A (middle panel).

[0246] 2. Chondrogenesis

[0247] 5×10 iMSCs cultured in M3 (3% HPLT) 5The cells were suspended in chondrogenic differentiation medium (DMEM-HG medium (Cat. No.: 11960-069, Thermo)) supplemented with 1 μM ascorbic acid, 100 nM dexamethasone (Cat. No.: D8893, Sigma), 1% ITS+ premix tissue culture supplement (Cat. No.: 354352, Corning), 10 ng / ml TGFβ1 (Cat. No.: 100-21-6, PeproTech), and 1% sodium pyruvate (Cat. No.: 11360-070, Thermo) with the lid open in a 15 mL centrifuge tube. The medium was replaced with the same medium every 3 days until day 28. On day 28, cell spheres were stained with Alcian Blue (Cat. No.: A105505, aladdin), and cell morphology is shown in Fig. 10A. Shown (right panel).

[0248] 3. Fat differentiation

[0249] iMSCs cultured in M3 (3% HPLT) were seeded into 6-well plates. When the culture medium reached 90% confluence, the medium was replaced with adipogenesis medium (DMEM-HG medium supplemented with 45 μM IBMX (Cat. No.: I5879, Sigma), 0.5 μM dexamethasone (Cat. No.: D8893, Sigma), 50 μM indomethacin (Cat. No.: I7378, Sigma), and 10% FBS). The medium was replaced with the same medium every 3 days until day 21. On day 21, adipogenesis cells were stained with Oil red O (Cat. No.: O0625, Sigma Aldrich), and cell morphology is shown in Fig. 10A (left panel).

[0250] The immunosuppressive effect of the iMSC was evaluated by performing a CFSE-based T cell proliferation assay according to the protocol described in Example 4 (Fig. 10B).

[0251] P3 iMSCs cultured in M3 (3% HPLT) medium were found to retain MSC characteristics, including triple lineage differentiation (Fig. 10A) and immunosuppressive effects (Fig. 10B).

[0252] Examples 6 to 10 above show that while M3 medium containing HPLT was able to support iMSC proliferation, it failed to stably maintain the iMSC stem cell phenotype (CD90 expression) during long-term proliferation due to a lack of ethanolamine and putrescine 2HCl, whereas M4 medium containing HPLT, ethanolamine, and putrescine 2HCl was able to more stably maintain the iMSC stem cell phenotype (CD90 expression) during long-term proliferation. This demonstrates that ethanolamine and putrescine 2HCl were important for stably maintaining the iMSC stem cell phenotype (CD90 expression) during long-term proliferation.

[0253] In addition, compared to M1, M4 was able to promote potent iMSC proliferation and provide an immunosuppressive effect on iMSCs without the use of animal-derived components.

[0254] In summary, when combining the results of Examples 6 to 10, it was found that M4 supports the long-term proliferation of iMSCs without losing the characteristics of iMSCs (including morphology, phenotype, differentiation potential, and immunomodulatory effects), which suggests high clinical applicability of M4.

[0255] Combining Examples 1 to 4 and Examples 6 to 10, it was confirmed that ethanolamine, putrescine 2HCl, and HPLT synergistically support the long-term proliferation of iMSCs.

[0256] Examples 11-13

[0257] This embodiment is It was performed to show that FGF2, HSA and / or heparin sodium can further promote iMSC proliferation.

[0258] Example 11

[0259] For example, M3 medium supplemented with 5% HPLT was used to verify the effect of FGF2 on iMSC proliferation. Cryopreserved iMSCs (RC01005, Nuwacell Co., Ltd.) were seeded in M1 medium at a density of 50,000 cells per well in 6-well plates for cell thawing. After 24 hours, the medium was replaced at a density of 2 ml per well with M1 medium, or with M3 medium (supplemented with 5% HPLT) without or additionally supplemented with FGF2 (Nuwacell Co., Ltd.) at different concentrations. M1 medium had the composition as described in Example 7. M3 medium (supplemented with 5% HPLT) had the same composition as that of Example 7. On the 4th day of culture, when the cells had confluenced, the number of iMSC cells was counted using a cell viability analyzer (Vi-CELL™XR, BECKMAN COULTER) according to the protocol described in Example 2.

[0260] As shown in Fig. 11, the number of iMSCs proliferated in M3 (5% HPLT) supplemented with additional FGF2 was significantly higher than that of iMSCs proliferated in M3 (5% HPLT) without FGF2 (the former being about 3.1 times or about 3.9 times the latter). Similar to the results in Fig. 5, FGF2 was also confirmed to significantly promote iMSC proliferation (Fig. 11).

[0261] Example 12

[0262] Considering that HPLT is expensive and its supply is limited, HSA (Chengdu Rongsheng Pharmaceuticals Co., Ltd., China) was added to reduce the amount of HPLT. For example, to verify the effect of HSA on iMSC proliferation, M3 medium containing a low concentration of HPLT (e.g., 0.5% HPLT) was used. Cryopreserved iMSCs (RC01005, Nuwacell Co., Ltd.) were seeded in M1 medium at a density of 1,000 cells per well in 6-well plates for cell thawing. The M1 medium had the composition as described in Example 6. After 24 hours, the medium was replaced at a rate of 2 ml per well with M3 medium (0.5% HPLT) supplemented with 4 mg / L untreated HSA (no dialysis) or dialyzed HSA, or without supplementation. M3 (0.5% HPLT) had the same composition as the M3 in Example 7, except for the concentration of HPLT. HSA dialysis was performed by placing HSA into a dialysis bag and immersing the dialysis bag in a DPBS (C14190500BT, Gibco) solution at 4°C overnight, with an HSA to DPBS ratio of 1:50 (v:v) and a pore size of 15 KD for the bag. The medium was replaced with the same medium every 3 days. The cell morphology on the 10th day of culture is shown in Fig. 12A, and the number of cells was counted using a cell viability analyzer (Vi-CELL™XR, BECKMAN COULTER) according to the protocol described in Example 5 (Fig. 12B).

[0263] As shown in Fig. 12A, compared to iMSC cells cultured in M3 (0.5% HPLT) without HSA, iMSC cells cultured in untreated or dialyzed HSA-containing M3 (0.5% HPLT) were found to retain their original morphological characteristics (such as spindle-shaped fibroblasts). In particular, iMSC cells cultured in M3 (0.5% HPLT) containing dialyzed HSA proliferated much better. Furthermore, the effect of HSA, particularly dialyzed HSA, on promoting the proliferation of iMSC cells was demonstrated through cell number analysis results (Fig. 12B). As shown in Fig. 12B, the number of iMSC cells cultured in M3 (0.5% HPLT) containing dialyzed HSA was approximately 2.3 times higher than the number of iMSC cells cultured in M3 (0.5% HPLT) containing untreated HSA.

[0264] Example 13

[0265] As an example, the effects of dialyzed HSA and heparin sodium on iMSC proliferation using M3 medium containing HPLT were further verified. Cryopreserved iMSCs (RC01005, Nuwacell Co., Ltd.) were seeded at 1,000 cells per well in 6-well M1 plates for cell thawing. M1 medium had the composition as described in Example 6. After 24 hours, the medium was replaced at a rate of 2 ml per well with M1 medium, or with M3 medium supplemented with 4 mg / L dialyzed HSA and 25 μg / ml heparin sodium (Cat. No.: A16198, Thermo Fisher), which had different HPLT concentrations. The medium was replaced with the same medium every 3 days. On the 10th day, the number of cultured cells was counted using a cell viability analyzer (Vi-CELL™XR, BECKMAN COULTER) according to the protocol described in Example 5. The results are shown in Fig. 13.

[0266] As confirmed by comparing the results of Fig. 13 with the results of Fig. 7 (iMSC histogram), the addition of dialyzed HSA could significantly lower the concentration of HPLT required for iMSC growth promotion. Additionally, Fig. 13 shows that heparin sodium can further promote iMSC growth.

[0267] In summary, Examples 11 to 13 show that the addition of HSA (particularly dialyzed HSA), FGF2 and / or heparin sodium can further promote iMSC proliferation, and that the addition of dialyzed HSA can significantly lower the HPLT concentration required to promote iMSC growth.

[0268] Examples 14-17

[0269] These embodiments were performed to further develop M5, a serum-free and exogen-free MSC proliferation medium, in order to more continuously support the long-term proliferation of iMSCs.

[0270] Example 14

[0271] Cryopreserved iMSCs (RC01005, Nuwacell Co., Ltd.) were seeded into 6-well plates in M1 medium at a density of 1,000 cells per well for cell thawing. The M1 medium had the composition as described in Example 1. After 24 hours, the medium was replaced with 2 ml per well of M5 medium containing HPLT or M5 medium containing HPLT at various concentrations (0.50%, 1%, 2%, or 3% by volume). M5 medium contains IMDM+DMEM / F12 (50% / 50%), 1 vol% Glutamax, 1-150 μg / ml (e.g., 120 μg / ml) Transferin, 5 μg / ml Insulin, 1-150 μg / ml (e.g., 110 μg / ml) Mg Ascorbate, 20 μM Ethanolamine, 10 μM Putrescine 2HCl, HPLT at suggested concentrations (0%, 0.5%, 1%, 2%, and 3 vol%), 4 ng / ml FGF2, 1 μM Hydrocortisone, 14 ng / ml Na2SeO3, 0.11 mg / ml Sodium Pyruvate, 2 mg / ml HSA, 50 μM MTG, 0.5-20 mM (e.g., 20 mM) Nicotinamide, and It contained 50 μg / ml of heparin sodium. iMSCs were cultured in the above medium for 10 days, and the medium was replaced with the same medium every 3 days. The number of cells was counted using a cell viability analyzer (Vi-CELL™XR, BECKMAN COULTER) according to the protocol described in Example 5.

[0272] As shown in Figure 14, similar to M2, the number of iMSC cells cultured in M5 containing HPLT was significantly higher than the number of iMSC cells cultured in M5 without HPLT (the former was about 6, 10, 13, or 14 times that of the latter). Likewise, HPLT was found to promote iMSC proliferation in M5.

[0273] Example 15

[0274] Cryopreserved iMSCs (RC01005, Nuwacell Co., Ltd.) were seeded in M1 medium at a rate of 50,000 cells per well in 6-well plates for cell thawing. After 24 hours, the medium was replaced with 2 ml per well of M1, M4, or M5 medium. M1 medium had the composition as described in Example 14 and was used as a control. M4 medium had the composition as described in Example 8. M5 medium had the composition as described in Example 14. The iMSCs were cultured and subcultured in the presented medium up to passage 9. On the 4th day of each passage, when the cells confluenced, the number of cells was counted using a cell viability analyzer (Vi-CELL™XR, BECKMAN COULTER) according to the protocol described in Example 2 (Fig. 15A). The cell morphology of passage 8 is shown in Fig. 15B.

[0275] At a high passage count (P8), iMSCs cultured in M4 and M5 maintained better original morphological characteristics of MSCs (similar to spindle-shaped fibroblasts), whereas iMSCs cultured in M1 did not (cells became larger and irregular) (Fig. 15B). iMSCs proliferated in M5 showed a lower PDT (population doubling time), which remained stable for a considerably long period of about 20–24 hours across all passages throughout long-term proliferation, whereas iMSCs cultured in M1 medium showed a higher PDT, which increased significantly from P3 (about 25 hours) to P9 (about 57 hours) throughout long-term proliferation (Fig. 15A). A lower PDT at each passage implies higher proliferation efficiency at each passage, while similar PDT at each passage implies that the proliferative capacity of the cells at each passage can be better maintained. Therefore, compared to M1 medium, M5 medium consistently supported robust growth of iMSCs throughout long-term proliferation.

[0276] Example 16

[0277] Cryopreserved iMSCs (RC01005, Nuwacell Co., Ltd.) were seeded in M1 medium at a density of 50,000 cells per well in 6-well plates for cell thawing. After 24 hours, the medium was replaced with 2 ml per well of M1, M4, or M5 medium. The M1, M4, and M5 media had the composition as described in Example 15. iMSCs (RC01005, Nuwacell Co., Ltd.) cultured in M1, M4, or M5 were harvested at passages 1, 4, and 7. Cells were stained with CD90-FITC antibody (BD Pharminge, 555595) and CD90 + The percentage of cells was detected using a flow cytometer according to the protocol described in Example 3 (Fig. 16).

[0278] As shown in Fig. 16, when iMSCs were cultured using M1, CD90-expressing cells were at 98.77% of P1 After rapidly decreasing to 84.14% of P4, it decreased to 81.55% of P7, demonstrating that M1 cannot stably maintain the stem cell phenotype of iMSCs from P1 to P7 during long-term iMSC proliferation. When iMSCs were cultured using M4, CD90 expression was stably maintained from passage 1 to passage 4 (99.59% to 98.78% for CD90-expressing cells), but upon reaching passage 7, CD90 + The proportion of cells decreased. On the other hand, M5 maintained CD90 expression more stably up to passage 7 compared to M1 and M4.

[0279] Example 17

[0280] iMSC (RC01005, Nuwacell Co., Ltd.) was cultured in M5 for 4 passages and tested for triline differentiation ability according to the protocol described in Example 10. In addition, iMSC (RC01005, Nuwacell Co., Ltd.) was cultured in M1 or M5 for 4 passages and tested for immunosuppressive effects according to the protocol described in Example 4. The M1 and M5 media had the composition as described in Example 15.

[0281] Similar to M1, P3 iMSCs cultured in M5 retained MSC characteristics, including triline differentiation (Fig. 17A) and immunosuppressive effects (Fig. 17B).

[0282] Examples 14 to 17 above show that during the long-term proliferation of iMSCs, M4 maintained the iMSC stem cell phenotype better than M1, and M5 maintained the iMSC stem cell phenotype more consistently compared to M4.

[0283] In addition, compared to M1, M5 was able to promote potent iMSC proliferation without the use of animal-derived components and provide an equivalent level of immunosuppressive effect to iMSCs.

[0284] In summary, when combining the results of Examples 14 to 17, it was confirmed that M5 supports the long-term proliferation of iMSCs more sustainably without losing MSC characteristics (including morphology, phenotype, differentiation potential, and immunomodulatory effects), which suggests high clinical applicability of M5.

[0285] Those skilled in the art will readily understand that the methods, compositions, and products described herein represent exemplary embodiments and are not intended to limit the scope of this disclosure. It will be obvious to those skilled in the art that various substitutions and modifications can be made to the contents disclosed herein without departing from the scope and spirit of this disclosure.

[0286] All patents and literature mentioned in the specification represent the level of a person skilled in the art to which this disclosure relates. All patents and literature are incorporated herein by reference to the same extent as each individual document specifically and individually indicates as incorporated by reference.

[0287] The present disclosure is not limited to the specific embodiments described in this application, which are intended as a single example of individual aspects of the present disclosure. As will be apparent to those skilled in the art, many modifications and variations to the present disclosure may be possible without departing from the spirit and scope of the invention. In addition to those listed herein, functionally equivalent badges and kits within the scope of the present disclosure will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to fall within the scope of the following claims. The present disclosure is limited only by the wording of the following claims and the full scope of equivalents recognized by such claims.

Claims

Claim 1 A culture medium comprising, as a culture medium: (a) a basic medium; (b) 5 to 30 μM of an ethanolamine-based compound; (c) 1 to 20 μM of a putrescine-based compound; and (d) 0.1 to 10 volume% of human platelet lysate (HPLT), wherein the culture medium is serum-free and xeno-free. Claim 2 A culture medium according to claim 1, wherein the ethanolamine-based compound comprises ethanolamine and the putrescine-based compound comprises putrescine dihydrochloride. Claim 3 A culture medium according to claim 1, further comprising transferrin. Claim 4 In paragraph 3, the culture medium is a culture medium in which transferrin is present at a concentration of 1 to 200 μg / ml. Claim 5 A culture medium according to claim 1, further comprising an insulin-based compound. Claim 6 In paragraph 5, the culture medium in which the insulin-based compound is present at a concentration of 1 to 15 μg / ml. Claim 7 A culture medium according to claim 1, further comprising an antioxidant. Claim 8 In claim 7, the culture medium in which the antioxidant is present at a concentration of 1 μg / mL to 200 μg / mL. Claim 9 A culture medium according to claim 1, further comprising glutamine or a derivative thereof. Claim 10 In claim 9, the culture medium in which glutamine or a derivative thereof is present at a concentration of 0.1 to 5 volume% in the culture medium. Claim 11 A culture medium according to claim 1, comprising 5 to 30 μM ethanolamine, 1 to 20 μM putrescine dihydrochloride, 0.1 to 10 volume% HPLT, 1 to 150 μg / ml transferrin, 1 to 10 μg / ml insulin, 1 to 150 μg / ml ascorbate, and 0.5 to 5 volume% glutamine in a basic medium. Claim 12 A culture medium according to claim 1, further comprising a growth factor. Claim 13 In paragraph 12, the growth factor is a culture medium selected from the group consisting of EGF, IGF, VEGF, PDGF, FGF2, TGFβ and any combination thereof. Claim 14 In paragraph 12, the growth factor is present in the culture medium at a concentration of 1 to 20 ng / ml. Claim 15 A culture medium according to claim 1, further comprising a corticosteroid compound. Claim 16 In paragraph 15, the culture medium is selected from the group consisting of hydrocortisone, corticosterone, dehydrocorticosterone, cortisone, and any combination thereof. Claim 17 In item 15, the culture medium in which the corticosteroid compound is present at a concentration of 0.1 to 5 μM. Claim 18 A culture medium according to claim 1, further comprising human serum albumin (HSA). Claim 19 In paragraph 18, HSA is a culture medium containing dialyzed HSA. Claim 20 In paragraph 18, the culture medium in which HSA is present at a concentration of 1 to 20 mg / ml. Claim 21 A culture medium according to claim 1, further comprising a heparin-based compound. Claim 22 In claim 21, the culture medium in which the heparin-based compound is present at a concentration of 1 to 150 μg / ml. Claim 23 A culture medium according to claim 11, further comprising 1 to 15 ng / ml of FGF2, 0.5 to 5 μM of hydrocortisone, 1 to 10 μM of lipoic acid, 1 to 30 μM of FeSO4, and 0.1 to 10 μM of Fe(NO3)3 in the basic medium. Claim 24 A culture medium according to claim 11, further comprising 1 to 15 ng / ml of FGF2, 0.5 to 5 μM of hydrocortisone, 5 to 20 ng / ml of Na2SeO3, 0.05 to 5 mg / ml of sodium pyruvate, 1 to 10 mg / ml of HSA, 10 to 150 μM of MTG, 0.5 to 20 mM of nicotinamide (NAM), and 10 to 100 μg / ml of heparin sodium in the basic medium. Claim 25 A culture medium according to claim 1, comprising 10 to 30 μM of ethanolamine, 1 to 15 μM of putrescine dihydrochloride, and 0.5 to 5 volumes of HPLT in a basic medium. Claim 26 A method for proliferating MSCs, comprising the step of contacting mesenchymal stem cells (MSCs) with the culture medium of claim 1. Claim 27 In paragraph 26, the method involves continuously growing MSCs over several passages. Claim 28 In paragraph 27, the method wherein the MSC is continuously propagated over at least four passages. Claim 29 In paragraph 27, the method wherein the MSC is continuously propagated over at least 7 passages. Claim 30 In paragraph 27, the method wherein MSCs are continuously propagated over at least 9 passages. Claim 31 delete

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