Serum-free, xeno-free culture medium for expanding the culture of mesenchymal stem cells.

Abstract

The disclosures described herein provide, in particular, serum-free xeno-free culture media and kits comprising them that can support the long-term expansion culture of mesenchymal stem cells (MSCs). This disclosure also provides a method for expanding the culture of mesenchymal stem cells (MSCs) using the disclosed culture medium, and a substantially homogeneous population of MSCs produced by the method.

Description

[Technical Field]

[0001] This disclosure generally relates to the field of cell culture technology, and more specifically to serum-free, xeno-free culture media and kits containing the same for the expansion culture of mesenchymal stem cells (MSCs). The disclosure also relates to methods for expanding the culture of mesenchymal stem cells (MSCs) using the disclosed culture media or kits, and to populations of MSCs produced by the disclosed methods. [Background technology]

[0002] Mesenchymal stem cells (MSCs) are stem cells that possess multipotency and self-renewal capabilities. MSCs can differentiate into a variety of cells, including osteoblasts, chondrocytes, and adipocytes. Furthermore, MSCs are known to have paracrine effects and cell adhesion interactions due to self-produced humoral factors. Based on these effects, MSCs exert the ability to repair and regenerate target tissues and cells, as well as to regulate immune responses, for example, in anti-inflammatory processes, thereby providing therapeutic effects against various diseases.

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

[0004] In clinical applications, there is a strong demand for a homogeneous and robust population with a large number of cells. Currently, numerous protocols have been developed for expanding the culture of MSCs in order to obtain a large number of them.

[0005] Currently, systems for large-scale MSC culture are primarily based on basal media supplemented with fetal bovine serum (FBS). However, FBS contains heterogeneous proteins and can carry bacteria, viruses, and protein infectious diseases. Furthermore, MSCs can phagocytose proteins present in the culture medium, such as bovine serum proteins (7mg-30mg / 10). 8 Several studies have shown that repeated infusion of MSCs (cells) into patients can lead to the production of anti-bovine protein antibodies and cell-mediated immune responses.

[0006] Numerous serum-free culture media have been developed for expanding the culture of MSCs. However, these media have not been able to reliably support the long-term expansion of MSCs. For example, MSCs with a high passage number not only lack proliferative capacity but also lose stem cell characteristics such as phenotype. [Overview of the project] [Problems that the invention aims to solve]

[0007] Therefore, there is still a need to provide improved culture media for large-scale cultivation of MSCs. [Means for solving the problem]

[0008] This disclosure provides, in particular, serum-free, xeno-free culture media, kits comprising the same, methods for expanding mesenchymal stem cells (MSCs) using the culture media or kit, and populations of MSCs produced by the disclosed methods.

[0009] One aspect of the present disclosure relates to a serum-free, xeno-free culture medium that can support the long-term expansion culture of mesenchymal stem cells (MSCs), comprising (a) a basal medium, (b) an ethanolamine compound, (c) a putrescine compound, and (d) human platelet lysate (HPLT).

[0010] In some embodiments, the ethanolamine-based compound is present in the culture medium at a concentration of 1 to 30 μM.

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

[0012] In some embodiments, the human platelet lysate is present in the culture medium at a concentration of 0.1 to 20% by volume.

[0013] In some embodiments, the ethanolamine-based compound contains ethanolamine, and the putrescine-based compound contains putrescine dihydrochloride.

[0014] In some embodiments, the culture medium further contains transferrin.

[0015] In some embodiments, transferrin is present in the culture medium at a concentration of 1 to 200 μg / ml.

[0016] In some embodiments, the culture medium further contains an insulin-based compound. In some embodiments, the insulin-based compound is present in the culture medium at a concentration of 1 to 15 μg / ml.

[0017] In some embodiments, the culture medium further contains an antioxidant. In some embodiments, the antioxidant is present in the culture medium at a concentration of 1 μg / mL to 200 μg / mL.

[0018] In some embodiments, the culture medium further contains glutamine or a derivative thereof. In some embodiments, glutamine or a derivative thereof is present in the culture medium at a concentration of 0.1 to 5% by volume.

[0019] In some embodiments, the culture medium contains 5 - 30 μM ethanolamine, 1 - 20 μM putrescine dihydrochloride, 0.1 - 10% by volume of HPLT, 1 - 150 μg / ml of transferrin, 1 - 10 μg / ml of insulin, 1 - 150 μg / ml of ascorbate, and 0.5 - 5% by volume of glutamine in a basal medium.

[0020] In some embodiments, the culture medium further contains a growth factor.

[0021] In some embodiments, the growth factor is selected from the group consisting of EGF, IGF, VEGF, PDGF, FGF2, TGFβ, and any combination thereof.

[0022] In some embodiments, the growth factor is present in the culture medium at a concentration of 1 - 20 ng / ml.

[0023] In some embodiments, the culture medium further contains a corticosteroid compound.

[0024] In some embodiments, the corticosteroid compound is selected from the group consisting of hydrocortisone, corticosterone, dehydrocorticosterone, cortisone, and any combination thereof.

[0025] <00001​​​​​​​​​​​​​​​​In some embodiments, the culture medium further comprises a heparin-based compound.

[0030] In some embodiments, the heparin compound is present in the culture medium at a concentration of 1 to 150 μg / ml.

[0031] In some embodiments, the culture medium further comprises one or more selected from the group consisting of lipoic acid, sulfate, iron(III) salt, selenite, pirubate, monothioglycerol (MTG), and nicotinamide compounds.

[0032] In some embodiments, the culture medium contains, in basal medium, 5–30 μM ethanolamine, 1–20 μM putrescine dihydrochloride, 0.1–10 vol% HPLT, 1–150 μg / ml transferrin, 1–10 μg / ml insulin, 1–150 μg / ml ascorbate, 0.5–5 vol% glutamine, 1–15 ng / ml FGF2, 0.5–5 μM hydrocortisone, 1–10 μM lipoic acid, 1–30 μM FeSO4, and 0.1–10 μM Fe(NO3)3.

[0033] In some embodiments, the culture medium contains, in basal medium, 5–30 μM ethanolamine, 1–20 μM putrescine dihydrochloride, 0.1–10% by volume HPLT, 1–150 μg / ml transferrin, 1–10 μg / ml insulin, 1–150 μg / ml ascorbate, 0.5–5% by volume glutamine, 1–15 ng / ml FGF2, 0.5–5 μM hydrocortisone, 5–20 ng / ml Na2SeO3, 0.05–5 mg / ml sodium pyruvate, 1–10 mg / ml HSA, 10–150 μM MTG, 0.5–20 mM nicotinamide (NAM), and 10–100 μg / ml heparin sodium.

[0034] Another aspect of this disclosure relates to a kit comprising the culture medium of this disclosure.

[0035] Another aspect of this disclosure relates to a method for expanding mesenchymal stem cells (MSCs), comprising contacting the MSCs with the culture medium of this disclosure.

[0036] In some embodiments, MSCs are continuously expanded and cultured over multiple passages, for example, over at least 4, 7, and 9 passages.

[0037] Another aspect of this disclosure relates to a substantially homogeneous population of MSCs produced by the method of this disclosure.

[0038] This specification describes several embodiments of the present disclosure by reference to the accompanying drawings, which are for illustrative purposes only. These embodiments are described in particular with reference to the drawings, emphasizing that the details shown are illustrative and for illustrative purposes only. In this regard, by considering this specification together with the drawings, it will become clear to those skilled in the art how embodiments of the present disclosure may be carried out. [Brief explanation of the drawing]

[0039] [Figure 1] Figure 1 shows bright-field images of iMSCs cultured in different media M1, SFM-M, PPRF, and MEM-FBS according to Example 1. [Figure 2A-2B] Figures 2A and 2B show the difference in the effect of M1 medium and M2 medium with a different HPLT concentration on the expansion culture of iMSCs according to Example 2. Figure 2A shows the cell morphology of the expanded cultured iMSCs, and Figure 2B shows the number of cells in the expanded cultured iMSCs. [Figure 3A-3B] Figures 3A and 3B show the difference in the effect of M1 medium and M2 medium on the long-term expansion culture of iMSCs according to Example 3. Figure 3A shows the cell number of iMSCs at passages 6, 7, and 8, and Figure 3B shows the CD90 expression of iMSCs at passages 1, 4, 5, and 7. [Figure 4A-4B]Figures 4A and 4B show the results of CFSE-based T cell proliferation assays of iMSCs cultured in M1 and M2 medium according to Example 4. Figure 4A shows the cell morphology of activated T cells cultured with or without expanded iMSCs, and Figure 4B shows the percentage of the divided T cell population for activated T cells cultured with or without expanded iMSCs. [Figure 5] Figure 5 shows the effects of different types of growth factors and different concentrations of hydrocortisone in the expansion culture medium on the proliferation of iMSCs according to Example 5. Here, F represents FGF2, FP represents the combination of FGF2(F) and PDFG-BB(P), FPT represents the combination of FGF2(F), PDFG-BB(P), and TGFβ1(T), and w / oG.F represents the case where no growth factors are present. [Figure 6A-6B] Figures 6A and 6B show the effects of different media I, II, III, IV, V, and M1 on the expansion culture of iMSCs according to Example 6. Figure 6A shows the cell morphology of the cultured iMSCs, and Figure 6B shows the cell number of the cultured iMSCs. [Figure 7] Figure 7 shows the difference in the effects of M1 medium and M3 medium with different HPLT concentrations on the proliferation of umbilical cord-derived mesenchymal stem cells (ucMSCs) and iMSCs according to Example 7. [Figure 8] Figure 8 shows the effects of different media M1, M3, and M4 on the growth of iMSCs according to Example 8. [Figure 9] Figure 9 shows the effects of different media M1, M3, and M4 on CD90 expression in expanded culture iMSCs according to Example 9. [Figure 10A-10B]Figures 10A and 10B show the results of the three-lineage differentiation and CFSE-based T cell proliferation assay of expanded cultured iMSCs according to Example 10. Figure 10A shows the morphology of adipocytes (left panel), skeletal cells (center panel), and chondrocytes (right panel) differentiated from iMSCs expanded in M3. Figure 10B shows the percentage of divided T cell populations for activated T cells and inactivated T cells, cultured in co-culture with expanded cultured iMSCs in M1 and M3, or cultured without expanded cultured iMSCs. [Figure 11] Figure 11 shows the effects of different media M1, M3, and M3 with additional FGF2 at different concentrations on iMSC growth according to Example 11. [Figures 12A-12B] Figures 12A and 12B show the effect of HSA in M3, particularly dialysis-containing HSA, on the expansion culture of iMSCs according to Example 12. Figure 12A shows the cell morphology of iMSCs expanded in M3 medium containing dialysis-containing or non-dialysis-containing HSA, and in M3 medium containing no HSA. Figure 12B shows the cell number of the iMSCs. [Figure 13] Figure 13 shows the effects of dialysis-induced heparin sodium in M3 with different HPLT concentrations on iMSC proliferation, as described in Example 13. [Figure 14] Figure 14 shows the effects of various HPLT concentrations in M5 on iMSC proliferation according to Example 14. [Figures 15A-15B] Figures 15A and 15B show the effects of various culture media on the cell morphology and population doubling time (PDT) of iMSCs during long-term expansion culture according to Example 15. Figure 15A shows the PDT curves of iMSCs from passage 3 to passage 9 during long-term expansion culture using M1 or M5, and Figure 15B shows the cell morphology of iMSCs at passage 8 during long-term expansion culture using M1, M4, or M5. [Figure 16] Figure 16 shows the effects of different media M1, M4, and M5 on CD90 expression in iMSCs during long-term expansion culture according to Example 16. [Figures 17A-17B]Figures 17A and 17B show the results of the three-lineage differentiation and CFSE-based T cell proliferation assay of expanded cultured iMSCs according to Example 17. Figure 17A shows the morphology of adipocytes (left panel), skeletal cells (center panel), and chondrocytes (right panel) differentiated from iMSCs expanded in M5. Figure 17B shows the percentage of divided T cell populations for activated T cells and inactivated T cells, cultured co-culturing with expanded cultured iMSCs in M1 and M5, or cultured without expanded cultured iMSCs. [Modes for carrying out the invention]

[0040] To provide a substantial understanding of this technology, please understand that certain aspects, forms, embodiments, variations, and features of this disclosure are described below with varying levels of detail.

[0041] Throughout this specification, any reference to “one embodiment,” “another embodiment,” “preferred embodiment,” “several embodiments,” or “some embodiments” means that certain features, structures, or characteristics described in relation to those embodiments are included in at least one or more embodiments of this disclosure. Furthermore, certain features, structures, or characteristics in one embodiment may be combined in any suitable manner with those in one or more other embodiments.

[0042] The disclosures described herein as exemplary may be adequately implemented in the absence of any one or more elements or limitations not specifically disclosed herein. Where used herein, the term “comprising” is intended to mean that the composition and method includes the enumerated elements, but does not exclude other elements. Where “consisting essentially of” is used to define a composition and method, it means that other elements that may have some essential importance to the composition or method are excluded. “Consisting of” means that elements of other components in trace amounts are excluded from the claimed composition, and other substantial method steps are excluded. Embodiments defined by each of these transitional terms are within the scope of this disclosure. Therefore, the methods and compositions are intended to include additional steps and components, or to include steps and compositions that are not essential, or to intend only the stated method steps or compositions. Furthermore, in each example herein, the terms “comprising,” “consisting essentially of,” and “consisting of” may be replaced with any of the other two terms.

[0043] This disclosure is not limited to any particular use, method, reagent, compound, composition, or biological system, and it is understood that such use, method, reagent, compound, composition, or biological system may vary. It is also understood that the terms used herein are intended solely to describe and not to limit any particular embodiment.

[0044] Unless otherwise defined, all technical and scientific terms used herein have meanings that are generally understood by those skilled in the art in which this disclosure pertains. However, for convenience and completeness, certain terms and their meanings are set forth below and throughout this specification.

[0045] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple subjects unless otherwise explicitly indicated by the context. Thus, for example, a reference to “the method” includes one or more methods and / or processes of the type described herein and / or of the type that would be obvious to a person skilled in the art who has read this disclosure.

[0046] The terms “about” or “approximately” mean a statistically meaningful range of values. Such a range may be within one decimal place of a given value or range, typically within 20%, more typically within 10%, and even more typically within 5%. The acceptable variation encompassed by the terms “about” or “approximately” depends on the particular system under consideration, which will be readily apparent to those skilled in the art. Whenever ranges are enumerated in this application, all integers within that range are construed as embodiments of the present disclosure.

[0047] As used herein, the terms “substantially” or “essentially” mean a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is approximately 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, volume, weight, or length. In some embodiments, the terms “essentially the same” or “substantially the same” mean a range of quantities, levels, values, numbers, frequency, percentage, dimensions, size, amount, weight, or length that is approximately the same as the reference quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length.

[0048] As used herein, the term “pluripotent stem cell” (PSC) refers to cells derived from the inner cell mass of a blastocyst. Pluripotent stem cells can be pluripotent and can give rise to all three major germ layers during development: ectoderm, endoderm, and mesoderm. Pluripotent stem cells can be of human origin (e.g., human PSCs or hPSCs). Pluripotent stem cells can be induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). ESCs (e.g., hESCs) and iPSCs (e.g., hiPSCs) are known in the art and can be readily obtained using conventional methods, e.g., methods described in the existing art, or commercially available products. Appropriate methods for producing iPSCs from somatic stem cells or pluripotent stem cells are well known to those skilled in the art. For example, iPSCs can be reliably produced from somatic cells by conventional reprogramming techniques.

[0049] As used herein, the terms “pluripotent” or “pluripotent” refer to cells that have the developmental ability 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 properties of a cell. Pluripotent characteristics include, but are not limited to, (i) morphology of pluripotent stem cells, (ii) unlimited self-renewal capacity, (iii) expression of pluripotent stem cell markers such as, but not limited to, SSEA1 (mouse only), SSEA3 / 4, SSEA5, TRA1-60 / 81, TRA1-85, 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 cell lineages (ectoderm, mesoderm and endoderm), (v) teratoma formation consisting of the three somatic cell lineages, and (vi) embryoid body formation consisting of cells from the three somatic cell lineages.

[0050] As used herein, the term “mesenchymal stem cell” or “MSC” refers to a multipotency stem cell capable of differentiating into cells such as adipocytes, osteocytes, chondrocytes, muscle cells, nerve cells, and cardiomyocytes. MSCs include primary MSCs and induced MSCs (also called iMSCs). Examples of primary MSCs include, for example, 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 can be differentiated from ESCs or iPSCs. As used herein, ESCs are obtained from human embryonic stem cells that have not been developed in vivo within 14 days of fertilization, or from commercially established human embryonic stem cell lines.

[0051] As used herein, the terms “broaden culture” or “broaden culture” mean increasing the number of MSCs (by 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) throughout the culture period.

[0052] As used herein, the term “culture medium” refers to a culture medium capable of supporting cell survival, growth, proliferation, maintenance, and / or differentiation in an in vitro environment. A culture medium may consist of a basal medium and one or more additives.

[0053] As used herein, the term “basal medium” refers to the basic components of a culture medium (e.g., differentiation medium or expansion medium) in contrast to one or more additives. Generally, the basal medium accounts for approximately 95–99% by volume of the culture medium (e.g., differentiation medium or expansion medium). In cell expansion mediums, the basal medium acts as a source of nutrients, hormones, and / or other factors that help amplify and / or proliferate cells. In cell differentiation mediums, the basal medium acts as a source of nutrients, hormones, and / or other factors that help differentiate cells.

[0054] As used herein, the term “additive” refers to an additive component of a culture medium (e.g., differentiation medium or expansion medium) in relation to its basal medium. In the context of this disclosure, the amount or concentration of all additive components or additives in a culture medium (e.g., expansion medium) is calculated based on the basal medium. All percentages mentioned in descriptions of the amount or concentration of additive components or additives are based on volume unless otherwise explicitly stated.

[0055] As used herein, the term “added” refers to the addition of additives for a culture medium (e.g., a widening culture medium) to its basal medium. Additives may be added to the basal medium of a culture medium before or during use.

[0056] As used herein, the term “serum-free” refers to the absence of human or animal serum. It should be noted that the function of serum in a culture protocol is to provide cultured cells with an environment similar to the in vivo environment (i.e., the environment in the organism from which the cells originate). However, the use of serum derived from either an animal source (e.g., bovine serum) or a human source (human serum) is limited by the significant variability in serum composition between donor individuals (from which the serum is obtained) and the risk of heterogeneity (when animal serum is used).

[0057] As used herein, the term "xenofree" means that the product contains no products of non-human animal origin.

[0058] As used herein, the term “serum replacement (SR)” refers to a defined formulation that replaces the function of serum by providing pluripotent stem cells or mesenchymal stem cells with components necessary for their growth and survival.

[0059] As used herein, the term “long-term expansion culture” refers to the continuous expansion culture of cells that are passaged through multiple passages. In the context of this disclosure, “long-term expansion culture” can be used interchangeably with “continuous expansion culture.”

[0060] The terms “seed” or “to be seeded,” as used herein, refer to the process of providing a cell culture to a bioreactor or another container. The cells may have been pre-amplified in another bioreactor or another container. Alternatively, the cells may have been frozen and thawed immediately before providing them to the bioreactor or container. The term refers to any number of cells, including single cells.

[0061] As used herein, the terms “passaging” or “to passage” refer to dividing cells in a culture vessel into two or more culture vessels, typically involving the addition of fresh culture medium. Passaging is typically performed when cells have reached a certain confluence under culture conditions.

[0062] As used herein, the term "in vitro" generally refers to activities performed outside the body of an organism.

[0063] As used herein, the term "in vivo" generally refers to activities that take place within a living organism.

[0064] 1. Culture medium According to one aspect of the present disclosure, a serum-free, xeno-free culture medium is provided which can support the long-term expansion culture of mesenchymal stem cells (MSCs), comprising (a) a basal medium, (b) an ethanolamine compound, (c) a putrescine compound, and (d) human platelet lysate (HPLT).

[0065] According to this disclosure, based on the synergistic effect of a combination of ethanolamine compounds, putrescine compounds, and HPLT, this culture medium maintains the stem cell phenotype (CD90) of MSCs even during long-term expansion culture. + This allows for the stable maintenance of the MSCs while significantly enhancing their expansion. Based on this, the culture medium according to this disclosure can support the long-term expansion of MSCs without substantially losing the characteristics of MSCs such as morphology, phenotype, differentiation potential, and immunomodulatory effects, and is therefore more suitable for the large-scale production of MSC products for clinical application.

[0066] Mesenchymal stem cells (MSCs) Any MSCs can be cultured in an expanded culture using the expanded culture medium of this 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 can be induced (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 expanded pluripotent stem cells (EPSCs). In some embodiments, iMSCs are ESC-derived MSCs. In some embodiments, iMSCs are iPSC-derived MSCs. In some embodiments, iMSCs are NPSC-derived MSCs. In some embodiments, MSCs are EPSC-derived MSCs. Commercially available MSCs can also be used in this disclosure.

[0067] ESCs (e.g., hESCs) and iPSCs (e.g., hiPSCs) are known in the art and can be readily obtained using conventional methods, such as those described in existing technologies, or commercially available products. For example, the CytoTune iPS 2.0 Sendai Reprogramming Kit (ThermoFisher Scientific) can be used to reliably generate induced pluripotent stem cells (iPSCs) from somatic cells, including PBMCs and T cells.

[0068] 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, bone marrow aspirate or biopsy material can be collected from a donor (e.g., a healthy donor), and MSCs can be isolated therefrom. Generally, mononuclear cells are typically isolated from bone marrow aspirate by gradient centrifugation, then seeded in 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 grown at 37°C under a humidified 5% CO2 atmosphere. Typically, non-adherent cells are removed after 24 hours (e.g., by washing with PBS-HSA solution). The culture medium is changed every 4 days. The culture should be nearly confluent after 2 weeks. MSCs are harvested using trypsin and replated as passage 1 cells. The cells can be maintained in culture for at least 8 passages, and the presence of MSC-related surface molecules can be routinely tested.

[0069] iPSCs (e.g., hiPSCs) can be differentiated using any common method for producing iMSCs. For example, to produce iMSCs from iPSCs, the differentiation method described in CN110592007B, which is incorporated in its entirety herein by reference, may be used.

[0070] MSCs can be characterized by the presence or absence of specific cell surface markers, such as CD90. MSCs can also be identified by functional assays, both in vitro and in vivo, particularly by assays concerning the ability of stem cells to produce multiple differentiated offspring and assays concerning responsiveness to canonical WNT signaling.

[0071] Basic culture medium As its name suggests, a basal medium can support cell survival, maintenance, growth, and proliferation as a culture medium and is a fundamental component of an expanded culture medium. Generally, a basal medium accounts for approximately 95-99% of the volume of the expanded culture medium. The basal medium used in the expanded culture medium of this disclosure may be a basal medium common in the art.

[0072] Examples of basal media include DMEM / F12 (e.g., Gibco catalog number C11330500BT), BME medium (e.g., Gibco catalog number 21010046 or Sigma-Aldrich catalog number B9638), IMDM medium (e.g., Gibco catalog number 12440053 or Sigma-Aldrich catalog number I3390), Eagle MEM medium (e.g., Minimum Essential Medium (MEM) developed by Harry Eagle, Sigma-Aldrich catalog numbers M2414 / M2279 / M5690), α-MEM medium (e.g., Gibco catalog number 12561056 or Sigma-Aldrich catalog number M0894), DMEM medium (e.g., Gibco catalog number 21068028), RPMI1640 medium (e.g., Gibco catalog number 11875093), Ham F12 medium (e.g., Gibco catalog number 11765054), or mixtures thereof. If the culture medium contains a mixture of two or more basal media, the relative ratio of those basal media can be arbitrary.

[0073] Ethanolamine compounds As used herein, “ethanolamine compounds” means ethanolamine, its derivatives, their salts, or mixtures thereof.

[0074] Ethanolamine (also known as 2-aminoethanol, monoethanolamine, ETA, or MEA) is an organic chemical compound with the formula HOCH2CH2NH2 or C2H7NO. This molecule is bifunctional, containing both a primary amine and a primary alcohol. Ethanolamine is commonly referred to as monoethanolamine or MEA to distinguish it from diethanolamine (DEA) and triethanolamine (TEA).

[0075] Examples of ethanolamine derivatives include, but are not limited to, substituted ethanolamines such as phosphatidylethanolamine. Examples of salts of ethanolamine and its derivatives include, but are not limited to, ethanolamine hydrochloride and ethanolamine hydrobromide, as well as hydrochloride or hydrobromide salts of substituted ethanolamines.

[0076] According to some embodiments, the ethanolamine compound used in the culture medium may be ethanolamine. According to some embodiments, the ethanolamine compound used in the culture medium may be ethanolamine hydrochloride.

[0077] According to this disclosure, the concentration of the ethanolamine compound is not particularly limited as long as it supports the long-term expansion culture of MSCs. According to some embodiments, the concentration of the ethanolamine compound is in the range of 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 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 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 preferred embodiments, the concentration of the ethanolamine compound is 5 to 30 μM.

[0078] Putrescine compounds As used herein, “putrescine compounds” means putrescine and its derivatives, salts thereof, or mixtures thereof.

[0079] Putrescine is an organic compound with the formula (CH2)4(NH2)2. It is commonly known as 1,4-butanediamine and is produced on an industrial scale by the hydrogenation of succinonitrile.

[0080] Examples of putrescine derivatives include, but are not limited to, substituted putrescine such as N-acetylputrescine. Examples of salts of putrescine and its derivatives include, but are not limited to, putrescine hydrochloride, putrescine dihydrochloride, putrescine hydrobromide, and dihydrochloride or dibromide of substituted putrescine such as N-acetylputrescine.

[0081] According to some embodiments, the putrescine-based compound used in the culture medium may be putrescine. According to some embodiments, the putrescine-based compound used in the culture medium may be putrescine dihydrochloride.

[0082] According to this disclosure, the concentration of the putrescine compound is not particularly limited as long as it supports the long-term expansion culture of MSCs. According to some embodiments, the concentration of the putrescine compound is in the range of 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 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 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 preferred embodiments, the concentration of the putrescine compound is 1 to 20 μM.

[0083] Human platelet lysate Human platelet lysates (HPLTs) are derived from human platelets. Human platelet lysates may be derived from healthy donor human platelets and are rich in growth factors. The human platelet lysates used in accordance with this disclosure are commercially available, such as PLTGold Human Platelet Lysate (Biological Industries, #PLTGOLD500R). However, human platelet lysates from other sources are also available and can be used in this disclosure. In this disclosure, human platelet lysates can substantially improve the cell expansion efficiency during long-term expansion culture.

[0084] According to this disclosure, the concentration of human platelet lysates is not particularly limited as long as it supports the long-term expansion culture of MSCs. According to some embodiments, the concentration of human platelet lysates 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 bodies These are volume%, 1-5 volume%, 1-10 volume%, 1-15 volume%, 1-20 volume%, 2-3 volume%, 2-4 volume%, 2-5 volume%, 2-10 volume%, 2-15 volume%, 2-20 volume%, 3-4 volume%, 3-5 volume%, 3-10 volume%, 3-15 volume%, 3-20 volume%, 4-5 volume%, 4-10 volume%, 4-15 volume%, or 4-20 volume%, 5-6 volume%, 5-7 volume%, 5-8 volume%, 5-9 volume%, 5-10 volume%, 5-15 volume%, or 5-20 volume%. According to some embodiments, the concentration of human platelet lysate is 0.1% by volume, 0.2% by volume, 0.3% by volume, 0.4% by volume, 0.5% by volume, 0.6% by volume, 0.7% by volume, 0.8% by volume, 0.9% by volume, 1% by volume, 2% by volume, 3% by volume, 4% by volume, 5% by volume, 6% by volume, 7% by volume, 8% by volume, 9% by volume, 10% by volume, 15% by volume, or 20% by volume. According to a preferred embodiment, the concentration of human platelet lysate is 0.1 to 10% by volume.

[0085] Additional reagents Depending on the requirements of this disclosure, one or more additional reagents may be optionally added to the culture medium before or during use. Examples of additional reagents include transferrin, insulin compounds, growth factors, corticoid compounds, human serum albumin (HSA), heparin compounds, glutamine or its derivatives, antioxidants, lipoic acid, sulfates, iron(III) salts, selenite, pirubate, monothioglycerol (MTG), and nicotinamide compounds.

[0086] transferrin 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.

[0087] According to this disclosure, the concentration of transferrin is not particularly limited as long as it supports the long-term expansion culture of MSCs. According to some embodiments, the concentration of transferrin 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 110 μg / ml, 1 to 120 μg / ml, 1 to 130 μg / ml, 1 to 140 μg / ml, 1 ~150μg / ml, 1~160μg / ml, 1~170μg / ml, 1~180μg / ml, 1~190μg / ml, 1~200μg / ml, 3~5μg / ml, 3~10μg / ml, 3~15μg / ml, 3~20μg / ml, 3~25μg / ml, 3~3 0μg / ml, 3~35μg / ml, 3~40μg / ml, 3~45μg / ml, 3~50μg / ml, 3~60μg / ml, 3~70μg / ml, 3~80μg / ml, 3~90μg / ml, 3~100μg / ml, 3~110μg / ml, 3~120μg / m l, 3~130μg / ml, 3~140μg / ml, 3~150μg / ml, 3~160μg / ml, 3~170μg / ml, 3~180μg / ml, 3~190μg / ml, 3~200μg / ml, 5~10μg / ml, 5~15μg / ml, 5~20μg / ml, 5~25μg / ml, 5~30μg / ml, 5~35μg / ml, 5~40μg / ml, 5~45μg / ml, 5~50μg / ml, 5~60μg / ml, 5~70μg / ml, 5~80μg / ml, 5~90μg / ml, 5~100μg / ml, 5~11 0μg / ml, 5~120μg / ml, 5~130μg / ml, 5~140μg / ml, 5~150μg / ml, 5~160μg / ml, 5~170μg / ml, 5~180μg / ml, 5~190μg / ml, 5~200μg / ml, 7~10μg / ml, 7~ 15μg / ml, 7~20μg / ml, 7~25μg / ml, 7~30μg / ml, 7~35μg / ml, 7~40μg / ml, 7~45μg / ml, 7~50μg / ml, 7~60μg / ml, 7~70μg / ml, 7~80μg / ml, 7~90μg / ml,7~100μg / ml, 7~110μg / ml, 7~120μg / ml, 7~130μg / ml, 7~140μg / ml, 7~150μg / ml, 7~160μg / ml, 7~170μg / ml, 7~180μ g / ml, 7~190μg / ml, 7~200μg / ml, 9~10μg / ml, 9~15μg / ml, 9~20μg / ml, 9~25μg / ml, 9~30μg / ml, 9~35μg / ml, 9~40μg / ml ml, 9~45μg / ml, 9~50μg / ml, 9~60μg / ml, 9~70μg / ml, 9~80μg / ml, 9~90μg / ml, 9~100μg / ml, 9~110μg / ml, 9~120μg / m l, 9-130μg / ml, 9-140μg / ml, 9-150μg / ml, 9-160μg / ml, 9-170μg / ml, 9-180μg / ml, 9-190μg / ml or 9-200μg / 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, 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 / m³.

[0088] Insulin-type compounds As used herein, “insulin-based compounds” means insulin, its analogues or derivatives thereof, or mixtures thereof.

[0089] According to some embodiments, the insulin-based compound includes insulin. According to some embodiments, the insulin-based compound may be an analog or derivative of insulin, such as insulin lispro, insulin aspart, and insulin glulisine.

[0090] According to this disclosure, the concentration of insulin-like compounds is not particularly limited, as long as it supports the long-term expansion culture of MSCs. According to some embodiments, the concentration of the insulin-like compound is 1 to 15 μg / ml, for example, 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 10 μg / ml, 9 to 15 μg / ml, or 10 to 15 μg / ml. According to some embodiments, the concentration of the insulin-like 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 preferred embodiments, the concentration of the insulin-like compound is 1 to 10 μg / ml.

[0091] Glutamine or its derivatives According to some embodiments, the culture medium further comprises glutamine or a derivative thereof.

[0092] According to some embodiments, glutamine or its derivatives may be glutamines such as L-glutamine or substituted glutamines such as alanyl-glutamine. Alanyl-glutamine is a chemical compound used in food nutrition supplementation, parenteral nutrition, and cell culture in the form of L-alanyl-L-glutamine. L-alanyl-L-glutamine is marketed by Thermo Fisher Scientific under the name GlutaMAX.

[0093] According to this disclosure, the concentration of glutamine or its derivatives is not particularly limited, as long as it supports the long-term expansion culture of MSCs. According to some embodiments, the concentration of glutamine or its derivatives 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 4 volume%, 0.4 to 5 volume%, 0.5 to 1 volume%, 0.5 to 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 derivatives is 0.1% by volume, 0.2% by volume, 0.3% by volume, 0.4% by volume, 0.5% by volume, 0.6% by volume, 0.7% by volume, 0.8% by volume, 0.9% by volume, 1% by volume, 2% by volume, 3% by volume, 4% by volume, or 5% by volume. According to preferred embodiments, the concentration of glutamine or its derivatives is 0.5 to 5% by volume.

[0094] antioxidants According to some embodiments, the culture medium further contains antioxidants.

[0095] As used herein, “antioxidant” refers to a compound that inhibits intracellular oxidation (usually occurring as auto-oxidation), which is a chemical reaction that can generate free radicals. The antioxidant may be ascorbic acid, its analogues, its derivatives, or salts thereof.

[0096] Antioxidants include ascorbic acid, ascorbates, magnesium salts such as L(+)-magnesium ascorbate, sodium salts such as L(+)-sodium ascorbate, and analogs or derivatives such as ascorbyl glucoside, 3-ethyl ascorbic acid, ascorbyl tetraisopalmitate, ascorbic acid phosphate salts, and ascorbyl palmitate.

[0097] According to some embodiments, the antioxidant is ascorbate. According to some embodiments, the antioxidant is sodium ascorbate or magnesium ascorbate.

[0098] According to this disclosure, the concentration of antioxidants is not particularly limited as long as it supports the long-term expansion culture of MSCs. According to some embodiments, the concentration of antioxidants 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 g / ml, 2~15μg / ml, 2~20μg / ml, 2~25μg / ml, 2~30μg / ml, 2~35μg / ml, 2~40μg / ml, 2~45μg / ml, 2~50μg / ml, 2~60μg / ml, 2~70μg / ml, 2~80μg / ml ml, 2~90μg / ml, 2~100μg / ml, 2~150μg / ml, 2~200μg / ml, 3~5μg / ml, 3~10μg / ml, 3~15μg / ml, 3~20μg / ml, 3~25μg / ml, 3~30μg / ml, 3~35μg / ml ml, 3~40μg / ml, 3~45μg / ml, 3~50μg / ml, 3~60μg / ml, 3~70μg / ml, 3~80μg / ml, 3~90μg / ml, 3~100μg / ml, 3~150μg / ml, 3~200μg / ml, 4~5μg / ml, 4~10μg / ml, 4~15μg / ml, 4~20μg / ml, 4~25μg / ml, 4~30μg / ml, 4~35μg / ml, 4~40μg / ml, 4~45μg / ml, 4~50μg / ml, 4~60μg / ml, 4~70μg / m l, 4~80μg / ml, 4~90μg / ml, 4~100μg / ml, 4~150μg / ml, 4~200μg / ml, 5~10μg / ml, 5~15μg / ml, 5~20μg / ml, 5~25μg / ml, 5~30μg / ml, 5~35μg / ml, 5-40 μg / ml, 5-45 μg / ml, 5-50 μg / ml, 5-60 μg / ml, 5-70 μg / ml, 5-80 μg / ml, 5-90 μg / ml, 5-100 μg / ml, 5-150 μg / ml, or 5-200 μg / ml.According to some embodiments, the antioxidant concentration 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, or 200 μg / ml. According to preferred embodiments, the antioxidant concentration is 1 to 150 μg / ml.

[0099] growth factors According to some embodiments, the culture medium further contains growth factors.

[0100] Growth factors are molecules that can stimulate a variety of cellular processes, including cell proliferation, cell migration, differentiation, and multicellular morphogenesis during development and tissue healing. Examples of growth factors include, but are not limited to, bone morphogenetic factor (BMP), epidermal growth factor (EGF), endothelial cell 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 this disclosure, the addition of one or more growth factors can clearly enhance the expansion of MSC culture.

[0101] 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.

[0102] Epidermal growth factor (EGF) refers to any polypeptide or variant of the epidermal growth factor (EGF) protein family that stimulates cell growth and differentiation. Typically, EGF exerts its activity by binding to the epidermal growth factor receptor. Therefore, variants of the EGF molecule that maintain biological activity, such as C-terminally truncated molecules or molecules truncated at the N-terminus, may be used in accordance with this disclosure.

[0103] The EGF used in the culture media of some embodiments of this disclosure may be purified EGF protein, synthetic EGF protein, or recombinant expression EGF protein. EGF can be obtained from various commercial sources.

[0104] 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 denoted as IGF-I) is primarily secreted by the liver as a result of stimulation by growth hormone (GH). IGF-1 is important for regulating both normal physiological functions and several pathological conditions, including cancer. While IGF-1 expression is necessary to achieve maximum growth, insulin-like growth factor 2 (IGF-2, sometimes denoted as IGF-II) is considered a major growth factor required for early development.

[0105] The IGF used in the culture medium of some embodiments of this disclosure may be purified IGF protein, synthetic IGF protein, or recombinant expression IGF protein. IGF can be obtained from various commercial sources.

[0106] Vascular endothelial growth factor (VEGF) is a signaling protein produced by many cells that stimulate blood vessel formation. VEGF is an important signaling protein involved in both vascularization (de novo formation of the embryonic circulatory system) and angiogenesis (growth of blood vessels from existing vascular structures).

[0107] The VEGF used in the culture medium of some embodiments of this disclosure may be purified VEGF protein, synthetic VEGF protein, or recombinant expression VEGF protein. VEGF can be obtained from various commercial sources.

[0108] Platelet-derived growth factor (PDGF) refers to one of four distinct isoforms of PDGF that activate cellular responses via two different receptors. These isoforms include A (observed as a homodimer called PDGF-AA and as part of a heterodimer with isoform B called PDGF-AB), B (observed as a homodimer called PDGF-BB and as part of a heterodimer with isoform A called PDGF-AB), C (observed as a homodimer called PDGF-CC), and D (observed as a homodimer called PDGF-DD). Therefore, as used herein, the term “PDGF” generally refers to the known PDGF homodimers and heterodimers (e.g., PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC, and PDGF-DD).

[0109] According to some embodiments, the PDGF used in the culture medium of some embodiments of this disclosure is PDGF-BB.

[0110] The PDGF used in the culture medium of some embodiments of this disclosure may be purified PDGF protein, synthetic PDGF protein, or recombinant expression PDGF protein. PDGF can be obtained from various commercial sources.

[0111] FGF2, also commonly known as basic fibroblast growth factor (FGF basic, bFGF, or FGF-β), is a member of the fibroblast growth factor family. The FGF2 used in the culture medium of some embodiments of this disclosure may be purified FGF2 protein, synthetic FGF2 protein, or recombinant expressed FGF2 protein.

[0112] Transforming growth factor beta (TGFβ) refers to any isoform of transforming growth factor beta (β), which functions in many cell types to regulate proliferation, differentiation, and other functions via the same receptor signaling pathway. TGFβ has a transformation-inducing effect and also acts as a negative autocrine growth factor.

[0113] According to some embodiments of this disclosure, TGFβ includes TGFβ1, TGFβ2, and / or TGFβ3.

[0114] 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 this disclosure may be purified TGFβ1 protein, synthetic TGFβ1 protein, or recombinant expression TGFβ1 protein. TGFβ1 is available from various commercial sources.

[0115] Transforming growth factor beta-2 (TGFβ2) is a polypeptide member of the transforming growth factor beta superfamily of cytokines. TGFβ2 plays a crucial role during embryonic development. The TGFβ2 used in the culture medium of some embodiments of this disclosure may be purified TGFβ2 protein, synthetic TGFβ2 protein, or recombinant expression TGFβ2 protein. TGFβ2 can be obtained from various commercial sources.

[0116] 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 this disclosure may be purified TGFβ3 protein, synthetic TGFβ3 protein, or recombinant expression TGFβ3 protein. TGFβ3 is available from various commercial sources.

[0117] According to this disclosure, the concentration of the growth factor is not particularly limited as long as it supports the long-term expansion culture 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.

[0118] corticoid compounds According to some embodiments, the culture medium further comprises a corticoid compound.

[0119] Corticoids (also known as corticosteroids) are a group of steroid hormones produced in the adrenal cortex of vertebrates, and their synthetic analogues. The two major classes of corticosteroids, glucocorticoids and mineralocorticoids, are involved in a wide range of physiological processes, including stress responses, immune responses, and the regulation of inflammation, carbohydrate metabolism, protein catabolism, blood electrolyte levels, and behavior. In this disclosure, corticoid compounds can enhance the proliferation of mesenchymal stem cells (MSCs).

[0120] According to some embodiments, the corticoid compound is selected from the group consisting of hydrocortisone, corticosterone, dehydrocorticosterone, cortisone, and any combination thereof. According to a preferred embodiment, the corticoid compound is hydrocortisone.

[0121] According to this disclosure, the concentration of the corticoid compound is not particularly limited as long as it supports the long-term expansion culture of MSCs. According to some embodiments, the concentration of the corticoid compound is 0.1-5 μM, for example 0.1-0.5 μM, 0.1-1 μM, 0.1-1.5 μM, 0.1-2 μM, 0.1-2.5 μM, 0.1-3 μM, 0.1-3.5 μM, 0.1-4 μM, 0.1-4.5 μM, 0.1-5 μM, 0.2-0.5 μM, 0.2-1 μM, 0.2-1.5 μM, 0.2-2 μM, 0.2-2.5 μM, 0.2-3 μM, 0.2-3.5 μM, 0.2-4 μM, 0.2-4.5 μM, 0.2-5 μM, 0.3-0.5 μM, 0.3-1 μM, 0.3-1 These ranges are 0.5 μM, 0.3~2 μM, 0.3~2.5 μM, 0.3~3 μM, 0.3~3.5 μM, 0.3~4 μM, 0.3~4.5 μM, 0.3~5 μM, 0.4~0.5 μM, 0.4~1 μM, 0.4~1.5 μM, 0.4~2 μM, 0.4~2.5 μM, 0.4~3 μM, 0.4~3.5 μM, 0.4~4 μM, 0.4~4.5 μM, 0.4~5 μM, 0.5~1 μM, 0.5~1.5 μM, 0.5~2 μM, 0.5~2.5 μM, 0.5~3 μM, 0.5~3.5 μM, 0.5~4 μM, 0.5~4.5 μM, or 0.5~5 μM. According to some embodiments, the concentration of the corticoid 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 preferred embodiments, the concentration of the corticoid compound is 0.5 to 5 μM.

[0122] Human serum albumin (HSA) According to some embodiments, the culture medium further comprises human serum albumin (HSA). Human serum albumin is serum albumin found in human blood. It is the most abundant protein in human plasma and constitutes about half of the serum proteins. In this disclosure, HSA can enhance the expansion culture of MSCs.

[0123] HSA may include dialysis-induced HSA or non-dialysis-induced HSA. According to a preferred embodiment, human serum albumin (HSA) is dialysis-induced HSA. Compared to non-dialysis-induced HSA, dialysis-induced HSA can further enhance the expansion culture of MSCs and can significantly reduce the HPLT concentration required to enhance the expansion culture of MSCs.

[0124] Dialyzed HSA can be prepared from HSA by dialysis. Specifically, HSA is placed in a dialysis bag, and the bag is placed overnight in a 4°C DPBS solution to perform dialysis. The ratio of HSA to DPBS is 1:50 (v:v), and the pore size of the bag is 15KD.

[0125] According to this disclosure, the concentration of human serum albumin is not particularly limited as long as it supports the long-term expansion culture of MSCs. According to some embodiments, the concentration of human serum albumin is 1 to 20 mg / ml, for example 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 preferred embodiments, the concentration of human serum albumin is 1 to 10 mg / ml.

[0126] Heparin compounds According to some embodiments, the culture medium further comprises a heparin-based compound.

[0127] As used herein, “heparin compounds” means heparin, its derivatives, salts thereof, or mixtures thereof.

[0128] Heparin is a highly sulfated glycosaminoglycan primarily produced and stored by mast cells, and is understood to have the highest net negative charge density of any known biological molecule. Its negative charge binds to positively charged heparin-binding domains present in numerous extracellular proteins. Such proteins include, for example, fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), bone morphogenetic proteins (BMPs), and large extracellular structural molecules such as fibronectin and laminin.

[0129] Examples of heparin derivatives include, but are not limited to, substituted heparins. Examples of salts of heparin or its derivatives include, but are not limited to, sodium heparin salts and lithium heparin salts, as well as salts of substituted heparin.

[0130] In this disclosure, heparin compounds can further enhance the expansion of MSC culture.

[0131] According to a preferred embodiment, the heparin compound is heparin sodium.

[0132] According to this disclosure, the concentration of the heparin compound is not particularly limited as long as it supports the long-term expansion culture of MSCs. According to some embodiments, the concentration of the heparin 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 ~150μg / ml, 2~5μg / ml, 2~10μg / ml, 2~15μg / ml, 2~20μg / ml, 2~25μg / ml, 2~30μg / ml, 2~35μg / ml, 2~40μg / ml, 2~45μg / ml, 2~50μg / ml, 2~60μg / m l, 2~70μg / ml, 2~80μg / ml, 2~90μg / ml, 2~100μg / ml, 2~110μg / ml, 2~120μg / ml, 2~130μg / ml, 2~140μg / ml, 2~150μg / ml, 3~5μg / ml, 3~10μg / ml, 3 ~15μg / ml, 3~20μg / ml, 3~25μg / ml, 3~30μg / ml, 3~35μg / ml, 3~40μg / ml, 3~45μg / ml, 3~50μg / ml, 3~60μg / ml, 3~70μg / ml, 3~80μg / ml, 3~90μg / m l, 3~100μg / ml, 3~110μg / ml, 3~120μg / ml, 3~130μg / ml, 3~140μg / ml, 3~150μg / ml, 4~5μg / ml, 4~10μg / ml, 4~15μg / ml, 4~20μg / ml, 4~25μg / ml, 4 ~30μg / ml, 4~35μg / ml, 4~40μg / ml, 4~45μg / ml, 4~50μg / ml, 4~60μg / ml, 4~70μg / ml, 4~80μg / ml, 4~90μg / ml, 4~100μg / ml, 4~110μg / ml, 4~120μ g / ml, 4~130μg / ml, 4~140μg / ml, 4~150μg / ml, 5~10μg / ml, 5~15μg / ml, 5~20μg / ml, 5~25μg / ml, 5~30μg / ml, 5~35μg / ml, 5~40μg / ml, 5~45μg / ml,5~50μg / ml, 5~60μg / ml, 5~70μg / ml, 5~80μg / ml, 5~90μg / ml, 5~100μg / ml, 5~110μg / ml, 5~120μg / m l, 5~130μg / ml, 5~140μg / ml, 5~150μg / ml, 10~15μg / ml, 10~20μg / ml, 10~25μg / ml, 10~30μg / ml, 10 ~35μg / ml, 10~40μg / ml, 10~45μg / ml, 10~50μg / ml, 10~60μg / ml, 10~70μg / ml, 10~80μg / ml, 10~90μ g / ml, 10-100 μg / ml, 10-110 μg / ml, 10-120 μg / ml, 10-130 μg / ml, 10-140 μg / ml, or 10-150 μg / ml. According to some embodiments, the concentration of the heparin 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, 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 preferred embodiments, the concentration of the heparin compound is 10 to 100 μg / ml.

[0133] In some embodiments, the culture medium further comprises one or more selected from the group consisting of lipoic acid, sulfate, iron(III) salt, selenite, pirubate, monothioglycerol (MTG), and nicotinamide compounds.

[0134] Lipoic acid According to some embodiments, the culture medium further comprises lipoic acid.

[0135] According to this disclosure, the concentration of lipoic acid is not particularly limited as long as it supports the long-term expansion culture of MSCs. According to some embodiments, the concentration of lipoic acid is 1 to 20 μM, for example 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.

[0136] Sulfate According to some embodiments, the culture medium further comprises a sulfate. Examples of sulfates include FeSO4, MgSO4, CuSO4, and ZnSO4. According to a preferred embodiment, the sulfate is FeSO4.

[0137] According to this disclosure, the sulfate concentration is not particularly limited as long as it supports the long-term expansion culture of MSCs. According to some embodiments, the sulfate concentration 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, 3~20μM, 3~25μM, 3~30μM, 3~35μM, 3~40μM, 3~45μM, 3~50μM, 4~5μM, 4~10μM, 4~15μM, 4~20μM, 4~25μM, 4~30μM, 4 ~35 μM, 4-40 μM, 4-45 μM, 4-50 μM, 5-10 μM, 5-15 μM, 5-20 μM, 5-25 μM, 5-30 μM, 5-35 μM, 5-40 μM, 5-45 μM, or 5-50 μM. According to some embodiments, the sulfate concentration 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 preferred embodiments, the sulfate concentration is 1 to 30 μM.

[0138] Iron(III) salts According to some embodiments, the culture medium further comprises an iron(III) salt. Examples of iron(III) salts include Fe(NO3)3, FeCl3, and iron(III) citrate. According to a preferred embodiment, the iron(III) salt is Fe(NO3)3.

[0139] According to this disclosure, the concentration of iron(III) salt is not particularly limited as long as it supports the long-term expansion culture of MSCs. According to some embodiments, the concentration of iron(III) 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. μM, 0.7~5μM, 0.7~10μM, 0.7~15μM, 0.7~20μM, 0.8~5μM, 0.8~10μM, 0.8~1 5μM, 0.8~20μM, 0.9~5μM, 0.9~10μM, 0.9~15μM, 0.9~20μM, 1~5μM, 1~10μM, 1~15μM, 1~20μM, 2~5μM, 2~10μM, 2~15μM, 2~20μM, 3~5μM, 3~10μM, 3~15μM, 3-20 μM, 4-5 μM, 4-10 μM, 4-15 μM, 4-20 μM, 5-10 μM, 5-15 μM, or 5-20 μM. According to some embodiments, the concentration of the iron(III) 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, 0.9 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 preferred embodiments, the concentration of the iron(III) salt is 0.1 to 10 μM.

[0140] Selenite According to some embodiments, the culture medium further comprises selenite. Examples of selenite include Na2SeO3 and K2SeO3. According to a preferred embodiment, the selenite is Na2SeO3.

[0141] According to this disclosure, the concentration of selenite is not particularly limited as long as it supports the long-term expansion culture 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 ~10ng / ml, 3~15ng / ml, 3~20ng / ml, 3~25ng / ml, 3~30ng / ml, 4~5ng / ml, 4~10ng / ml, 4~15ng / ml, 4~20 ng / ml, 4~25ng / ml, 4~30ng / ml, 5~10ng / ml, 5~15ng / ml, 5~20ng / ml, 5~25ng / ml, or 5~30ng / ml. According to some embodiments, the selenite concentration 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 ng / ml. According to a preferred embodiment, the selenite concentration is 5 to 20 ng / ml.

[0142] Pilbert 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.

[0143] According to this disclosure, the concentration of pirubate is not particularly limited as long as it supports the long-term expansion culture of MSCs. According to some embodiments, the concentration of pirubate 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, 0.03~5mg / ml, 0.03~7.5mg / ml, 0.03~10mg / ml, 0.05~1mg / ml, 0.05~2mg / ml, 0.05~3mg / ml, 0.05~4mg / ml, 0.05~5mg / ml, 0.05~7.5mg / ml, 0.05~10mg / ml, 0.075~1mg / ml, 0.075~2mg / ml, 0.075~3mg / ml, 0.075~4mg / ml, 0.075~5mg / ml, 0.075~7.5mg / ml, 0.075~10mg / ml, 0.1~1mg / ml, 0.1~2mg / ml, 0.1~3mg / ml, 0.1~4mg / ml, 0.1~5mg / ml, 0.1~7.5mg / ml, 0.1~10mg / ml, 0.25~1mg / ml, 0.25~2mg / ml, 0.25~3mg / ml, 0.25~4mg / ml, 0.25~5mg / ml, 0.25~7.5mg / ml, 0.25~10mg / ml, 0.5~1mg / ml, 0.5~2mg / ml , 0.5~3mg / ml, 0.5~4mg / ml, 0.5~5mg / ml, 0.5~7.5mg / ml, 0.5~10mg / ml, 0.75~1mg / ml, 0.75~2mg / ml, 0.75~3mg / ml, 0.75~4mg / ml, 0.75~5mg / ml, 0.75~7.5mg / m, 0.75~10mg / ml, 1~2mg / ml, 1~3mg / ml, 1~4mg / ml, 1~5mg / ml, 1~7.5mg / m, or 1~10mg / ml.According to some embodiments, the concentration of pirubate 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 preferred embodiments, the concentration of pirubate is 0.05 to 5 mg / ml.

[0144] Monothioglycerol (MTG) According to some embodiments, the culture medium further comprises monothioglycerol (MTG).

[0145] According to this disclosure, the concentration of MTG is not particularly limited as long as it supports the long-term expansion culture 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 30μM, 2~35μM, 2~40μM, 2~45μM, 2~50μM, 2~60μM, 2~70μM, 2~80μM, 2~90μM, 2~100μM, 2~150μM, 2~200μM, 3~5μM, 3~10 μM, 3~15μM, 3~20μM, 3~25μM, 3~30μM, 3~35μM, 3~40μM, 3~45μM, 3~50μM, 3~60μM, 3~70μM, 3~80μM, 3~90μM, 3~100μM, 3~150μM, 3~200μM, 4~5μM, 4~10μM, 4~15μM, 4~20μM, 4~25μM, 4~30μM, 4~35μM, 4~40μM, 4~45μM, 4~50μM, 4~60μM, 4~7 0μM, 4~80μM, 4~90μM, 4~100μM, 4~150μM, 4~200μM, 5~10μM, 5~15μM, 5~20μM, 5~25μM, 5~30μM, 5~35μM, 5~40μM, 5~45 μM, 5~50μM, 5~60μM, 5~70μM, 5~80μM, 5~90μM, 5~100μM, 5~150μM, 5~200μM, 10~15μM, 10~20μM, 10~25μM, 10~30μM, 1 0-35 μM, 10-40 μM, 10-45 μM, 10-50 μM, 10-60 μM, 10-70 μM, 10-80 μM, 10-90 μM, 10-100 μM, 10-150 μM, or 10-200 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 a preferred embodiment, the concentration of MTG is 10 to 150 μM.

[0146] Nicotinamide (NAM) compounds According to some embodiments, the culture medium further comprises a nicotinamide compound.

[0147] As used herein, “nicotinamide compounds” means nicotinamide, its analogues, metabolites of nicotinamide or nicotinamide analogues, such as NAD, NADH, and NADPH, their salts, or mixtures thereof.

[0148] According to embodiments of this disclosure, the nicotinamide compound may be selected from the group consisting of nicotinamide, nicotinamide analogs, nicotinamide metabolites, nicotinamide analog metabolites, and derivatives thereof.

[0149] Nicotinamide is an amide-type niacin, and both belong to the vitamin B3 family. They are precursors to nicotinamide adenine dinucleotide (NAD) and act as coenzymes in several cellular processes, including energy metabolism and DNA repair. Nicotinamide can be converted to nicotinamide mononucleotide (NMN) by nicotinamide phosphoribosyltransferase (NAMPT), which then converts to NAD by nicotinamide mononucleotide adenylyltransferase (NMNAT). + It will be converted.

[0150] As used herein, “nicotinamide analog” refers to any molecule known to act similarly to nicotinamide. Examples of nicotinamide analogs include, but are not limited to, nicotinthioamides (thiol analogs of nicotinamide) and nicotinic acid. Examples of nicotinamide derivatives include, but are not limited to, substituted nicotinamide compounds and substituted nicotinthioamides, as well as N-substituted nicotinamide compounds and N-substituted nicotinthioamides.

[0151] Examples of salts of nicotinamide, its analogues, its metabolites, and derivatives include, but are not limited to, nicotinamide hydrochloride, nicotinamide hydrobromide, and salts thereof of substituted nicotinamides.

[0152] According to a preferred embodiment, the nicotinamide compound is nicotinamide.

[0153] According to this disclosure, the concentration of the nicotinamide compound is not particularly limited as long as it supports the long-term expansion culture of MSCs. According to some embodiments, the concentration of the nicotinamide compound is 0.1 to 30 mM, for example 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 It is in the range of ~20mM, 0.75~25mM, 0.75~30mM, 1~5mM, 1~10mM, 1~15mM, 1~20mM, 1~25mM, 1~30mM, 2~5mM, 2~10mM, 2~15mM, 2~20mM, 2~25mM, 2~30mM, 3~5mM, 3~10mM, 3~15mM, 3~20mM, 3~25mM, 3~30mM, 4~5mM, 4~10mM, 4~15mM, 4~20mM, 4~25mM, 4~30mM, 5~10mM, 5~15mM, 5~20mM, 5~25mM, or 5~30mM. According to some embodiments, the concentration of the nicotinamide 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, 0.9 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 preferred embodiments, the concentration of the nicotinamide compound is 0.5 to 20 μM.

[0154] In some embodiments, the culture medium contains, in basal medium, 5-30 μM of an ethanolamine compound, 1-20 μM of a putrescine compound, 0.1-10% by volume of HPLT, 1-150 μg / ml of transferrin, 1-10 μg / ml of an insulin compound, 1-150 μg / ml of an antioxidant, and 0.5-5% by volume of glutamine or a derivative thereof.

[0155] In some embodiments, the culture medium contains, in basal medium, 5–30 μM ethanolamine, 1–20 μM putrescine dihydrochloride, 0.1–10 vol% HPLT, 1–150 μg / ml transferrin, 1–10 μg / ml insulin, 1–150 μg / ml ascorbate, and 0.5–5 vol% glutamine.

[0156] In some embodiments, the culture medium contains, in basal medium, 5–30 μM ethanolamine, 1–20 μM putrescine dihydrochloride, 0.1–10 vol% HPLT, 1–150 μg / ml transferrin, 1–10 μg / ml insulin, 1–150 μg / ml ascorbate, 0.5–5 vol% glutamine, 1–15 ng / ml FGF2, 0.5–5 μM hydrocortisone, 1–10 μM lipoic acid, 1–30 μM FeSO4, and 0.1–10 μM Fe(NO3)3.

[0157] In some embodiments, the culture medium contains, in basal medium, 5–30 μM ethanolamine, 1–20 μM putrescine dihydrochloride, 0.1–10% by volume HPLT, 1–150 μg / ml transferrin, 1–10 μg / ml insulin, 1–150 μg / ml ascorbate, 0.5–5% by volume glutamine, 1–15 ng / ml FGF2, 0.5–5 μM hydrocortisone, 5–20 ng / ml Na2SeO3, 0.05–5 mg / ml sodium pyruvate, 1–10 mg / ml HSA, 10–150 μM MTG, 0.5–20 mM nicotinamide (NAM), and 10–100 μg / ml heparin sodium.

[0158] In some embodiments, this culture medium provides a high seeding density (e.g., 5 × 10) for MSCs. 4 The culture medium provides a magnification of approximately 13 to 15 times (cells / well). In some embodiments, this culture medium provides a high seeding density (e.g., 5 × 10⁶) for MSCs. 4(Cells / well), providing a magnification of about 15 to about 30 times. In contrast, the control medium provides a magnification of about 8 to about 10 times for MSCs at the same high seeding density (e.g., 5×10 4 (Cells / well).

[0159] In some embodiments, the present culture medium provides a magnification of about 650 to about 1×10 3 times for MSCs at a low seeding density (e.g., 1×10 3 (Cells / well). In some embodiments, the present culture medium provides a magnification of about 1×10 3 (Cells / well) to about 4.5×10 3 times for MSCs at a low seeding density (e.g., 1×10 3 (Cells / well). In contrast, the control medium provides a magnification of about 500 to about 600 times for MSCs at the same low seeding density (e.g., 1×10 3 (Cells / well).

[0160] In some embodiments, the present culture medium stably supports long-term expansion culture of MSCs with a flat PDT curve of about 20 hours to about 24 hours. In contrast, the control medium provides a steep PDT curve for MSCs from about 25 hours to about 57 hours during long-term expansion culture.

[0161] 2. Kit According to one aspect of the present invention, there is provided a kit comprising the above-described culture medium disclosed herein. The culture medium can be filled in a suitable packaging material such as a container. The kit can optionally include a specification of the culture medium or the components therein, or an instruction manual for the culture medium or the components therein.

[0162] The container of the kit can generally be a vial, flask, bottle or any other container capable of properly containing the components. If there are more than one component in the kit, the kit can also include second, third and other additional containers for separately containing the additional components. However, various combinations of the components may be included in one container.

[0163] In some embodiments, the culture medium is filled into a single container. In some embodiments, the components of the culture medium are filled into different containers separately.

[0164] The components of the kit may be provided as one or more solutions. However, the components of the kit may also be provided as dry powders. If the components are provided as dry powders, the powders can be reconstituted by adding a suitable solvent.

[0165] 3. Method for expanding culture According to one aspect of this disclosure, a method for expanding mesenchymal stem cells (MSCs) is provided, comprising contacting MSCs with a culture medium disclosed herein.

[0166] In some embodiments, this method involves raising MSCs from approximately 500 cells / well to approximately 1 × 10⁶ cells. 5 The method further includes seeding at a cell / well density. In some embodiments, the method involves seeding MSCs at approximately 5 × 10⁶ 4 Cells / well ~ approximately 1 x 10⁻⁶ 5 The method further includes seeding at a high density of cells / well. In some embodiments, the method involves seeding MSCs at a density of approximately 500 cells / well to approximately 1 × 10⁶ cells / well. 3 This further includes seeding at a low cell / well density.

[0167] In some embodiments, the method further includes replacing the expansion culture medium with the same medium every few days during expansion culture. In some embodiments, the method further includes replacing the expansion culture medium with the same medium every three days during expansion culture.

[0168] In some embodiments, MSCs are continuously expanded for multiple passages. In some embodiments, MSCs are continuously expanded for at least two passages. In some embodiments, MSCs are continuously expanded for at least three passages. In some embodiments, MSCs are continuously expanded for at least four passages. In some embodiments, MSCs are continuously expanded for at least five passages. In some embodiments, MSCs are continuously expanded for at least six passages. In some embodiments, MSCs are continuously expanded for at least seven passages. In some embodiments, MSCs are continuously expanded for at least eight passages. In some embodiments, MSCs are continuously expanded for at least nine passages.

[0169] According to this disclosure, MSCs produced by the methods disclosed herein are a substantially homogeneous cell population even when the MSCs are continuously expanded and cultured over multiple passages, such as at least 4, 7, or 9 passages. Specifically, MSCs produced by the methods disclosed herein can maintain MSC characteristics such as morphology, phenotype, differentiation potential, and immunomodulatory effects, even when the MSCs are continuously expanded and cultured over multiple passages.

[0170] 4. Cell population According to one aspect of this disclosure, a substantially homogeneous population of MSCs produced by the method of this disclosure is provided.

[0171] According to some embodiments, a substantially homogeneous population of MSCs is a population of cells 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, a substantially homogeneous population of MSCs is a population of cells at passage 2, passage 3, passage 4, passage 5, passage 6, passage 7, passage 8, or passage 9.

[0172] According to some embodiments, the population of MSCs of the present disclosure may be cryopreserved or stored for further expansion culture and / or differentiation.

[0173] According to this disclosure, a substantially homogeneous population of MSCs is CD90 during long-term expansion culture. + The percentage of cells is high. In some embodiments, a substantially homogeneous population of MSCs has at least 90% CD90 + Includes cells. In some embodiments, a substantially homogeneous population of MSCs contains at least 95% CD90 + Includes cells. In some embodiments, a substantially homogeneous population of MSCs contains at least 99% CD90 + Includes cells. In some embodiments, a substantially homogeneous population of MSCs is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% CD90 + Contains cells.

[0174] Examples Some embodiments of the present invention will be demonstrated below with reference to examples. However, it should be understood that these examples are illustrative only and are not intended to be entirely definitive regarding the conditions and scope of the present invention.

[0175] Furthermore, unless otherwise described in detail, the following examples were performed using standard techniques that are well known and commonly used to those skilled in the art. As stated above, the following examples are provided for illustrative purposes only and should not be construed as limiting the scope of the present invention.

[0176] material All reagents and equipment used throughout the examples of this disclosure are commercially available. Some of the reagents used in the examples are listed below.

[0177] [Table 1]

[0178] [Table 1-2]

[0179] Examples 1-4 This example was conducted to demonstrate that HPLT is crucial for supporting the long-term expansion culture of iMSCs.

[0180] Example 1 Frozen iMSCs (RC01005, Nuwacell Co., Ltd.) were thawed by seeding 1000 cells / well in M1 medium into 6-well plates. After 24 hours, the above medium was replaced with 2 ml / well of one of the following media: M1, SFM-M, PPRF, or MEM-FBS. The M1 medium contained IMDM (+NaHCO3) / F12 (50% / 50%), 2 vol% Ultroser G (serum substitute (SR)), 1 vol% Glutamax, 1-150 μg / ml (e.g., 120 μg / ml) transferrin, 5 μg / ml insulin, 1-150 μg / ml (e.g., 110 μg / ml) magnesium ascorbate, 20 μM ethanolamine, and 10 μM putrescine 2HCl. SFM-M contained IMDM / F12 (50% / 50%), 1 vol% Glutamax, 5 μg / ml insulin, 5 μg / ml transferrin, 50 μg / ml magnesium 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, 1 ng / ml IL-3, and 1 ng / ml GM-SCF. PPRF medium contained F12, 2 vol% Glutamax, 25 μg / ml insulin, 25 μg / ml transferrin, 50 μg / ml magnesium ascorbate, 55.9 μM putrescine 2HCl, 20.5 mM NaHCO3, 4.9 mM HEPES, 4 mg / ml HSA, 0.10% chemically defined lipids, 36 ng / ml hydrocortisone, 5.66 ng / ml progesterone, 2 ng / ml FGF2, and 1 ng / ml TGFβ1. MEM-FBS medium contained 90% (v / v) αMEM and 10% (v / v) FBS. During subsequent cell culture, the medium was replaced with the same medium every three days. On day 10 of culture, cells were observed and photographed using a phase-contrast microscope.Figure 1 shows the cell morphology of iMSCs cultured in the different culture media described above.

[0181] As shown in Figure 1, cells cultured in M1 and MEM-FBS expanded well, but cells cultured in other media (SFM-M and PPRF) did not expand. Although the inventors demonstrated in the above examples that M1 medium and MEM-FBS medium can support the expansion culture of iMSCs, the Ultroser G contained as the main component in M1 contains animal-derived components, and the FBS contained in MEM-FBS medium is also animal-derived serum, which was not a good choice for clinical application. Therefore, developing serum-free, xeno-free media for the expansion culture of MSCs (e.g., iMSCs) would have great clinical significance.

[0182] Example 2 Frozen iMSCs (RC01005, Nuwacell Co., Ltd.) were thawed by seeding 50,000 cells / well in M1 medium into a 6-well plate. After 24 hours, the medium was replaced with 2 ml / well of either M1 or M2 medium. M1 medium had the composition described in Example 1 and was used as a control. Compared to M1 medium, M2 medium had the same composition except that HPLT was used instead of Ultrasar G. M2 medium contained HPLT at the indicated concentrations (1 vol%, 2 vol%, 3 vol%, 4 vol%, or 5 vol%). On day 4 of culture, cells were digested with sorase (RP01021, Nuwacell Co., Ltd.) for 3 minutes and collected. They were then centrifuged, the supernatant was removed, and the cells were resuspended in the corresponding culture medium. The number of iMSC cells was counted using a viable-dead-cell analyzer (Vi-CELL® XR, BECKMAN COULTER). Prior to digestion, cells were observed and photographed using a phase-contrast microscope. The cell morphology of the iMSCs is shown in Figure 2A, and the number of iMSC cells is shown in Figure 2B.

[0183] As shown in Figure 2A, iMSC cells cultured in M2 containing HPLT were found to expand well and retain their original morphological characteristics (spindle-shaped fibroblast-like) similar to those cultured in M1 medium. As shown in Figure 2B, the number of iMSC cells cultured in M2 containing HPLT was significantly higher than the number of iMSC cells cultured in M1 containing Ultraser G (the former was approximately 1.5 to 3 times higher than the latter), demonstrating that replacing Ultraser G with HPLT not only avoids the use of animal-derived components but also greatly improves the expansion efficiency of iMSC cells. In addition, Figure 2B shows that differences in HPLT concentration can have some effect on the expansion efficiency of iMSC cells.

[0184] Example 3 Frozen iMSCs (RC01005, Nuwacell Co., Ltd.) were thawed by seeding 50,000 cells / well in M1 medium into a 6-well plate. After 24 hours, the medium was replaced with 2 ml / well of M1 or M2 (3% HPLT). M1 medium had the composition 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 continuously cultured and passaged. The number of cells in each passage from passage 6 to passage 8 was counted using a viable and dead cell analyzer (Vi-CELL® XR, BECKMAN COULTER) according to the protocol described in Example 2 (Figure 3A).

[0185] iMSCs at passages 1, 4, 5, and 7 were stained with CD90-FITC antibody (BD Pharminge, 555595), and CD90 + The percentage of cells was detected by flow cytometry analysis (Figure 3B). Briefly, the cells were digested with solase (RP01021, Nuwacell Co., Ltd.) for 3 minutes, resuspended in assay buffer (0.5% BSA and 0.5 mM EDTA in DPBS), and the density was set to 5 × 10⁻⁶. 5 ~1 × 10 6The cells were prepared in tubes. After incubation with CD90-FITC antibody at 4°C for 30 minutes, the samples were washed with assay buffer, the assay buffer was removed, and the samples were resuspended in 100 μl of assay buffer. Using a flow cytometer (CytoFLEX, BECKMAN COULTER), the CD90 antibody was measured. + The percentage of cells was analyzed.

[0186] As shown in Figure 3A, iMSCs cultured in M2 had a higher cell count than iMSCs cultured in M1 for all P6, P7, and P8 cell stages, demonstrating that replacing Ultraser G with HPLT can improve expansion efficiency for iMSC cells at each passage, particularly for high passage iMSC cells. As shown in Figure 3B, when iMSCs were cultured using M1, CD90-expressing cells rapidly decreased from 99.81% in P1 to 88.87% in P4, 70.04% in P5, and 44.37% in P7, indicating that M1 cannot stably maintain the stem cell phenotype of iMSCs during long-term expansion culture. In particular, CD90-expressing cells decreased more rapidly during long-term expansion culture of iMSCs. In contrast, when iMSCs were cultured using M2, the proportion of CD90-expressing cells was more stably maintained from P1 to P7. Therefore, the data in Figures 3A and 3B demonstrate that replacing Ultroser G with HPLT improves cell expansion efficiency for each passage and more stably maintains the stem cell phenotype of iMSCs during long-term expansion culture. In addition, when iMSCs were cultured using M2, several small peaks appeared on the left side during long-term expansion culture of iMSCs (Figure 3B).

[0187] Example 4 The immunosuppressive effect of iMSCs was evaluated using a CFSE-based T cell proliferation assay. To expand T cell culture, PBMCs were activated for 4 days with immobilized CD3 antibody (biogerm, 05121-25-100) and CD28 antibody (biogerm, 10311-25-100) in TPA medium (T cell proliferation assay medium: RPMI1640 + 10% (v / v) FBS + 1% (v / v) Glutamax) supplemented with 100 IU / ml IL-2 (Jiangsu Jinsili Pharmaceutical Co., Ltd, China). Activated T cells were collected, then stained with CFSE (Ab145291, Abcam), and cultured for 5 days in co-culture with two types of iMSCs cultured according to Example 3, or without those iMSCs. Specifically, cell proliferation was inhibited by treating passage 3 iMSCs with mitomycin C before co-culturing with T cells. Next, T cells were added to the treated iMSCs in a 1:1 ratio. The cell morphology on day 5 of co-culturing is shown in Figure 4A. Following the protocol described in Example 3, the percentage of divided T cell populations (dark gray, low CFSE population) was detected by flow cytometry (Figure 4B).

[0188] As shown in Figure 4A, cell division of T cells cultured without iMSCs was strongly activated, resulting in a significant decrease in CFSE fluorescence intensity (the peak shifted to the left), while T cell expansion with iMSCs was significantly inhibited (the peak hardly shifted) (Figure 4B). In addition, as indicated by the percentage of divided T cell population, iMSCs cultured in M2 had an immunosuppressive effect comparable to those cultured in M1. Therefore, the data in Figure 4 demonstrate that P3 iMSCs cultured in M2 retain an immunosuppressive effect on T cells.

[0189] Examples 1-4 above demonstrate that while M1 medium containing ethanolamine and putrescine 2HCl can support the expansion culture of iMSCs, it lacks HPLT and therefore cannot stably maintain the stem cell phenotype (CD90 expression) of iMSCs during long-term expansion culture. On the other hand, M2 medium containing ethanolamine, putrescine 2HCl, and HPLT can stably maintain the stem cell phenotype (CD90 expression) of iMSCs during long-term expansion culture. This demonstrates that HPLT is extremely important for stably maintaining the stem cell phenotype of iMSCs during long-term expansion culture.

[0190] In addition, compared to M1 medium, M2 medium avoided the use of animal-derived components, promoted robust iMSC proliferation, and provided iMSCs with a comparable immunosuppressive effect.

[0191] In summary, by combining the results of Examples 1-4, it was found that M2 supports long-term expansion culture of iMSCs without loss of MSC characteristics (including morphology, phenotype, differentiation potential, and immunomodulatory effects). This demonstrates the high potential of M2 for clinical applications.

[0192] Example 5 This example was performed to demonstrate that FGF2 and / or hydrocortisone further enhance the expansion culture of iMSCs.

[0193] Frozen iMSCs (RC01005, Nuwacell Co., Ltd.) were thawed by seeding 1000 cells / well in M1 medium into a 6-well plate. After 24 hours, the medium was replaced with 2 ml / well of the following medium: M1; or M2 (5% HPLT), which had one or more growth factors (F: FGF2 (10 ng / ml, Nuwacell Co., Ltd.); P: PDFG-BB (10 ng / ml, catalog number: 100-14B-100, Peprotech); T: TGFβ1 (1 ng / ml, catalog number: 100-21, Peprotech)) added, or not, in the presence or absence of hydrocortisone (1 μM, catalog number: 4098, Tocris). Medium M1 had the composition described in Example 1 and was used as a control. M2 (5% HPLT) had the same composition as described in Example 2. The culture medium was replaced with the same medium every three days. On day 10 of culture, the cells were digested with sorase (RP01021, Nuwacell Co., Ltd.) for 3 minutes and collected, then centrifuged, the supernatant was removed, and the cells were resuspended in the corresponding medium. The number of iMSC cells was counted using a viable and dead cell analyzer (Vi-CELL® XR, BECKMAN COULTER) (Figure 5).

[0194] As shown in Figure 5, the number of iMSC cells cultured in M2 (5% HPLT) with FGF2 alone, a combination of FGF2 and PDFG-BB or TGFβ1, or a combination of FGF2, PDFG-BB and TGFβ1, was significantly higher than that of iMSC cells cultured in M1 (the former being approximately 4.5 to 9 times higher than the latter), and was far superior to iMSC cells cultured in M2 (5% HPLT) without any growth factors (the former being approximately 2.3 to 3.75 times higher than the latter). This demonstrates that the addition of one or more growth factors can clearly enhance the expansion of iMSC culture. Furthermore, the addition of hydrocortisone can enhance iMSC proliferation, especially in the presence of the indicated growth factors, and the addition of hydrocortisone clearly enhanced the expansion of iMSC culture.

[0195] Examples 6-10 These examples were conducted to demonstrate that ethanolamine and putrescine 2HCl are crucial for supporting the long-term expansion culture of iMSCs.

[0196] Example 6 Frozen iMSCs (RC01005, Nuwacell Co., Ltd.) were thawed by seeding 1000 cells / well in M1 medium into a 6-well plate. After 24 hours, the medium was replaced with 2 ml / well of the following media: I, II, III, IV, V, or M1. The M1 medium had the composition described in Example 1. Medium I contained IMDM base (+NaHCO3) (50%), Ham F12 (50%), 1–150 μg / ml (e.g., 110 μg / ml) of magnesium ascorbate, 20 μM of ethanolamine, 23.2 nM of progesterone, 10 μM of putrescine 2HCl, 5 μg / ml of insulin, 1–150 μg / ml (e.g., 120 μg / ml) of transferrin, 100 μM of 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 of hydrocortisone, 1 vol% of glutamac, 10 ng / ml of FGF2, 10 ng / ml of PDGF-BB, and 4 mg / ml of HSA. Medium II had the same composition as Medium I, except that 10% by volume of KOSR was added to Medium I. Medium III had the same composition as Medium I, except that 2% by volume of B27 was added to Medium I. Medium IV had the same composition as Medium I, except that 2% by volume of B27 and 1% by volume of N2 were added to Medium I. Medium V contained IMDM base (+NaHCO3) (100%), 1-150 μg / ml (e.g., 110 μg / ml) of magnesium 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 three days. The cell morphology of the culture on day 10 is shown in Figure 6A, and the cell number was counted using a viable and dead cell analyzer (Vi-CELL® XR, BECKMAN COULTER) according to the protocol described in Example 5 (Figure 6B).

[0197] As shown in Figure 6, of media I, II, III, IV, and V, only medium V (also called EPM) was able to support the expansion culture of iMSCs and was significantly superior to M1. Similar to M1, medium V also contained BIT9500, a serum substitute that is not a good option for clinical application.

[0198] Example 7 Frozen ucMSCs (RC02003, Nuwacell Co., Ltd.) and iMSCs (RC01005, Nuwacell Co., Ltd.) were thawed by seeding 50,000 cells / well in M1 medium into 6-well plates. After 24 hours, the medium was replaced with 2 ml / well of either M1 or M3 medium. Medium M1 had the composition described in Example 6 and was used as a control. Medium M3 had the same composition as the EPM shown in Example 6, except that HPLT was used instead of BIT9500. Medium M3 contained HPLT at the indicated concentrations (1 vol%, 2 vol%, 3 vol%, 4 vol%, or 5 vol%). On day 4 of culture, when the cells reached confluence, the number of iMSC cells was counted using a viable and dead cell analyzer (Vi-CELL® XR, BECKMAN COULTER) according to the protocol described in Example 2.

[0199] As shown in Figure 7, the number of ucMSCs or iMSCs cultured in M3 containing HPLT was substantially greater than the number of ucMSCs or iMSCs cultured in M1 containing Ultroser G. Therefore, it was found that HPLT completely replaces BIT9500 in EPM and has the effect of increasing the proliferation of both ucMSCs and iMSCs in a concentration-dependent manner. This demonstrates that M3 medium can support the expansion of MSCs (iMSCs and ucMSCs) due to the presence of HPLT.

[0200] Example 8 Frozen iMSCs (RC01005, Nuwacell Co., Ltd.) were thawed by seeding 50,000 cells / well in M1 medium into a 6-well plate. After 24 hours, the medium was replaced with 2 ml / well of the following media: M1, M3 (3% HPLT), and M4. Mediums M1 and M3 (3% HPLT) had the same composition as shown in Example 7 and were used as controls. Compared to M3 (3% HPLT), M4 had the same composition except that it contained an additional 10 μM putrescine 2HCl and 20 μM ethanolamine. On day 4 of culture, when the cells reached confluence, the number of iMSC cells was counted using a viable and dead cell analyzer (Vi-CELL® XR, BECKMAN COULTER) according to the protocol described in Example 2.

[0201] As shown in Figure 8, the number of iMSC cells cultured in M4 was not only significantly better than that cultured in M1 (the former being about 1.5 times greater than the latter), but also slightly greater than that cultured in M3. This demonstrates that M4 can support the expansion of iMSC culture.

[0202] Example 9 Frozen iMSCs (RC01005, Nuwacell Co., Ltd.) were thawed by seeding 50,000 cells / well in M1 medium into a 6-well plate. After 24 hours, the medium was replaced with 2 ml / well of the following media: M1, M3 (3% HPLT), or M4. The iMSCs were cultured in the indicated medium and passaged for 4 generations. M1, M3 (3% HPLT), and M4 had the composition of Example 8. P1 or P4 cells were stained with CD90-FITC antibody (BD Pharminge, 555595), and CD90 + The percentage of cells was detected by flow cytometry according to the protocol described in Example 3.

[0203] Surprisingly, the M4 is CD90 +As indicated by the higher percentage of cells, the stem cell phenotype of iMSCs was better maintained with M4 than with either M1 or M3 (Figure 9). Specifically, as shown in Figure 9, the CD90-expressing cells cultured using M1 rapidly decreased from 98.63% in P1 to 89.67% in P4, and the CD90-expressing cells cultured using M3 rapidly decreased from 96.88% in P1 to 80.74% in P4. This indicates that M1 and M3 were unable to stably maintain the stem cell phenotype of iMSCs during long-term expansion culture. In contrast, the proportion of CD90-expressing cells cultured using M4 was more stably maintained from P1 to P4. Therefore, the data in Figure 9 demonstrate that the inclusion of ethanolamine and putrescine 2HCl can stably maintain the stem cell phenotype of iMSCs during long-term expansion culture.

[0204] Example 10 Frozen iMSCs (RC01005, Nuwacell Co., Ltd.) were thawed by seeding 50,000 cells / well in M1 medium into a 6-well plate. After 24 hours, the medium was replaced with 2 ml / well of either M1 or M3 (3% HPLT). M1 and M3 (3% HPLT) had the composition of Example 9. iMSCs (RC01005, Nuwacell Co., Ltd.) were cultured in M3 (3% HPLT) and subculturised for 4 generations, and characterized as follows:

[0205] 1. Osteogenic differentiation iMSCs of P3 cultured in M3 (3% HPLT) were seeded in 6-well plates. When the culture reached 90% confluence, the medium was replaced with osteogenic differentiation medium (αMEM medium (catalog number: 11900-024, thermo) supplemented with 20 vol% FBS (catalog number: SH30071.03, Hyclone), 1% Glutamax, 50 μg / ml ascorbic acid, and 5 mM β-glycerophosphate (catalog number: G9422, sigma)). The medium was replaced with the same medium every three days until day 21. On day 21, cells differentiated to osteogenicity were stained with Alizarin Red (catalog number: 0223, ScienCell®). Cell morphology is shown in Figure 10A (center panel).

[0206] 2.Chondrogenic differentiation 5 × 10⁶ cells cultured in M3 (3% HPLT) 5 iMSCs were suspended in 15 mL centrifuge tubes with the caps loosened in chondrogenesis differentiation medium (DMEM-HG medium (catalog number: 11960-069, Thermo) supplemented with 1 M ascorbic acid, 100 nM dexamethasone (catalog number: D8893, Sigma), 1% ITS+ premix tissue culture supplement (catalog number: 354352, Corning), 10 ng / ml TGFβ1 (catalog number: 100-21-6, PeproTech), and 1% sodium pyruvate (catalog number: 11360-070, Thermo). The medium was replaced with the same medium every three days until day 28. On day 28, cell spheres were stained with Alcian blue (catalog number: A105505, Aladdin). Cell morphology is shown in Figure 10A (right panel).

[0207] 3.Adipogenic differentiation iMSCs cultured in M3 (3% HPLT) were seeded in 6-well plates. When the culture reached 90% confluence, the medium was replaced with adipogenic differentiation medium (DMEM-HG medium supplemented with 45□M IBMX (catalog no.: I5879, Sigma), 0.5□M dexamethasone (catalog no.: D8893, Sigma), 50□M indomethacin (catalog no.: I7378, Sigma), and 10% FBS). The medium was replaced with the same medium every three days until day 21. On day 21, cells differentiated to be adipogenic were stained with Oil Red O (catalog no.: O0625, Sigma Aldrich). Cell morphology is shown in Figure 10A (left panel).

[0208] To evaluate the immunosuppressive effect of the above iMSCs, a CFSE-based T cell proliferation assay was performed according to the protocol described in Example 4 (Figure 10B).

[0209] iMSCs of P3 cultured in M3 (3% HPLT) medium were found to retain MSC characteristics, including triphyletic differentiation (Figure 10A) and immunosuppressive effects (Figure 10B).

[0210] Examples 6-10 demonstrate that while M3 medium containing HPLT can support the expansion culture of iMSCs, it lacks ethanolamine and putrescine 2HCl, making it unable to stably maintain the stem cell phenotype (CD90 expression) of iMSCs during long-term expansion culture. On the other hand, M4 medium containing HPLT, ethanolamine, and putrescine 2HCl can more stably maintain the stem cell phenotype (CD90 expression) of iMSCs during long-term expansion culture. This demonstrates that ethanolamine and putrescine 2HCl are extremely important for stably maintaining the stem cell phenotype (CD90 expression) of iMSCs during long-term expansion culture.

[0211] In addition, compared to M1, M4 avoided the use of animal-derived components, promoted robust iMSC proliferation, and exerted immunosuppressive effects on iMSCs.

[0212] In summary, by combining the results from Examples 6-10, it was found that M4 supports long-term expansion culture of iMSCs without loss of iMSC characteristics (including morphology, phenotype, differentiation potential, and immunomodulatory effects). This demonstrates the high potential of M4 for clinical applications.

[0213] By combining Examples 1-4 with Examples 6-10, it was found that ethanolamine, putrescine 2HCl, and HPLT synergistically support the long-term expansion culture of iMSCs.

[0214] Examples 11-13 These examples were conducted to demonstrate that FGF2, HSA, and / or heparin sodium can further enhance the expansion culture of iMSCs.

[0215] Example 11 As an example, to verify the effect of FGF2 on the expansion culture of iMSCs, M3 medium containing 5% HPLT was used. Frozen iMSCs (RC01005, Nuwacell Co., Ltd.) were thawed by seeding 50,000 cells / well in M1 medium in a 6-well plate. After 24 hours, the medium was replaced with 2 ml / well of the following: M1; or M3 (containing 5% HPLT) with additional FGF2 (Nuwacell Co., Ltd.) at different concentrations, or without FGF2. M1 medium had the composition described in Example 7. M3 (5% HPLT) had the same composition as in Example 7. On day 4 of culture, when the cells reached confluence, the number of iMSC cells was counted using a viable and dead cell analyzer (Vi-CELL® XR, BECKMAN COULTER) according to the protocol described in Example 2.

[0216] As shown in Figure 11, the number of iMSC cells cultured in M3 (5% HPLT) with additional FGF2 was significantly higher than the number of iMSC cells cultured in M3 (5% HPLT) without FGF2 (the former was approximately 3.1 times or 3.9 times higher than the latter). Similar to the results in Figure 5, FGF2 was also found to significantly enhance the expansion of iMSC culture (Figure 11).

[0217] Example 12 Considering the high price and limited supply of HPLT, HSA (Chengdu Rongsheng Pharmaceuticals Co., Ltd., China) was added to reduce the amount of HPLT. As an example, to verify the effect of HSA on the expansion culture of iMSCs, M3 medium with a low concentration of HPLT (e.g., 0.5% HPLT) was used. Frozen iMSCs (RC01005, Nuwacell Co., Ltd.) were thawed by seeding 1000 cells / well in M1 medium into a 6-well plate. M1 medium had the composition described in Example 6. After 24 hours, the medium was replaced with 2 ml / well of the following medium: M3 (0.5% HPLT) with 4 mg / L of untreated HSA (undialysis) or dialysis HSA further added, or without them. M3 (0.5% HPLT) had the same composition as M3 in Example 7, except for the concentration of HPLT. HSA dialysis was performed by placing HSA in a dialysis bag and immersing the bag overnight in a 4°C DPBS (C14190500BT, Gibco) solution. The ratio of HSA to DPBS was 1:50 (v:v), and the pore size of the bag was 15KD. The culture medium was replaced with the same medium every three days. The cell morphology of the culture on day 10 is shown in Figure 12A, and the number of cells was counted using a viable and dead cell analyzer (Vi-CELL® XR, BECKMAN COULTER) according to the protocol described in Example 5 (Figure 12B).

[0218] As shown in Figure 12A, iMSC cells cultured in M3 (0.5% HPLT) containing untreated or dialyzed HSA were found to expand compared to iMSC cells cultured in M3 (0.5% HPLT) without HSA, while maintaining their intrinsic morphological characteristics (spindle-shaped fibroblast-like). In particular, iMSC cells cultured in M3 (0.5% HPLT) containing dialyzed HSA grew much better. In addition, the effect of HSA, especially dialyzed HSA, on enhancing the expansion culture of iMSC cells was also demonstrated by cell count analysis (Figure 12B). As shown in Figure 12B, the cell count of iMSCs expanded in M3 (0.5% HPLT) containing dialyzed HSA was approximately 2.3 times that of iMSCs expanded in M3 (0.5% HPLT) containing untreated (undialyzed) HSA.

[0219] Example 13 As an example, to further investigate the effects of dialysis-induced heparin sodium and heparin sodium on the expansion culture of iMSCs, M3 medium containing HPLT was used. Frozen iMSCs (RC01005, Nuwacell Co., Ltd.) were thawed by seeding 1000 cells / well in M1 medium into a 6-well plate. The M1 medium had the composition described in Example 6. After 24 hours, the medium was replaced with 2 ml / well of the following mediums: M1; or M3, which had different HPLT concentrations and further contained 4 mg / L dialysis-induced heparin sodium or 4 mg / L dialysis-induced heparin sodium and 25 μg / ml heparin sodium (catalog number: A16198, Thermo fisher). The medium was replaced with the same medium every three days. The number of cells on day 10 of culture was counted using a viable-dead-cell analyzer (Vi-CELL® XR, BECKMAN COULTER) according to the protocol described in Example 5. The results are shown in Figure 13.

[0220] As confirmed by comparing the results in Figure 13 with those in Figure 7 (iMSC histogram), the addition of dialysis-induced heparin sodium significantly reduced the HPLT concentration required to promote iMSC growth. In addition, Figure 13 demonstrates that heparin sodium can further enhance iMSC growth.

[0221] In summary, Examples 11-13 demonstrated that the addition of HSA (particularly dialysis-induced HSA), FGF2, and / or heparin sodium can further enhance the expansion culture of iMSCs, and that the addition of dialysis-induced HSA can significantly reduce the HPLT concentration required to promote iMSC growth.

[0222] Examples 14-17 These examples were conducted with the aim of further developing serum-free, xeno-free MSC expansion culture medium M5 to more sustainably support the long-term expansion culture of iMSCs.

[0223] Example 14 Frozen iMSCs (RC01005, Nuwacell Co., Ltd.) were thawed by seeding 1000 cells / well in M1 medium into a 6-well plate. The M1 medium had the composition described in Example 1. After 24 hours, the medium was replaced with M5 medium without 2 ml / well of HPLT or with M5 medium containing different concentrations of HPLT (0.50 vol%, 1 vol%, 2 vol%, or 3 vol%). M5 medium contained IMDM+DMEM / F12 (50% / 50%), 1 vol% Glutamax, 1–150 μg / ml (e.g., 120 μg / ml) transferrin, 5 μg / ml insulin, 1–150 μg / ml (e.g., 110 μg / ml) magnesium ascorbate, 20 μM ethanolamine, 10 μM putrescine 2HCl, HPLT at the indicated concentrations (0 vol%, 0.5 vol%, 1 vol%, 2 vol%, 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 50 μg / ml sodium heparin. iMSCs were cultured in the above medium for 10 days, with the medium being replaced with the same medium every three days. The number of cells was counted using a viable and dead cell analyzer (Vi-CELL® XR, BECKMAN COULTER) according to the protocol described in Example 5.

[0224] 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 being approximately 6, 10, 13, or 14 times higher than the latter). Similarly, HPLT in M5 was found to promote iMSC proliferation.

[0225] Example 15 Frozen iMSCs (RC01005, Nuwacell Co., Ltd.) were thawed by seeding 50,000 cells / well in M1 medium into a 6-well plate. After 24 hours, the medium was replaced with 2 ml / well of the following media: M1, M4, or M5. M1 medium had the composition described in Example 14 and was used as a control. M4 medium had the composition described in Example 8. M5 medium had the composition described in Example 14. iMSCs were cultured in the indicated media and passed up to passage 9. For each passage, on day 4 of culture, when the cells reached confluence, the cell count was counted using a viable-dead-cell analyzer (Vi-CELL® XR, BECKMAN COULTER) according to the protocol described in Example 2 (Figure 15A). The cell morphology at passage 8 is shown in Figure 15B.

[0226] At high passages (P8), iMSCs cultured in M4 and M5 medium better maintained the intrinsic morphological characteristics of MSCs (spindle-shaped fibroblast-like), whereas iMSCs cultured in M1 medium failed to do so (cells became large and irregular) (Figure 15B). iMSCs cultured in M5 medium had a shorter PDT (population doubling time), which was substantially maintained at approximately 20-24 hours throughout the entire long-term expansion culture, while iMSCs cultured in M1 medium had a longer PDT, which significantly increased from P3 (approximately 25 hours) to P9 (approximately 57 hours) throughout the entire long-term expansion culture (Figure 15A). A lower PDT in each passage indicates higher expansion efficiency in each passage, while similar PDTs in each passage indicate better maintenance of expansion ability in each passage. Therefore, compared to M1 medium, M5 medium consistently supported robust iMSC growth throughout the entire long-term expansion culture.

[0227] Example 16 Frozen iMSCs (RC01005, Nuwacell Co., Ltd.) were thawed by seeding 50,000 cells / well in M1 medium into a 6-well plate. After 24 hours, the medium was replaced with 2 ml / well of the following media: M1, M4, or M5. The M1, M4, and M5 media had the compositions described in Example 15. iMSCs (RC01005, Nuwacell Co., Ltd.) cultured in M1, M4, or M5 were collected at passages 1, 4, and 7. The cells were stained with CD90-FITC antibody (BD Pharminge, 555595) and CD90 + The percentage of cells was detected by flow cytometry according to the protocol described in Example 3 (Figure 16).

[0228] As shown in Figure 16, when iMSCs were cultured using M1, CD90-expressing cells rapidly decreased from 98.77% in P1 to 84.14% in P4, and then to 81.55% in P7, indicating that M1 cannot stably maintain the stem cell phenotype of iMSCs from P1 to P7 during long-term expansion culture of iMSCs. 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), and then up to passage 7. + The percentage of cells decreased. In contrast, M5 maintained CD90 expression more stably up to passage 7 compared to M1 and M4.

[0229] Example 17 iMSCs (RC01005, Nuwacell Co., Ltd.) were cultured in M5 medium for 4 passages, and their differentiation potential into three lineages was examined according to the protocol described in Example 10. Furthermore, iMSCs (RC01005, Nuwacell Co., Ltd.) were cultured in M1 or M5 medium for 4 passages, and their immunosuppressive effects were examined according to the protocol described in Example 4. The M1 and M5 media had the compositions described in Example 15.

[0230] Similar to M1, iMSCs from P3 cultured in M5 retained MSC characteristics, including triphyletic differentiation (Figure 17A) and immunosuppressive effects (Figure 17B).

[0231] Examples 14-17 demonstrate that during long-term expansion culture of iMSCs, M4 better maintains the stem cell phenotype of iMSCs than M1, and that M5 maintains the stem cell phenotype of iMSCs more sustainably compared to M4.

[0232] In addition, compared to M1, M5 avoided the use of animal-derived components, promoted robust iMSC proliferation, and provided iMSCs with a comparable immunosuppressive effect.

[0233] In summary, by combining the results from Examples 14-17, it was found that M5 more sustainably supports the long-term expansion culture of iMSCs without losing the characteristics of MSCs (including morphology, phenotype, differentiation potential, and immunomodulatory effects). This demonstrates the high potential of M5 for clinical applications.

[0234] It will be readily apparent to those skilled in the art that the methods, compositions, and products described herein represent exemplary embodiments and are not intended to limit the scope of the disclosure. It will also be readily apparent to those skilled in the art that various substitutions and modifications can be made to the disclosure disclosed herein without departing from the scope and spirit of the disclosure.

[0235] All patents and publications referenced herein represent the level of expertise of those skilled in the art to which this disclosure relates. All patents and publications are incorporated herein by reference to the same extent as if each individual publication specifically and individually indicated that it is incorporated herein by reference.

[0236] This disclosure is not limited by the specific embodiments described in this application, which are intended as single examples of individual aspects of this disclosure. Numerous modifications and variations can be made without departing from the essence and scope of this disclosure, as will be apparent to those skilled in the art. Functionally equivalent media and kits within the scope of this disclosure, in addition to those listed herein, will be apparent to those skilled in the art from the above description. Such modifications and variations are intended to be included within the appended claims. This disclosure should be limited only by the language of the appended claims and the entire scope of equivalents to which such claims apply.

Claims

1. A serum-free, xeno-free culture medium that can support the long-term expansion culture of mesenchymal stem cells (MSCs), comprising (a) a basal medium, (b) an ethanolamine compound, (c) a putrescine compound, and (d) human platelet lysate (HPLT).

2. The culture medium according to claim 1, wherein the ethanolamine compound is present in the culture medium at a concentration of 1 to 30 μM.

3. The culture medium according to any one of claims 1 to 2, wherein the putrescine compound is present in the culture medium at a concentration of 1 to 30 μM.

4. The culture medium according to any one of claims 1 to 3, wherein the HPLT is present in the culture medium at a concentration of 0.1 to 20% by volume.

5. The culture medium according to any one of claims 1 to 3, wherein the ethanolamine compound comprises ethanolamine and the putrescine compound comprises putrescine dihydrochloride.

6. A culture medium according to any one of claims 1 to 5, further comprising transferrin.

7. The culture medium according to claim 6, wherein the transferrin is present in the culture medium at a concentration of 1 to 200 μg / ml.

8. A culture medium according to any one of claims 1 to 7, further comprising an insulin-based compound.

9. The culture medium according to claim 8, wherein the insulin-like compound is present in the culture medium at a concentration of 1 to 15 μg / ml.

10. A culture medium according to any one of claims 1 to 9, further comprising an antioxidant.

11. The culture medium according to claim 10, wherein the antioxidant is present in the culture medium at a concentration of 1 μg / mL to 200 μg / mL.

12. A culture medium according to any one of claims 1 to 11, further comprising glutamine or a derivative thereof.

13. The culture medium according to claim 12, wherein the glutamine or its derivative is present in the culture medium at a concentration of 0.1 to 5% by volume.

14. The culture medium according to any one of claims 1 to 13, wherein the basal medium contains 5 to 30 μM of ethanolamine, 1 to 20 μM of putrescine dihydrochloride, 0.1 to 10% by 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% by volume of glutamine.

15. A culture medium according to any one of claims 1 to 14, further comprising a growth factor.

16. The culture medium according to claim 15, wherein the growth factor is selected from the group consisting of EGF, IGF, VEGF, PDGF, FGF2, TGFβ and any combination thereof.

17. The culture medium according to claim 15 or claim 16, wherein the growth factor is present in the culture medium at a concentration of 1 to 20 ng / ml.

18. A culture medium according to any one of claims 1 to 17, further comprising a corticoid compound.

19. The culture medium according to claim 18, wherein the corticoid compound is selected from the group consisting of hydrocortisone, corticosterone, dehydrocorticosterone, cortisone, and any combination thereof.

20. The culture medium according to claim 18 or claim 19, wherein the corticoid compound is present in the culture medium at a concentration of 0.1 to 5 μM.

21. A culture medium according to any one of claims 1 to 19, further comprising human serum albumin (HSA).

22. The culture medium according to claim 21, wherein the HSA includes dialysis HSA.

23. The culture medium according to claim 21 or claim 22, wherein the HSA is present in the culture medium at a concentration of 1 to 20 mg / ml.

24. A culture medium according to any one of claims 1 to 23, further comprising a heparin-based compound.

25. The culture medium according to claim 24, wherein the heparin compound is present in the culture medium at a concentration of 1 to 150 μg / ml.

26. A culture medium according to any one of claims 1 to 25, further comprising one or more selected from the group consisting of lipoic acid, sulfate, iron(III) salt, selenite, pirubate, monothioglycerol (MTG), and nicotinamide compounds.

27. The basal medium contains 5-30 μM ethanolamine, 1-20 μM putrescine dihydrochloride, 0.1-10% by volume HPLT, 1-150 μg / ml transferrin, 1-10 μg / ml insulin, 1-150 μg / ml ascorbate, 0.5-5% by volume glutamine, 1-15 ng / ml FGF2, 0.5-5 μM hydrocortisone, 1-10 μM lipoic acid, and 1-30 μM FeSO4. ４ , and 0.1 to 10 μM Fe (NO ３ ) ３ A culture medium according to any one of claims 1 to 26, including the above.

28. The basal medium contains 5–30 μM ethanolamine, 1–20 μM putrescine dihydrochloride, 0.1–10% by volume HPLT, 1–150 μg / ml transferrin, 1–10 μg / ml insulin, 1–150 μg / ml ascorbate, 0.5–5% by volume glutamine, 1–15 ng / ml FGF2, 0.5–5 μM hydrocortisone, and 5–20 ng / ml Na ２ SeO ３ A culture medium according to any one of claims 1 to 26, comprising 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.

29. A method for expanding the culture of mesenchymal stem cells (MSCs), comprising contacting the MSCs with a culture medium described in any one of claims 1 to 28.

30. The method according to claim 29, wherein the MSCs are continuously expanded and cultured over multiple passages, for example, over at least four passages, seven passages, and nine passages.

31. A substantially homogeneous population of MSCs produced by the method of claim 29 or claim 30.

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