Umbilical cord mesenchymal stem cell serum-free medium

Abstract

The invention discloses an umbilical cord mesenchymal stem cell serum-free culture medium which is composed of a basic culture medium and an additive, and the basic culture medium is DMEM / F12. The additive is prepared from albumin, transferrin, insulin, a fibroblast growth factor (FGF), an epidermal growth factor (EGF), dexamethasone, glutamine, ascorbic acid, recombinant human III type collagen and recombinant human XVII type collagen. According to the UCMSCs obtained by culturing the umbilical cord mesenchymal stem cells by using the serum-free culture medium, the cellular morphology and surface marker expression are maintained; the umbilical cord mesenchymal stem cell serum-free culture medium has the advantages that the cell proliferation capacity, the monoclonal performance capacity and the three-line differentiation capacity are enhanced, the umbilical cord mesenchymal stem cell serum-free culture medium only contains 12 raw materials, the compounding process is simple, the cost is low, the raw materials of the umbilical cord mesenchymal stem cell serum-free culture medium are clear in quality, and the umbilical cord mesenchymal stem cell serum-free culture medium can be used for culturing clinical-grade UCMSCs.

Description

Technical Field

[0001] The invention relates to the technical field of stem cell culture, and in particular to a serum-free culture medium for umbilical cord mesenchymal stem cells. Background Art

[0002] Mesenchymal stem cells (MSCs) have become the most clinically studied cells in the world due to their advantages such as wide sources, self-renewal, multidirectional differentiation, and immune regulation. Among them, umbilical cord-derived MSCs (UCMSCs) have the following advantages: 1. Rich sources, easy collection, and no adverse effects on donors; 2. Strong proliferation ability and can be used multiple times; 3. Genetically stable, not prone to mutation, and safe and reliable to use.

[0003] The original content of UCMSCs in the body is very small. In order to meet the application of clinical treatment, it is crucial to develop a system that can meet its large-scale in vitro expansion and culture. Among them, the culture medium is an important condition for in vitro cell culture. At present, UCMSCs culture medium is mainly divided into two categories: serum culture medium and serum-free culture medium. Traditional serum culture medium usually contains bovine serum, which has complex composition, large batch-to-batch differences, and low safety. Serum-free culture medium does not contain serum or animal-derived components, has clear composition, stable quality, small batch-to-batch differences, and high safety. It is a necessary condition for the clinical transformation of cell therapy.

[0004] At present, the following problems still exist regarding the patented technology of umbilical cord mesenchymal stem cell serum-free culture medium:

[0005] 1. Contains complex ingredients such as lysates, hydrolysates, and extracts, which increase batch differences, such as: CN202410132642, CN202310674353.9, CN202211066738.9, CN202111228806.2, CN202110609716.1, CN202110585323.1, CN202110503142.X, CN202010664927.0, CN2 02010399128.5, CN202010182051.6, CN201910795751.X, CN201910500463.7, CN201810646178.1, CN201710306912.5, CN201710306911.0, CN201610578158.6, CN201510920507.3, CN201510919017.1, etc.;

[0006] 2. There are many additive ingredients (nearly / more than 20 kinds), which increases the complexity of the process and the cost of the product, such as: CN202310319497.2, CN202111301022.8, CN202110983717.2, CN202110638431.0, CN202110220798.0, CN202011507568.4, CN202011102735.7, CN201911219419.5, CN201910748103.9, CN201811341164.5 , CN201811115805.5, CN201711383518.8, CN201710484790.9, CN201611075132.6, CN201610560103.2, CN201610513565.9 , CN201510430188.8, CN201410394557.8, CN201310583547.4, CN201310180222.1, CN201210350602.0, CN201210350601.6;

[0007] 3. All relevant patents do not clearly define the raw material standards, making it impossible to control product quality from the source.

[0008] To this end, the present invention provides a serum-free culture medium for umbilical cord mesenchymal stem cells, which has simple and clear ingredients, stable and controllable quality, and excellent cell culture performance. The serum-free culture medium is easy to scale up and produce, and the UCMSCs obtained by culture can be further applied to the treatment of clinical diseases. Summary of the invention

[0009] In view of the shortcomings of the prior art, the present invention improves the prior art and provides a serum-free culture medium for umbilical cord mesenchymal stem cells. The present invention is achieved through the following technical solutions:

[0010] The invention discloses a serum-free culture medium for umbilical cord mesenchymal stem cells. The culture medium consists of a basic culture medium and an additive. The basic culture medium is DMEM / F12; the additive consists of albumin, transferrin, insulin, fibroblast growth factor FGF, epidermal growth factor EGF, dexamethasone, glutamine, ascorbic acid, recombinant human type III collagen and recombinant human type XVII collagen.

[0011] As a further improvement, the concentrations of the components in the additive of the present invention are albumin: 0.1~10 mg / mL, transferrin: 0.005~0.5 mg / mL, insulin: 0.005~0.05 mg / mL, FGF: 0.005~0.05 mg / L, EGF: 0.05~0.5 mg / L, dexamethasone: 0.05~0.5 mg / L, glutamine: 0.5~5 mg / mL, ascorbic acid: 0.05~0.5 mg / mL, recombinant human type III collagen: 0.001~0.05 mg / mL, and recombinant human type XVII collagen: 0.001~0.05 mg / mL.

[0012] As a further improvement, the raw materials of the basal culture medium and additives described in the present invention are pharmaceutical grade, API grade, pharmaceutical excipient grade, or cell culture grade. Pharmaceutical grade should be given priority, followed by API grade and pharmaceutical excipient grade, and finally cell culture grade.

[0013] As a further improvement, the raw material levels of each component in the additive described in the present invention are: albumin: pharmaceutical or pharmaceutical excipient level; transferrin: cell culture level; insulin: pharmaceutical or raw material level; FGF: pharmaceutical level; EGF: pharmaceutical level; dexamethasone: pharmaceutical or raw material level; glutamine: pharmaceutical or raw material level; ascorbic acid: pharmaceutical or raw material level; recombinant human type III collagen: pharmaceutical excipient level; recombinant human type XVII collagen: cell culture level.

[0014] The present invention provides a serum-free culture medium for umbilical cord mesenchymal stem cells, which has simple and clear ingredients, stable and controllable quality, and excellent cell culture performance. The serum-free culture medium is easy to scale up and produce, and the UCMSCs obtained by culture can be further applied to the treatment of clinical diseases.

[0015] The beneficial effects of the present invention are:

[0016] (1) The umbilical cord mesenchymal stem cell serum-free culture medium is composed of albumin, transferrin, insulin, fibroblast growth factor FGF, epidermal growth factor EGF, dexamethasone, glutamine, ascorbic acid, recombinant human type III collagen, and recombinant human type XVII collagen added to DMEM / F12 basic culture medium. Among them, DMEM / F12 provides basic substances such as amino acids, vitamins, inorganic salts, glucose, etc. for UCMSCs; and has the function of maintaining the pH stability of the culture medium. Albumin can bind to fatty acids and metal ions to maintain their stability and reduce cytotoxicity; and has an antioxidant effect and delays cell aging. Transferrin can regulate iron ion transport and metabolism; maintain the balance and homeostasis of multiple metal ions in cells; maintain cell growth and proliferation; and act as an extracellular antioxidant to delay cell aging. Insulin can regulate the metabolism of fat and protein; and promote cell growth. FGF and EGF can promote cell proliferation; and maintain cell pluripotency. Dexamethasone can promote cell proliferation. Glutamine participates in protein synthesis and nucleic acid metabolism; promotes nitrogen storage and transfer into cells; ascorbic acid promotes cell proliferation; and has an antioxidant effect, delaying cell aging. Recombinant type III and type XVII collagen can enhance cell adhesion and promote cell proliferation. The scientific combination and ratio of the culture medium formula enables it to effectively promote the proliferation of UCMSCs in vitro and maintain cell performance. Among them, the combination of "albumin and transferrin", "FGF, EGF and dexamethasone", "glutamine and ascorbic acid", and "recombinant human type III collagen and recombinant human type XVII collagen" within the following concentration range supports and interacts with each other, and is more conducive to the enhancement of cell activity; albumin: 0.1~10mg / mL, transferrin: 0.005~0.5mg / mL; FGF: 0.005~0.05mg / L, EGF: 0.05~0.5mg / L, dexamethasone: 0.05~0.5mg / L; glutamine: 0.5~5mg / mL, ascorbic acid: 0.05~0.5mg / mL; recombinant human type III collagen: 0.001~0.05mg / mL, recombinant human type XVII collagen: 0.001~0.05mg / mL.

[0017] (2) The UCMSCs cultured using the serum-free medium for umbilical cord mesenchymal stem cells maintained their cell morphology and surface marker expression; compared with the traditional serum medium, the cell proliferation ability, monoclonal performance and tri-lineage differentiation ability were enhanced.

[0018] (3) The umbilical cord mesenchymal stem cell serum-free culture medium contains only 12 raw materials, has a simple compounding process, is easy to scale up for production, and has low cost.

[0019] (4) The raw material quality of the umbilical cord mesenchymal stem cell serum-free culture medium is clear, the preparation process is simple, and the quality of the culture medium is stable and controllable, and it can be used to culture clinical-grade UCMSCs. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a diagram showing the cell activity detection results of Example 1;

[0021] Figure 2 This is a diagram showing the cell activity detection results of Example 2;

[0022] Figure 3 This is a diagram showing the cell activity detection results of Example 3;

[0023] Figure 4 This is a diagram showing the cell activity detection results of Example 4;

[0024] Figure 5 This is a diagram showing the cell activity detection results of Example 5;

[0025] Figure 6 This is a diagram showing the morphological observation results of passaged cells in Example 6;

[0026] Figure 7 This is a diagram showing the results of cell monoclonal ability detection in Example 7;

[0027] Figure 8 This is a graph showing the results of the three-lineage differentiation ability test of cells in Example 7. DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further described below by means of specific embodiments:

[0029] Example 1

[0030] The specific implementation of this embodiment is as follows:

[0031] 1. Weigh each component according to the proportion in Table 1 and add it to DMEM / F12 basal medium (unit: mg / mL).

[0032] Table 1 is a serum-free medium additive formula table of Example 1

[0033]

[0034] 2. Filter the prepared culture medium using a filter with a pore size of 0.22 microns.

[0035] 3. Inoculate umbilical cord mesenchymal stem cells into 96-well plates and culture them using filtered culture medium.

[0036] 4. Use commercially available mesenchymal stem cell serum-free culture medium and DMEM / F12 serum culture medium containing 10% fetal bovine serum as controls.

[0037] 5. After 72 hours of culture, the cell activity was detected by MTT method.

[0038] 6. Cell activity test results Figure 1 As shown in the above examples and comparative cases, it can be seen that albumin: 0.1~10mg / mL, transferrin: 0.005~0.5mg / mL, albumin and transferrin concentrations within this range are beneficial to enhancing cell activity; while concentrations of the two components below or above the range will lead to reduced cell activity.

[0039] Example 2

[0040] The specific implementation of this embodiment is as follows:

[0041] 1. Weigh each component according to the proportion in Table 2 and add it to DMEM / F12 basal medium (unit: mg / mL).

[0042] Table 2 is a table of serum-free medium additives in Example 2

[0043]

[0044] 1. Filter the prepared culture medium using a filter with a pore size of 0.22 μm.

[0045] 2. Inoculate umbilical cord mesenchymal stem cells into 96-well plates and culture them using filtered culture medium.

[0046] 3. Use commercially available mesenchymal stem cell serum-free culture medium and DMEM / F12 serum culture medium containing 10% fetal bovine serum as controls.

[0047] 4. After 72 hours of culture, the cell activity was detected by MTT method.

[0048] 5. Cell activity test results Figure 2 As shown. Through the above examples and comparative cases, it can be obtained that FGF: 0.005~0.05mg / L, EGF: 0.05~0.5mg / L, dexamethasone: 0.05~0.5mg / L; the addition of FGF, EGF and dexamethasone is beneficial to enhancing cell activity; the concentrations of the three components within the range are beneficial to enhancing cell activity; the concentrations of the three components above the range all lead to reduced cell activity.

[0049] Example 3

[0050] The specific implementation of this embodiment is as follows:

[0051] 1. Weigh each component according to the proportion in Table 3 and add it to DMEM / F12 basal medium (unit: mg / mL).

[0052] Table 3 is a table of serum-free medium additives in Example 3

[0053]

[0054] 2. Filter the prepared culture medium using a filter with a pore size of 0.22 microns.

[0055] 3. Inoculate umbilical cord mesenchymal stem cells into 96-well plates and culture them using filtered culture medium.

[0056] 4. Use commercially available mesenchymal stem cell serum-free culture medium and DMEM / F12 serum culture medium containing 10% fetal bovine serum as controls.

[0057] 5. After 72 hours of culture, the cell activity was detected by MTT method.

[0058] 6. Cell activity test results Figure 3 As shown. Through the above examples and comparative cases, it can be obtained that glutamine: 0.5~5mg / mL, ascorbic acid: 0.05~0.5mg / mL, the addition of glutamine and ascorbic acid is beneficial to enhance cell activity; the concentration of the two components within the above range is beneficial to enhance cell activity; the concentration of the two components above the above range will lead to reduced cell activity.

[0059] Example 4

[0060] The specific implementation of this embodiment is as follows:

[0061] 1. Weigh each component according to the proportion in Table 4 and add it to DMEM / F12 basal medium (unit: mg / mL).

[0062] Table 4 is a table of serum-free medium additives in Example 4

[0063]

[0064] 2. Filter the prepared culture medium using a filter with a pore size of 0.22 microns.

[0065] 3. Inoculate umbilical cord mesenchymal stem cells into 96-well plates and culture them using filtered culture medium.

[0066] 4. Use commercially available mesenchymal stem cell serum-free culture medium and DMEM / F12 serum culture medium containing 10% fetal bovine serum as controls.

[0067] 5. After 72 hours of culture, the cell activity was detected by MTT method.

[0068] The results of cell viability assay were as follows Figure 4As shown in Figure 2, the addition of recombinant human type III and type XVII collagen is beneficial to enhance cell activity.

[0069] Example 5

[0070] The specific implementation of this embodiment is as follows:

[0071] 1. Weigh each component according to the proportion in Table 5 and add it to DMEM / F12 basal medium (unit: mg / mL).

[0072] Table 5 is a table of serum-free medium additives in Example 5

[0073]

[0074] 2. Filter the prepared culture medium using a filter with a pore size of 0.22 microns.

[0075] 3. Inoculate umbilical cord mesenchymal stem cells into 96-well plates and culture them using filtered culture medium.

[0076] 4. Two commercially available serum-free culture media for mesenchymal stem cells (purchased from Cell Science & Technology Institute INC (CSTI) and Youkang Biotechnology (Beijing) Co., Ltd., respectively) and DMEM / F12 serum culture medium containing 10% fetal bovine serum were used as controls.

[0077] 5. After 72 hours of culture, the cell activity was detected by MTT method.

[0078] The results of cell viability assay were as follows Figure 5 As shown, the serum-free medium of Example 5 of the present invention has a better effect on enhancing cell activity than commercially available serum-free medium and traditional serum medium.

[0079] Example 6

[0080] The specific implementation of this embodiment is as follows:

[0081] 1. Weigh each component according to the proportion in Table 6 and add it to DMEM / F12 basal medium (unit: mg / mL).

[0082] Table 6 is a table of serum-free medium additives in Example 6

[0083]

[0084] 2. Filter the prepared culture medium using a filter with a pore size of 0.22 microns.

[0085] 3. Inoculate the first generation umbilical cord mesenchymal stem cells into culture bottles and culture them using filtered culture medium.

[0086] 4. After 72 hours, observe the cell morphology, digest the cells, and subculture them.

[0087] 5. Use the filtered culture medium to culture the second generation umbilical cord mesenchymal stem cells.

[0088] 6. Repeat step 4.

[0089] 7. Use the filtered culture medium to culture the second generation umbilical cord mesenchymal stem cells. After 72 hours, observe the cell morphology.

[0090] 8. Use DMEM / F12 serum culture medium containing 10% fetal bovine serum as a control simultaneously with step 7; after 72 hours of culture, digest the cells and label the cells with CD34, CD45, CD72, CD90, and CD105 antibodies; use flow cytometry to detect the expression of cell surface markers.

[0091] The cell morphology observation results are as follows Figure 6 When the umbilical cord mesenchymal stem cells were cultured in the serum-free medium of Example 6 to the third generation, the cells still showed the typical MSC spindle-shaped morphology and the cells were in good condition.

[0092] The results of cell surface marker detection are shown in Table 7. The cell surface markers of the umbilical cord mesenchymal stem cells cultured in the serum-free medium of Example 6 meet the MSCs standard, that is, CD34 and CD45 are negative, and CD73, CD90, and CD105 are positive.

[0093] Table 7 is a table of cell surface marker expression detection results

[0094]

[0095] Example 7

[0096] The specific implementation of this embodiment is as follows:

[0097] 1. Weigh each component according to the proportion in Table 8 and add it to DMEM / F12 basal medium (unit: mg / mL).

[0098] Table 8 is a table of serum-free medium additives in Example 7

[0099]

[0100] 2. Filter the prepared culture medium using a filter with a pore size of 0.22 microns.

[0101] 3. Inoculate the umbilical cord mesenchymal stem cells into 6-well plates and culture flasks. The cell inoculation density in the 6-well plates is 50 cells / well, and culture is performed using filtered culture medium.

[0102] 4. Use DMEM / F12 serum medium containing 10% fetal bovine serum as a control.

[0103] After 14 days of cell culture in 5- and 6-well plates, the cells were stained with gentian violet and the number of monoclonal clones was counted.

[0104] 6. After culturing the cells in the culture flask for 72 hours, the cells were digested and re-seeded in a 24-well plate and cultured using bone induction medium, cartilage induction medium, and fat induction medium, respectively.

[0105] 7. After 14 days of bone induction culture, the cells were stained with Alizarin red; after 14 days of fat induction, the cells were stained with Oil red; after 21 days of cartilage induction, the cells were stained with Alcian blue.

[0106] The results of cell monoclonal formation ability test are as follows Figure 7 Compared with the traditional serum culture medium, the umbilical cord mesenchymal stem cells cultured in the serum-free culture medium of Example 7 have enhanced monoclonal formation ability.

[0107] The results of the cell tri-lineage differentiation ability test are as follows Figure 8 Compared with the traditional serum culture medium, the umbilical cord mesenchymal stem cells cultured with the serum culture medium of Example 7 have enhanced osteogenic, adipogenic and chondrogenic differentiation abilities.

[0108] The embodiments described above are only descriptions of the preferred implementation modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A serum-free culture medium for umbilical cord mesenchymal stem cells, characterized in that: The culture medium consists of a basic culture medium and additives, wherein the basic culture medium is DMEM / F12; and the additives consist of albumin, transferrin, insulin, fibroblast growth factor FGF, epidermal growth factor EGF, dexamethasone, glutamine, ascorbic acid, recombinant human type III collagen and recombinant human type XVII collagen.

2. The umbilical cord mesenchymal stem cell serum-free culture medium according to claim 1, characterized in that The concentrations of the components in the additive are albumin: 0.1-10 mg / mL, transferrin: 0.005-0.5 mg / mL, insulin: 0.005-0.05 mg / mL, FGF: 0.005-0.05 mg / L, EGF: 0.05-0.5 mg / L, dexamethasone: 0.05-0.5 mg / L, glutamine: 0.5-5 mg / mL, ascorbic acid: 0.05-0.5 mg / mL, recombinant human type III collagen: 0.001-0.05 mg / mL, and recombinant human type XVII collagen: 0.001-0.05 mg / mL.

3. The umbilical cord mesenchymal stem cell serum-free culture medium according to claim 1 or 2, characterized in that: The raw materials of the basic culture medium and additives are of pharmaceutical grade, raw material grade, pharmaceutical excipient grade, or cell culture grade.

4. The umbilical cord mesenchymal stem cell serum-free culture medium according to claim 3, characterized in that The raw material levels of the ingredients in the additive are as follows: albumin: pharmaceutical or pharmaceutical excipient level; transferrin: cell culture level; Insulin: drug or API grade; FGF: drug grade; EGF: pharmaceutical grade; dexamethasone: pharmaceutical or API grade; glutamine: pharmaceutical or API grade; ascorbic acid: pharmaceutical or API grade; recombinant human type III collagen: pharmaceutical excipient grade; recombinant human type XVII collagen: cell culture grade.

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