Serum-free culture medium as well as preparation method and application thereof

Abstract

The invention relates to a serum-free culture medium and a preparation method and application thereof in the technical field of cell culture. The serum-free culture medium comprises a basic culture medium and the following components in final concentration: 5 to 15 ng / mL of dexamethasone, 200 to 600 ng / mL of hydrocortisone, 20 to 200 [mu] g / mL of transferrin, 0.2 to 1 mg / mL of human serum albumin, 40 to 70 [mu] M of beta-mercaptoethanol, 5 to 12 [mu] M of glutathione, 10 to 65 mg / L of ascorbic acid, 1 to 10 ng / mL of EGF, 1 to 20 ng / mL of IGF-1, 1 to 10 ng / mL of bFGF and 1 to 10 ng / mL of PDGF-BB. The basic culture medium is IMDM. According to the serum-free culture medium, an excellent culture effect can still be obtained by adding fewer culture medium components, the industrial production complexity is greatly reduced, the industrial production cost is reduced, and large-scale clinical requirements can be better met.

Description

Technical Field

[0001] This application relates to the field of cell culture technology, and in particular to serum-free culture media, their preparation methods, and applications. Background Technology

[0002] Mesenchymal stem cells (MSCs) are a type of adult stem cell originating from the mesoderm, widely found in various tissues and organs such as bone marrow, adipose tissue, umbilical cord, and placenta. Their core biological characteristics include: first, multipotent differentiation potential, capable of differentiating into mesoderm-derived cells such as osteoblasts, chondrocytes, and adipocytes under specific induction conditions, and also capable of differentiating across germ layers into nerve cells, cardiomyocytes, etc.; second, immunomodulatory capacity, inhibiting T lymphocyte proliferation and regulating dendritic cell maturation by secreting cytokines such as transforming growth factor-β (TGF-β) and interleukin-10 (IL-10), thereby reducing immune rejection; and third, paracrine function, releasing bioactive substances including cytokines, chemokines, and exosomes, participating in the regulation of the tissue repair microenvironment and promoting the regeneration of damaged tissues. These characteristics make MSCs a core research subject in regenerative medicine and cell therapy.

[0003] In recent years, significant progress has been made in the clinical research and application of mesenchymal stem cells (MSCs), covering multiple disease areas. In the treatment of degenerative diseases, studies on MSCs for osteoarthritis have shown that they can delay articular cartilage degeneration by differentiating into chondrocytes and inhibiting the activity of cartilage matrix degrading enzymes. In the field of neurological diseases, animal experiments and early clinical studies on ischemic stroke and Alzheimer's disease have shown that MSCs can promote nerve cell survival and improve neural circuit connectivity through paracrine effects. In immune-related diseases, MSCs have been used as adjuvant therapy for graft-versus-host disease (GVHD) and systemic lupus erythematosus, reducing tissue damage caused by excessive immune activation. To date, thousands of MSC-related clinical studies have been conducted globally, with some studies entering Phase III clinical trials, demonstrating broad prospects for clinical translation.

[0004] Recent research indicates that umbilical cord-derived mesenchymal stem cells (MSCs) not only serve as an ideal substitute for bone marrow MSCs but also possess greater application potential. Umbilical cord MSCs express various molecular markers specific to embryonic stem cells and exhibit characteristics such as high differentiation potential, strong proliferative capacity, low immunogenicity, convenient sourcing, lack of ethical restrictions, and ease of industrial-scale preparation. Therefore, they have the potential to become the most promising pluripotent stem cells for clinical applications.

[0005] Currently, the following culture protocols for umbilical cord-derived mesenchymal stem cells have been reported:

[0006] One literature describes a culture medium for human mesenchymal stem cells. This medium is based on DMEM / F12 and supplemented with the following components: human serum albumin, human transferrin, human insulin, fibronectin, cholesterol, sodium selenite, hydrocortisone, putrescine, β-mercaptoethanol, ethanolamine, L-ascorbic acid, bFGF, EGF, PDGF-BB, SCF, nicotinamide, and acalcidin. The concentrations of each component in the basal medium are: human serum albumin 10-20 g / L, human transferrin 5-20 mg / L, human insulin 1-10 mg / L, fibronectin 1-5 mg / L, cholesterol 5-30 μg / mL, sodium selenite 30-50 nmol / L, hydrocortisone 10-50 μg / L, putrescine 5-30 mg / L, and β-mercaptoethanol 5 × 10⁻⁶. -5 M, ethanolamine 1-3 μg / mL, L-ascorbic acid 1-50 mg / L, bFGF 5-10 ng / mL, EGF 5-10 ng / mL, PDGFBB 5-10 ng / mL, SCF 5-10 ng / mL, nicotinamide 5-10 mM, acalcidin 1-3 mM.

[0007] Another literature describes a method for culturing mesenchymal stem cells using a serum-free culture medium. The mesenchymal stem cells are umbilical cord mesenchymal stem cells. The serum-free culture medium consists of the following components: non-essential amino acids: 1 wt%; glutamine: 2 mM; hydrocortisone: 50 μg / L; dexamethasone: 10 μg / L; polyvinyl alcohol: 2-10 g / L; recombinant human insulin: 10 mg / L; recombinant human transferrin: 5.5 mg / L; recombinant human epidermal growth factor: 20 μg / L; recombinant human basic fibroblast growth factor: 20 μg / L; recombinant human Wnt-3a protein: 20 μg / L; recombinant human fibronectin: 50 μg / L; recombinant human laminin: 20 μg / L; L-glutathione: 4 mg / L; L-ascorbic acid: 50 mg / L; β-mercaptoethanol: 2.5 mg / L; ethanolamine: 0.2 g / L; Pluronic acid. F-68: 500 mg / L; Tween 80: 22 mg / L; Cholesterol: 2.2 mg / L; Adenine: 10 mg / L; Sodium selenite: 0.00067 mg / L; Basal culture medium: balance.

[0008] However, current mesenchymal stem cell culture medium formulations are complex, involve many types of raw materials, have high protein content, and are costly. Therefore, this application is filed. Summary of the Invention

[0009] Based on this, one or more embodiments of this application provide serum-free culture media, their preparation methods, and applications. These include the following technical solutions:

[0010] One or more embodiments of this application provide a serum-free culture medium, which includes a basal culture medium and components at the following final concentrations:

[0011] Dexamethasone 5-15 ng / mL,

[0012] Hydrocortisone 200-600 ng / mL

[0013] Transferrin 20-200 μg / mL

[0014] Human serum albumin 0.2-1 mg / mL,

[0015] β-Mercaptoethanol 40-70 μM

[0016] Glutathione 5-12μM,

[0017] Ascorbic acid 10-65 mg / L,

[0018] EGF 1-10 ng / mL,

[0019] IGF-1 1-20 ng / mL

[0020] bFGF 1-10 ng / mL,

[0021] PDGF-BB 1-10 ng / mL;

[0022] The basal culture medium is IMDM.

[0023] In some embodiments of this application, the serum-free culture medium comprises the components at the following final concentrations:

[0024] Dexamethasone 8-10 ng / mL,

[0025] Hydrocortisone 350-450 ng / mL

[0026] Transferrin 50-70 μg / mL

[0027] Human serum albumin is 0.4-0.8 mg / mL.

[0028] β-Mercaptoethanol 55-65 μM

[0029] Glutathione 8-11 μM,

[0030] Ascorbic acid 55-65 mg / L,

[0031] EGF 1-10 ng / mL,

[0032] IGF-1 2-8 ng / mL,

[0033] bFGF 4-7 ng / mL,

[0034] PDGF-BB 1-3 ng / mL;

[0035] The basal culture medium is IMDM.

[0036] One or more embodiments of this application provide a method for culturing mesenchymal stem cells, wherein the method uses the serum-free culture medium described above to culture mesenchymal stem cells.

[0037] In some embodiments of this application, the culture includes primary culture.

[0038] In some embodiments of this application, the primary culture conditions include: culturing at 36-38°C and 4.5%-5.5% (v / v) CO2 for 10-14 days.

[0039] In some embodiments of this application, primary culture is carried out until the confluence of the crawling cells reaches 80%-90%.

[0040] In some embodiments of this application, cultivation includes subculturing.

[0041] In some embodiments of this application, the conditions for subculture include: culturing at 36-38°C and 4.5%-5.5% (v / v) CO2, with each generation lasting 48-96 hours.

[0042] In some embodiments of this application, the cells are passaged to any one of generations P3 to P10.

[0043] In some embodiments of this application, the mesenchymal stem cells are derived from the umbilical cord, amnion, placenta, or bone marrow.

[0044] Compared with traditional technologies, this application has the following advantages:

[0045] This application provides a novel serum-free culture medium for MSC culture. Compared to traditional MSC culture media, this serum-free medium contains fewer culture medium components, including reduced protein usage, while still achieving excellent culture results. This significantly reduces the complexity and cost of industrial production, better meeting large-scale clinical needs. The culture medium avoids introducing excessive exogenous substances, reducing the safety risks of in vitro stem cell culture and ensuring that cultured mesenchymal stem cells meet the quality and safety standards for clinical application. This medium is suitable for culturing both primary and passaged mesenchymal stem cells, exhibiting versatility. Furthermore, cell expansion is high and stem cell viability is well maintained when cultured using this medium. Attached Figure Description

[0046] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0047] Figure 1 The image shown is a primary cell emergence diagram in serum-free culture medium 1.

[0048] Figure 2 The image shown is a primary cell emergence diagram in serum-free culture medium 1-3.

[0049] Figure 3 The image shown is a primary cell growth curve in serum-free medium 1-4.

[0050] Figure 4 The figure shows the statistical results of cell number under different serum-free culture conditions in the passage culture of umbilical cord mesenchymal stem cells.

[0051] Figure 5 The results show the statistical results of cell viability under different serum-free culture conditions during passage culture of umbilical cord mesenchymal stem cells.

[0052] Figure 6 The image shown is a graph of positive results from flow cytometry detection in serum-free culture medium 1-P5 generation.

[0053] Figure 7 The image shown is a negative result of flow cytometry detection in serum-free culture medium 1-P5 generation. Detailed Implementation

[0054] The present application will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0055] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0056] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0057] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0058] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0059] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0060] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0061] In this document, terms such as “preferred,” “better,” “more suitable,” and “ideal” are merely used to describe implementation methods or examples that achieve better results, and should be understood not to limit the scope of protection of this application.

[0062] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0063] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0064] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0065] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0066] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0067] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0068] In this application, %(w / w) and wt% both represent weight percentage, %(v / v) refers to volume percentage, and %(w / v) refers to mass-volume percentage.

[0069] All references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0070] A first aspect of this application provides a serum-free culture medium, the serum-free culture medium comprising a basal culture medium and components at the following final concentrations:

[0071] Dexamethasone 5-15 ng / mL,

[0072] Hydrocortisone 200-600 ng / mL

[0073] Transferrin 20-200 μg / mL

[0074] Human serum albumin 0.2-1 mg / mL,

[0075] β-Mercaptoethanol 40-70 μM

[0076] Glutathione 5-12μM,

[0077] Ascorbic acid 10-65 mg / L,

[0078] EGF 1-10 ng / mL,

[0079] IGF-1 1-20 ng / mL

[0080] bFGF 1-10 ng / mL,

[0081] PDGF-BB 1-10 ng / mL;

[0082] The basal culture medium is IMDM.

[0083] In some embodiments of this application, the serum-free culture medium comprises the components at the following final concentrations:

[0084] Dexamethasone 8-10 ng / mL,

[0085] Hydrocortisone 350-450 ng / mL

[0086] Transferrin 50-70 μg / mL

[0087] Human serum albumin is 0.4-0.8 mg / mL.

[0088] β-Mercaptoethanol 55-65 μM

[0089] Glutathione 8-11 μM,

[0090] Ascorbic acid 55-65 mg / L,

[0091] EGF 1-10 ng / mL,

[0092] IGF-1 2-8 ng / mL,

[0093] bFGF 4-7 ng / mL,

[0094] PDGF-BB 1-3 ng / mL;

[0095] The basal culture medium is IMDM.

[0096] A second aspect of this application provides a method for culturing mesenchymal stem cells, wherein the method uses the serum-free culture medium described above to culture the mesenchymal stem cells.

[0097] In some embodiments of this application, the culture includes primary culture.

[0098] In some embodiments of this application, the primary culture conditions include: culturing at 36-38°C (e.g., 36, 36.5, 37, 37.5, 38°C) and 4.5%-5.5% (v / v, e.g., 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%) CO2 for 10-14 days (e.g., 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14 days).

[0099] In some embodiments of this application, primary culture is carried out until the confluence of the crawling cells reaches 80%-90%.

[0100] In some embodiments of this application, cultivation includes subculturing.

[0101] In some embodiments of this application, the subculture conditions include: culturing at 36-38°C (e.g., 36, 36.5, 37, 37.5, 38°C) and 4.5%-5.5% (v / v, e.g., 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%) CO2, with each generation lasting 48-96 hours (e.g., 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 81, 84, 86, 88, 90, 92, 94, 96 hours).

[0102] In some embodiments of this application, the cells are passaged to any one of generations P3 to P10.

[0103] In some embodiments of this application, the mesenchymal stem cells are derived from the umbilical cord, amnion, placenta, or bone marrow.

[0104] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0105] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0106] Example 1: Primary migration and passage culture of umbilical cord mesenchymal stem cells

[0107] 1. Primary migration of umbilical cord mesenchymal stem cells

[0108] (1) Remove the umbilical cord from the preservation solution and rinse the surface with washing solution, i.e., DPBS (Dubor's phosphate buffer) containing 25 mg / L gentamicin. Cut off about 0.5 cm from both ends of the umbilical cord and transfer the remaining cord to a new dish. Use sterile forceps to remove as much cord blood as possible. If there is residual blood, cut the umbilical cord into 5 cm pieces and continue to remove the blood.

[0109] (2) Transfer the umbilical cord tissue segment processed in step (1) to a 50mL centrifuge tube, add an appropriate amount of cleaning solution (25mg / L gentamicin + DPBS), shake and invert, let stand and soak for 5min, repeat 3 times, and try to remove blood stains.

[0110] (3) Transfer the umbilical cord tissue segment treated in step (2) to a 50mL centrifuge tube, add an appropriate amount of DPBS, shake and invert, let stand and soak for 5 minutes, repeat 2-3 times, and try to remove blood stains and gentamicin residue.

[0111] (4) Immerse the umbilical cord tissue segment treated in step (3) in the prepared serum-free culture medium to keep the tissue moist. Take out a piece of umbilical cord tissue segment and place it in a culture dish. Use sterile surgical scissors to cut along the umbilical vein and remove the inner wall of the vein (remove as completely as possible).

[0112] (5) Turn the umbilical cord tissue processed in step (4) upside down, remove the amnion layer and the umbilical artery, and what remains is Wharton's glue (if there is residual blood clots, it can be rinsed again with DPBS).

[0113] (6) Use a sterile scalpel to process Wharton's jelly into 3-5 mm tissue blocks, transfer them to a 150 mm culture dish, spread 30-35 tissue blocks evenly, and let stand for 15 min. Add 20 mL of serum-free culture medium and incubate at 37℃ in a 5% CO2 incubator (record as day 1). After 2 days, add more to 30 mL.

[0114] (7) Change the medium as needed (every 72 hours), replacing with fresh serum-free culture medium. Observe and label the cell emergence. Results showed that using 1-serum-free culture medium, cells emerged on day 5. Figure 1 As shown; using serum-free medium 2 (1), fewer tissue cells migrated out on day 10; using serum-free medium 3 (1), tissue cells migrated out on day 5, as... Figure 2 As shown; using serum-free culture medium 1-4, the tissue cells migrated out on day 6, as... Figure 3 As shown.

[0115] (8) When the confluence of the extruded cells reaches 80%-90%, remove the umbilical cord tissue block, discard the original serum-free culture medium, and digest and collect the primary cells. Primary cells were harvested on day 13 using serum-free culture medium 1; on day 14 using serum-free culture medium 2; on day 14 using serum-free culture medium 3; and on day 14 using serum-free culture medium 4. The statistical results of the harvested primary cells are shown in Table 2.

[0116] In the above steps, the serum-free culture media are shown as 1-serum-free culture medium 1 to 1-serum-free culture medium 4 below. The formulations of 1-serum-free culture medium 1 and 1-serum-free culture medium 2 are shown in Table 1. The preparation steps are as follows: Prepare the serum-free culture media according to the formulations in the table below. After each component is dissolved according to its corresponding characteristics, filter it through a 0.22μm filter to remove bacteria, and store it at 4℃ in the dark for later use.

[0117] Table 1. Serum-free culture medium formulation

[0118]

[0119] 1- Serum-free culture medium 3: Commercially available serum-free MSC culture medium 1; Manufacturer: Suzhou Huachen; Product number: HC-UC02R+HC-UC02S.

[0120] 1- Serum-free culture medium 4: Commercially available serum-free MSC culture medium 2; Manufacturer: Beijing Youkang; Product No.: NC0106+NC0106.S.

[0121] The primary cell collection data for each serum-free culture medium are summarized in the table below:

[0122] Table 2

[0123]

[0124] In this embodiment: the total number of cells is determined using an automated cell counter; cell viability = number of viable cells / total number of cells × 100%, where viable cells have intact cell membranes, preventing trypan blue from entering the cell and thus remaining colorless and transparent. Dead cells or cells with damaged membranes have increased cell membrane permeability, allowing trypan blue to enter the cell and bind to intracellular components, causing the cell to appear blue.

[0125] Culture results showed that serum-free medium 2 almost did not support the migration of primary cells, and a sufficient number of primary mesenchymal stem cells could not be obtained. Primary cells migrated in serum-free medium 1, serum-free medium 3 and serum-free medium 4. Moreover, the number of primary cells that migrated in serum-free medium 1 was significantly higher than that in serum-free medium 3, and the number of cells that migrated in serum-free medium 3 was higher than that in serum-free medium 4. The cells harvested from the three serum-free media were spindle-shaped, uniform in size and clear in outline.

[0126] Figure 1 The image shown is a primary cell emergence diagram in serum-free culture medium 1.

[0127] Figure 2 The image shown is a primary cell emergence diagram in serum-free culture medium 1-3.

[0128] Figure 3 The image shown is a primary cell growth curve in serum-free medium 1-4.

[0129] 2. Passaging of umbilical cord mesenchymal stem cells

[0130] P2 generation UC-MSCs (umbilical cord mesenchymal stem cells) were resuscitated and seeded at a density of 7000 / cm² in T25 culture flasks and cultured in a 37℃ 5% CO2 incubator. After 72 h of culture, cell morphology was observed under a microscope and photographed for preservation. Cell count, viability, and cell diameter were recorded after trypsin digestion. The obtained cells were then continuously seeded at a density of 7000 / cm² in T25 culture flasks for continuous passage culture, and data from P3 to P5 passage culture were collected. The culture medium used for passage culture was the same as that in Section 1 (1-serum-free medium 1 to 1-serum-free medium 4).

[0131] The summary results are shown in Table 3 below. Figure 4 and Figure 5 The results showed that cells grew faster and had a higher viability in serum-free medium 1, demonstrating a significant advantage over the other three serum-free media.

[0132] Table 3

[0133]

[0134] 3. Phenotypic identification of umbilical cord mesenchymal stem cells

[0135] Resuscitated P2 generation UC-MSCs were seeded at a density of 7000 / cm² in T25 culture flasks and cultured at 37℃ in a 5% CO2 incubator. After 72 hours of culture, cells were collected from each group by trypsin digestion and seeded again at a density of 7000 / cm² in T25 culture flasks for continuous passage culture. Cells from each group were collected at P5 and P10, and surface markers CD73, CD90, CD105, CD14, CD19, CD34, CD45, and HLA-DR were detected by flow cytometry.

[0136] The flow cytometry results are shown in Tables 4 and 5. Figure 6 and Figure 7 As shown, the results indicate that the positive markers CD73, CD90, and CD105 in the four serum-free culture media are all ≥95%, while the negative markers CD14, CD19, CD34, CD45, and HLA-DR are all ≤2%, all of which meet the standards for surface markers of umbilical cord mesenchymal stem cells and can meet the needs of clinical application of umbilical cord mesenchymal stem cells.

[0137] Table 4

[0138]

[0139] Table 5

[0140]

[0141] Figure 6 The image shown is a graph of positive results from flow cytometry detection of serum-free culture medium 1 P5 generation.

[0142] Figure 7 The image shown is a negative result of flow cytometry detection in serum-free culture medium 1 P5 generation.

[0143] Example 2

[0144] This example is a variation of Example 1, differing only in the formulation of the serum-free culture medium. Details are shown in Tables 6 and 7 below:

[0145] Table 6

[0146]

[0147] Table 7

[0148]

[0149] Referring to Example 1, primary culture was performed using the method described in Section 1 of Example 1, and subculture was performed using the method described in Section 2 of Example 1. The only difference was the use of serum-free culture medium; all other procedures were the same. The results are as follows:

[0150] Table 8

[0151]

[0152] Table 8 shows that using serum-free medium 1 and serum-free medium 2, the tissue cells emerged on day 5, and could be harvested on day 10, with a total harvested cell count of 80.00 × 10⁻⁶. 5 cells, 81.10×10 5 Cells; using serum-free medium 3 and serum-free medium 4, the time for tissue cells to emerge was slightly later than that using serum-free medium 1 and serum-free medium 2, the culture time was longer and the number of harvested cells was slightly lower; while using serum-free medium 5 to serum-free medium 9, the emergence of tissue cells was significantly delayed, and even with the extension of culture time, the number of harvested cells was relatively low.

[0153] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0154] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A serum-free culture medium, characterized in that, The serum-free culture medium comprises a basal medium and the following components at final concentrations: Dexamethasone 5-15 ng / mL, Hydrocortisone 200-600 ng / mL Transferrin 20-200 μg / mL Human serum albumin 0.2-1 mg / mL, β-Mercaptoethanol 40-70 μM Glutathione 5-12μM, Ascorbic acid 10-65 mg / L, EGF 1-10 ng / mL, IGF-1 1-20 ng / mL bFGF 1-10 ng / mL, PDGF-BB 1-10 ng / mL; The basal culture medium is IMDM.

2. The serum-free culture medium according to claim 1, characterized in that, The serum-free culture medium comprises the following components at the following final concentrations: Dexamethasone 8-10 ng / mL, Hydrocortisone 350-450 ng / mL Transferrin 50-70 μg / mL Human serum albumin is 0.4-0.8 mg / mL. β-Mercaptoethanol 55-65 μM Glutathione 8-11 μM, Ascorbic acid 55-65 mg / L, EGF 1-10 ng / mL, IGF-1 2-8 ng / mL, bFGF 4-7 ng / mL, PDGF-BB 1-3 ng / mL; The basal culture medium is IMDM.

3. A method for culturing mesenchymal stem cells, characterized in that, The culture method uses the serum-free culture medium according to any one of claims 1 to 2 to culture mesenchymal stem cells.

4. The method for culturing mesenchymal stem cells according to claim 3, characterized in that, Culture includes primary culture.

5. The method for culturing mesenchymal stem cells according to claim 4, characterized in that, The conditions for primary culture include: culturing at 36-38℃ and 4.5%-5.5% (v / v) CO2 for 10-14 days.

6. The method for culturing mesenchymal stem cells according to claim 5, characterized in that, Primary culture until the cells reach 80%-90% confluence.

7. The method for culturing mesenchymal stem cells according to any one of claims 3 to 6, characterized in that, Cultivation includes generational cultivation.

8. The method for culturing mesenchymal stem cells according to claim 7, characterized in that, The conditions for subculture include: culturing at 36-38℃ and 4.5%-5.5% (v / v) CO2, with each generation lasting 48-96 hours.

9. The method for culturing mesenchymal stem cells according to claim 8, characterized in that, Transplanted to any generation from P3 to P10.

10. The method for culturing mesenchymal stem cells according to any one of claims 3 to 6 and 8 to 9, characterized in that, The mesenchymal stem cells are derived from the umbilical cord, amnion, placenta, or bone marrow.

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