WO2021185198A1 - 一种无血清、无异源成分的间充质干细胞培养基及其应用 - Google Patents

一种无血清、无异源成分的间充质干细胞培养基及其应用 Download PDF

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WO2021185198A1
WO2021185198A1 PCT/CN2021/080728 CN2021080728W WO2021185198A1 WO 2021185198 A1 WO2021185198 A1 WO 2021185198A1 CN 2021080728 W CN2021080728 W CN 2021080728W WO 2021185198 A1 WO2021185198 A1 WO 2021185198A1
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mesenchymal stem
stem cells
medium
stem cell
human
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佟曼
付丽娜
薛博涵
马静
辛文
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北京全式金生物技术有限公司
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
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    • C12N2500/00—Specific components of cell culture medium
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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Definitions

  • the present invention relates to the technical field of cell culture. More specifically, it relates to a serum-free and heterologous-free mesenchymal stem cell culture medium and its application.
  • Mesenchymal stem cells are a type of adult stem cells with self-replication, renewal and multi-differentiation potential, which are widely present in various tissues of fetuses and adults.
  • mesenchymal stem cells In addition to self-renewal, mesenchymal stem cells have unique immune regulation and tissue regeneration functions.
  • mesenchymal stem cells regulate immune cells through interaction with immune cells and paracrine soluble factors, mediate the balance of immune response, and help the body form a microenvironment that is conducive to the repair or regeneration of diseased tissue cells.
  • Immune regulation of mesenchymal stem cells includes inhibition of T lymphocyte activation, reduction of B cell activation and proliferation and antibody secretion, inhibition of natural killer cell proliferation, and inhibition of dendritic cell maturation and antigen presentation.
  • mesenchymal stem cells can differentiate into osteoblasts, chondrocytes, adipocytes, liver parenchymal cells, muscle cells and neurons and other functional cells, and participate in the reconstruction of damaged tissues and organs.
  • mesenchymal stem cell transplantation for the treatment of various diseases registered on the Clinical trails website worldwide, including mesenchymal stem cell transplantation for the treatment of graft versus host disease (GVHD) (ClinicalTrials.gov Identifier: NCT00366145), Osteonecrosis of Femoral Head (ONFH) (ClinicalTrials.gov Identifier: NCT01700920), Osteoarthritis (OA) (ClinicalTrials.gov Identifier: NCT01809), Long Beili Syndrome (Romberg's Disease) (ClinicalTrials.gov Identifier: NCT02494752), etc., have achieved significant effects.
  • GVHD graft versus host disease
  • the therapeutic mechanism mainly depends on the immune regulation and tissue regeneration functions of mesenchymal stem cells.
  • the occurrence of graft-versus-host disease is mainly related to activated donor T cells.
  • Activated T cells produce the inflammatory factor IFN ⁇ after the immune response to the antigen, and the transplanted mesenchymal stem cells can respond to IFN ⁇ , inhibit the proliferation of T cells by secreting immune regulatory factors such as PGE2, and realize the treatment of graft-versus-host disease (Polchert D, Sobinsky J, Douglas G, Kidd M, Moadsiri A, Reina E, Genrich K, Mehrotra S, Setty S, Smith Bet al: IFN-gamma activation of mesenchymal stem cells for treatment and prevention of graft of Europe journal of immunology 2008, 38(6):1745-1755.).
  • the application of mesenchymal stem cells to the treatment of ONFH, OA, and Romberg's Disease depends to a certain extent on the osteogenesis, cartilage, and
  • mesenchymal stem cells As important seed cells in basic medicine, regenerative medicine, clinical medicine, tissue engineering and other fields, they meet the requirements of industrial transformation and clinical application, and can better maintain the immune regulation function and multi-directional function of mesenchymal stem cells.
  • An efficient in vitro expansion system with differentiation potential is a key factor to support the transformation of the mesenchymal stem cell industry and promote the research and development of new mesenchymal stem cell drugs.
  • the commonly used mesenchymal stem cell culture medium contains a certain concentration of fetal bovine serum/animal protein. Fetal bovine serum/animal protein is a heterogeneous protein, which can be swallowed by mesenchymal stem cells during culture.
  • serum-free and heterologous mesenchymal stem cell culture media such as the widely used StemPro TM MSC SFM XenoFree (Cat. No. A1067501) from Thermo Fisher in the United States.
  • the serum-free mesenchymal stem cell culture media that have been developed have the following shortcomings: 1) Complex components. There are as many as ten or more kinds of ingredients, which leads to poor stability between batches of the medium and difficult quality control; 2) The immune regulation function of mesenchymal stem cells cannot be maintained well.
  • the cultured mesenchymal stem cells secrete the immunoregulatory factor PGE2, and the ability to inhibit the proliferation of PBMC cells is weak; 3) the mesenchymal stem cells cannot well maintain the multidirectional differentiation potential of the mesenchymal stem cells.
  • the cultured mesenchymal stem cells differentiate into osteoblasts, chondrocytes, and adipocytes with low efficiency. These problems have largely restricted the industrial transformation and clinical application of mesenchymal stem cells.
  • An object of the present invention is to provide a mesenchymal stem cell culture medium, which is serum-free and free of heterologous components, can significantly improve the proliferation ability of mesenchymal stem cells, and can also maintain the growth of mesenchymal stem cells. Immunomodulatory function and multi-directional differentiation potential.
  • Another object of the present invention is to provide the application of the above-mentioned mesenchymal stem cell culture medium in culturing mesenchymal stem cells.
  • the present invention first provides a serum-free and heterologous mesenchymal stem cell culture medium.
  • the mesenchymal stem cell culture medium of the present invention includes a basal medium and a supplement, the basal medium is DMEM/F12 medium, and the supplement is human platelet lysate (hPL) and/or human lipid Plasma transfer protein 2 (lipocalin 2 or LCN2 protein [Homo sapiens]).
  • the basal medium is DMEM/F12 medium
  • the supplement is human platelet lysate (hPL) and/or human lipid Plasma transfer protein 2 (lipocalin 2 or LCN2 protein [Homo sapiens]).
  • the mesenchymal stem cell culture medium of the present invention may only consist of a basal medium and supplements, or may contain other components; wherein, the supplements may only consist of human platelet lysate and human lipocalin 2, Other components can also be included.
  • the volume content of the human platelet lysate in the mesenchymal stem cell culture medium is 1%-20%, and the content of the human lipocalin 2 in the mesenchymal stem cell culture medium is 10-200 ⁇ g /mL.
  • the volume content of the human platelet lysate in the mesenchymal stem cell culture medium is 2%-10%, and the human lipocalin 2 is cultured in the mesenchymal stem cell
  • the content of the base is 20-100 ⁇ g/mL.
  • the volume content of the human platelet lysate in the mesenchymal stem cell culture medium is 5%, and the human lipocalin 2 is in the mesenchymal stem cell culture medium.
  • the content is 50 ⁇ g/mL.
  • the pH of the mesenchymal stem cell culture medium is 7.3-7.5, and the osmotic pressure is 300-350 mOsm/kg.
  • the present invention further provides the application of the above-mentioned mesenchymal stem cell culture medium in culturing mesenchymal stem cells or in the preparation of products for culturing mesenchymal stem cells.
  • the culture may be primary isolation culture and/or subculture expansion culture.
  • mesenchymal stem cell culture medium in any of the following is also within the protection scope of the present invention:
  • the improvement of the immunoregulatory ability of mesenchymal stem cells is embodied in that culturing mesenchymal stem cells with the medium of the present invention can enhance the secretion of immunoregulatory factor PGE2 by mesenchymal stem cells and inhibit the proliferation of PBMC cells.
  • the improved ability of mesenchymal stem cell differentiation is mainly reflected in the use of the medium of the present invention to culture mesenchymal stem cells can improve the efficiency of the directional differentiation of mesenchymal stem cells into osteocytes, chondrocytes, and adipocytes.
  • the present invention further provides the application of human platelet lysate and/or human lipocalin 2 in culturing mesenchymal stem cells or in preparing products for culturing mesenchymal stem cells.
  • the culture may be primary isolation culture and/or subculture expansion culture.
  • the improvement of the immunoregulatory ability of mesenchymal stem cells is embodied in that culturing mesenchymal stem cells with the medium of the present invention can enhance the secretion of immunoregulatory factor PGE2 by mesenchymal stem cells and inhibit the proliferation of PBMC cells.
  • the improved ability of mesenchymal stem cell differentiation is mainly reflected in the use of the medium of the present invention to culture mesenchymal stem cells can improve the efficiency of the directional differentiation of mesenchymal stem cells into osteocytes, chondrocytes, and adipocytes.
  • the mesenchymal stem cells are human mesenchymal stem cells, which may be mesenchymal stem cells derived from human umbilical cord, human bone marrow, or human fat, that is, human umbilical cord mesenchymal stem cells, human bone marrow mesenchymal stem cells and/or human Adipose mesenchymal stem cells.
  • the product can be a reagent or a kit.
  • the formula is simple (contains only 3 components), the batch is stable, and the cost is low, which can greatly improve the in vitro proliferation ability of mesenchymal stem cells. Meet the requirements of industrial transformation.
  • the serum-free and heterologous mesenchymal stem cell culture medium of the present invention does not contain heterologous substances, does not have the risk of introducing animal-derived pathogenic microorganisms, and meets the safety requirements of clinical applications.
  • the serum-free and heterologous mesenchymal stem cell culture medium of the present invention can enhance the biological effects of mesenchymal stem cells, including improving the immune regulation ability and multidirectional differentiation function matching the indication, which is in line with the effectiveness of clinical application Sexual requirements.
  • Figure 1 shows the use of the serum-free and heterologous mesenchymal stem cell culture medium of Examples 1-5, the control serum-containing medium of Example 6 and the control serum-free medium of Example 7 to treat human umbilical cord mesenchymal stem cells respectively.
  • Figure 2 shows the use of the serum-free and heterologous mesenchymal stem cell medium in Example 3, the control serum-containing medium in Example 6 and the control serum-free medium in Example 7 on human bone marrow mesenchymal stem cells. Diagram of the cumulative cell population doubling number of continuous culture for 5 generations.
  • Figure 3 shows the use of the serum-free and heterologous mesenchymal stem cell medium in Example 3, the control serum-containing medium in Example 6 and the control serum-free medium in Example 7 to compare human adipose-derived mesenchymal stem cells.
  • Figure 4 shows the continuous cultivation of human umbilical cord mesenchyme using the serum-free and heterologous mesenchymal stem cell medium of Example 3, the control serum-containing medium of Example 6 and the control serum-free medium of Example 7 respectively Cell morphology map of stem cells, human bone marrow mesenchymal stem cells, and human adipose-derived mesenchymal stem cells, scale: 100 ⁇ m.
  • Figure 5 shows the continuous cultivation of human umbilical cord mesenchymal stem cells using the serum-free and heterologous mesenchymal stem cell medium of Example 3 and the control serum-free medium of Example 7 respectively.
  • Figure 6 shows the use of the serum-free and heterologous mesenchymal stem cell medium of Example 3 and the control serum-containing medium of Example 6, respectively, and the control serum-free medium of Example 7 for continuous cultivation of human umbilical cord mesenchyme
  • P2 represents the proportion of non-proliferating cells
  • P3 represents the proportion of proliferating cells.
  • Fig. 7 is a diagram showing the continuous culture of human umbilical cord mesenchymal stem cells using the serum-free and heterologous mesenchymal stem cell medium in Example 3 and the control serum-free medium in Example 7 with Alizarin Red staining , Scale: 100 ⁇ m, ***p ⁇ 0.001.
  • Figure 8 shows the use of the serum-free and heterologous mesenchymal stem cell medium of Example 3 and the control serum-free medium of Example 7 to continuously culture human umbilical cord mesenchymal stem cells for chondrogenic differentiation and alicin blue staining. , Scale: 100 ⁇ m, **p ⁇ 0.01.
  • Figure 9 is a diagram showing the continuous adipogenic differentiation of human umbilical cord mesenchymal stem cells using the serum-free and heterologous mesenchymal stem cell medium of Example 3 and the control serum-free medium of Example 7 respectively. Oil Red O staining , Scale: 100 ⁇ m, ***p ⁇ 0.001.
  • the mesenchymal stem cell culture medium has complex components and cannot well maintain the in vitro expansion capacity, immunoregulatory function and multidirectional differentiation potential of mesenchymal stem cells.
  • the present invention provides a serum-free and heterologous mesenchymal stem cell Medium.
  • the serum-free and heterologous mesenchymal stem cell culture medium of the present invention includes a basic culture medium and additives.
  • the basic medium is DMEM/F12 medium, which contains basic substances such as carbohydrates, amino acids, vitamins and inorganic salts, and provides an energy source for the metabolism of mesenchymal stem cells.
  • the additives are human platelet lysate and human lipocalin 2, wherein the human platelet lysate is rich in various growth factors, including PDGF, VEGF, EGF, etc., which can replace fetal bovine serum to support the growth of mesenchymal stem cells ; Human lipocalin 2 has many functions such as anti-oxidation, promoting cell proliferation, and inhibiting stem cell senescence.
  • human lipocalin 2 has not been used in the cultivation of mesenchymal stem cells, and it regulates the mesenchyme in in vitro culture.
  • the role of stem cell proliferation, maintenance of immunoregulatory function and multi-directional differentiation potential has not yet been definitively concluded.
  • the inventors of the present invention have discovered that the mesenchymal stem cell culture medium formulated with DMEM/F12 medium, human platelet lysate and human lipocalin 2 has the function of promoting cell proliferation and efficiently maintaining cellular immune regulation on mesenchymal stem cells. And the effect of multi-directional differentiation potential, the effect is especially significant at a specific concentration.
  • the volume content of the human platelet lysate in the mesenchymal stem cell medium is 1-20%, and the content of the human lipocalin 2 in the mesenchymal stem cell medium is 10-200 ⁇ g/ mL.
  • the volume content of the human platelet lysate in the mesenchymal stem cell culture medium can be 1%, 5%, 10%, 20%, etc. or any range between the volume content, and the human lipocalin 2 is in
  • the content of the mesenchymal stem cell culture medium is 10 ⁇ g/mL, 50 ⁇ g/mL, 100 ⁇ g/mL, 200 ⁇ g/mL, etc. or any range between the content.
  • the serum-free and heterologous component-free mesenchymal stem cell culture medium of the present invention has simple components and is applied to the cultivation of mesenchymal stem cells. It has the following advantages: first, it can improve the cell proliferation ability of mesenchymal stem cells, which is specifically embodied in : The cumulative cell population doubling number of human umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, and adipose-derived mesenchymal stem cells cultured using the culture medium of the present invention is 1.82, 1.18, 1.82, 1.18, and 1.82 of those of the control serum-containing medium and the control serum-free medium, respectively 1.66 and 1.67, 1.54, 1.71 times; second, it can improve the immune regulation ability of mesenchymal stem cells, secrete the immunoregulatory factor PGE2 and inhibit the proliferation of PBMC cells, which is embodied in: use under the co-stimulation of TNF ⁇ and IFN ⁇ The PGE2 secretion of human umbilical
  • Serum medium and control serum-free medium are 1.35 times and 2.62 times; third, it can improve the multi-differentiation ability of mesenchymal stem cells, and the efficiency of directed differentiation into osteocytes, chondrocytes, and adipocytes is significantly improved, which is specifically reflected in :
  • the positive staining areas of human umbilical cord mesenchymal stem cells cultured in the culture medium of the present invention for osteogenic (alizarin red), cartilage (alixian blue) and adipogenic (oil red O) differentiation are those of the control serum-free medium. 1.98, 2.63, 3.25 times.
  • the materials in the following examples are all commonly used materials in the field, and can be obtained from commercial sources. Unless otherwise specified, the cell culture conditions in the following examples are 37°C and 5% CO 2 .
  • the experimental data in the following examples are expressed as mean ⁇ standard deviation, analyzed by GraghPad Prism 5 statistical software, *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001.
  • the sources of reagents, cells and antibodies used in the examples of the present invention are as follows:
  • Hypoxanthine Na hypoxanthine monosodium salt
  • Linoleic Acid 280.0 0.042 Lipoic Acid ( ⁇ -Lipoic Acid) 206.0 0.105 Phenol Red 376.4 8.1
  • Putrescine 2HCl putrescine dihydrochloride 161.0 0.081 Thymidine 242.0 0.365
  • StemPro TM MSC SFM XenoFree Thermo Fisher company, the article number is A1067501.
  • the medium contains: StemPro TM MSC SFM basal medium (product number A13829-01) and StemPro TM MSC SFM XenoFree additive (product number A11577-01).
  • StemPro TM MSC SFM basal medium product number A13829-01
  • StemPro TM MSC SFM XenoFree additive product number A11577-01.
  • Human lipocalin 2 Abcam company, the catalog number is ab95007.
  • Human lipocalin 2 is a protein encoded by a DNA sequence with Genbank number AAH33089.1 (updated 09-JUN-2008), and consists of 198 amino acid residues.
  • Fetal bovine serum Thermo Fisher company, article number 16140071.
  • PGE2 Prostaglandin E2 (PGE2) ELISA test kit, Abcam company, the catalog number is ab133055.
  • CD90-FITC antibody Thermo Fisher, article number 11-0909-41.
  • CD73-FITC antibody Thermo Fisher, catalog number 11-0739-41.
  • CD105-PE antibody Thermo Fisher, article number 12-1057-41.
  • CD34-FITC antibody Thermo Fisher, article number 11-0349-41.
  • CD45-PE antibody Biolegend, product number 304007.
  • HLA-DR-PE antibody Biolegend, catalog number 307605.
  • the method for primary isolation and culture of human umbilical cord mesenchymal stem cells in the following examples includes the following steps:
  • the cells crawling out of the edge of the tissue block are the primary umbilical cord mesenchymal stem cells.
  • the method for subculture expansion and culture of human umbilical cord mesenchymal stem cells, human bone marrow mesenchymal stem cells, and human adipose-derived mesenchymal stem cells includes the following steps:
  • the diluted Fibronectin cannot be stored for a long time. Please dilute before use.
  • Example 1 A serum-free and heterologous-free mesenchymal stem cell culture medium
  • a serum-free, heterologous-free mesenchymal stem cell culture medium which is composed of DMEM/F12 medium (1 ⁇ ), human platelet lysate and human lipocalin 2.
  • the human platelet lysates
  • the volume content of the liquid in the mesenchymal stem cell culture medium is 20%, and the content of the human lipocalin 2 in the mesenchymal stem cell culture medium is 200 ⁇ g/mL.
  • the pH of the mesenchymal stem cell culture medium is 7.38, and the osmotic pressure is 341 mOsm/kg.
  • Example 2 A serum-free and heterologous-free mesenchymal stem cell culture medium
  • a serum-free, heterologous-free mesenchymal stem cell culture medium which is composed of DMEM/F12 medium (1 ⁇ ), human platelet lysate and human lipocalin 2.
  • the human platelet lysates The volume content of the liquid in the mesenchymal stem cell culture medium is 10%, and the content of the human lipocalin 2 in the mesenchymal stem cell culture medium is 100 ⁇ g/mL.
  • the pH of the mesenchymal stem cell culture medium is 7.35, and the osmotic pressure is 335 mOsm/kg.
  • Example 3 A serum-free and heterologous mesenchymal stem cell culture medium
  • a serum-free, heterologous-free mesenchymal stem cell culture medium which is composed of DMEM/F12 medium (1 ⁇ ), human platelet lysate and human lipocalin 2.
  • the human platelet lysates
  • the volume content of the liquid in the mesenchymal stem cell culture medium is 5%, and the content of the human lipocalin 2 in the mesenchymal stem cell culture medium is 50 ⁇ g/mL.
  • the pH of the mesenchymal stem cell culture medium is 7.42, and the osmotic pressure is 346 mOsm/kg.
  • Example 4 A serum-free and heterologous-free mesenchymal stem cell culture medium
  • a serum-free, heterologous-free mesenchymal stem cell culture medium which is composed of DMEM/F12 medium (1 ⁇ ), human platelet lysate and human lipocalin 2.
  • the human platelet lysates
  • the volume content of the liquid in the mesenchymal stem cell culture medium is 5%, and the content of the human lipocalin 2 in the mesenchymal stem cell culture medium is 10 ⁇ g/mL.
  • the pH of the mesenchymal stem cell culture medium is 7.46, and the osmotic pressure is 337 mOsm/kg.
  • Example 5 A serum-free and heterologous-free mesenchymal stem cell culture medium
  • a serum-free, heterologous-free mesenchymal stem cell culture medium which is composed of DMEM/F12 medium (1 ⁇ ), human platelet lysate and human lipocalin 2.
  • the human platelet lysates
  • the volume content of the liquid in the mesenchymal stem cell culture medium is 1%, and the content of the human lipocalin 2 in the mesenchymal stem cell culture medium is 10 ⁇ g/mL.
  • the pH of the mesenchymal stem cell culture medium is 7.37, and the osmotic pressure is 329 mOsm/kg.
  • Example 6 A serum-containing mesenchymal stem cell culture medium (control serum-containing culture medium)
  • a serum-containing mesenchymal stem cell culture medium is composed of DMEM/F12 medium (1 ⁇ ) and fetal bovine serum, and the volume content of the fetal bovine serum in the mesenchymal stem cell culture medium is 10%.
  • the pH of the mesenchymal stem cell culture medium is 7.44, and the osmotic pressure is 349 mOsm/kg.
  • Example 7 A serum-free and heterologous-free mesenchymal stem cell culture medium (control serum-free medium)
  • a serum-free and heterologous-free mesenchymal stem cell culture medium wherein the culture medium is StemPro TM MSC SFM XenoFree.
  • the medium is composed of StemPro TM MSC SFM basal medium and StemPro TM MSC SFM XenoFree additive.
  • the volume content of the StemPro TM MSC SFM basal medium in the mesenchymal stem cell medium is 99%, and the StemPro TM
  • the volume content of the MSC SFM XenoFree additive in the mesenchymal stem cell culture medium is 1%.
  • the pH of the mesenchymal stem cell culture medium is 7.35, and the osmotic pressure is 323 mOsm/kg.
  • Example 8 Comparative test of in vitro expansion ability of human umbilical cord mesenchymal stem cells
  • Human umbilical cord mesenchymal stem cells were used as the test cells, and the serum-free and heterologous mesenchymal stem cell culture medium of Example 1, Example 2, Example 3, Example 4 and Example 5 was used as the test cell Medium, using the serum-containing mesenchymal stem cell medium of Example 6 as the control serum-containing medium and the serum-free and heterologous mesenchymal stem cell medium of Example 7 as the control serum-free medium,
  • the human umbilical cord mesenchymal stem cells were cultured continuously for 10 generations according to the above-mentioned human umbilical cord mesenchymal stem cell passage expansion culture method in the present invention, and the live cell count data of each generation when the human umbilical cord mesenchymal stem cells were cultured with different media were recorded and calculated Cumulative population doubling level (CPDL) is used to compare the in vitro proliferation ability of human umbilical cord mesenchymal stem cells with different media.
  • CPDL Cumulative population doubling level
  • Example 9 Comparative experiment on the in vitro expansion ability of human bone marrow mesenchymal stem cells
  • Human bone marrow mesenchymal stem cells were used as test cells, the serum-free and heterologous mesenchymal stem cell medium of Example 3 was used as the test medium, and the serum-containing mesenchymal stem cells of Example 6 were used as the test medium.
  • the medium was used as a control serum-containing medium and the serum-free and heterologous mesenchymal stem cell medium of Example 7 was used as a control serum-free medium, according to the above-mentioned human bone marrow mesenchymal stem cell subculture expansion culture method in the present invention
  • Culture human bone marrow mesenchymal stem cells for 5 consecutive generations record the live cell count data of each generation when human bone marrow mesenchymal stem cells are cultured in different media, and calculate the cumulative cell population doubling number to compare the effects of different media on human bone marrow mesenchyme The ability of stem cells to proliferate in vitro.
  • the results of the cumulative cell population doubling number of human bone marrow mesenchymal stem cells cultured continuously for 5 generations using different culture media are shown in Figure 2.
  • the in vitro proliferation ability in the heterologous mesenchymal stem cell culture medium is the strongest.
  • the in vitro proliferation ability in the control serum medium of Example 6 is average, and the in vitro proliferation ability in the control serum-free medium of Example 7 is the strongest. weak.
  • the cumulative cell population doubling number of human bone marrow mesenchymal stem cells cultured using the medium of Example 3 was 1.18 and 1.54 times that of the control serum-containing medium and the control serum-free medium, respectively.
  • Example 10 Comparative experiment on the in vitro expansion ability of human adipose-derived mesenchymal stem cells
  • Human adipose-derived mesenchymal stem cells were used as test cells, the serum-free and heterologous mesenchymal stem cell medium of Example 3 was used as the test medium, and the serum-containing mesenchymal stem cells of Example 6 were used as the test medium.
  • the medium was used as a control serum-containing medium and the serum-free and heterologous mesenchymal stem cell medium of Example 7 was used as a control serum-free medium, according to the above-mentioned human adipose-derived mesenchymal stem cell generation expansion culture method of the present invention Continuously culture human adipose-derived mesenchymal stem cells for 4 generations, record the live cell count data of each generation when human adipose-derived mesenchymal stem cells are cultured in different media, and calculate the cumulative cell population doubling number to compare the effects of different media on human adipose-derived mesenchymal stem cells The in vitro proliferation ability.
  • Example 11 Cell morphology comparison experiment of culturing human umbilical cord mesenchymal stem cells, human bone marrow mesenchymal stem cells, and human adipose mesenchymal stem cells
  • the serum-free and heterologous mesenchymal stem cell medium of Example 3 was used as the test medium, and the serum-containing mesenchymal stem cell medium of Example 6 was used as the control serum-containing medium and Example 7 Serum-free and heterologous mesenchymal stem cell medium as a control serum-free medium, according to the present invention, the above-mentioned human umbilical cord mesenchymal stem cells, human bone marrow mesenchymal stem cells, and human adipose mesenchymal stem cells are subcultured and expanded Cultivation method Human umbilical cord mesenchymal stem cells, human bone marrow mesenchymal stem cells, and human adipose-derived mesenchymal stem cells were cultured for 5 consecutive passages, respectively at the 6th passage on the 3rd day (P6, D3) and the 7th passage on the 4th day (P7).
  • Example 4 Using the serum-free and heterologous mesenchymal stem cell medium of Example 3, the control serum-free medium of Example 6 and the control serum-containing medium of Example 7 for continuous cultivation of human umbilical cord mesenchymal stem cells and human bone marrow The cell morphology of mesenchymal stem cells and human adipose-derived mesenchymal stem cells is shown in Figure 4.
  • Example 3 the cell morphology of continuously cultured human umbilical cord mesenchymal stem cells, human bone marrow mesenchymal stem cells, and human adipose mesenchymal stem cells in the serum-free and heterologous mesenchymal stem cell medium was spindle-shaped. The volume is smaller and equal, and it grows in a whirlpool shape.
  • Example 12 Comparative experiment on identification of surface markers of human umbilical cord mesenchymal stem cells by flow cytometry
  • the serum-free and heterologous mesenchymal stem cell medium of Example 3 was used as the test medium, and the serum-containing mesenchymal stem cell medium of Example 6 was used as the control serum-containing medium and Example 7
  • the serum-free and heterologous mesenchymal stem cell culture medium is used as the control serum-free culture medium.
  • human umbilical cord mesenchymal stem cell subculture expansion culture method in the present invention human umbilical cord mesenchymal stem cells are continuously cultured for 9 generations .
  • Example 13 Comparative experiment of PGE2 secretion ability of human umbilical cord mesenchymal stem cells under the stimulation of inflammatory factors
  • the serum-free and heterologous mesenchymal stem cell medium of Example 3 was used as the test medium, and the serum-free and heterologous mesenchymal stem cell medium of Example 7 was used as a control serum-free culture.
  • the human umbilical cord mesenchymal stem cells are continuously cultured for 5 generations.
  • kit positive control well TA
  • kit negative control well Second Edition
  • kit positive control well TA
  • kit negative control well Blank
  • kit non-specific binding control well NBS
  • the amount of PGE2 secreted by human umbilical cord mesenchymal stem cells cultured in the serum-free and heterologous mesenchymal stem cell medium of Example 3 was 14.79 times that of the control serum-free medium.
  • the results show that the human umbilical cord mesenchymal stem cells cultured in the serum-free and heterologous mesenchymal stem cell medium of Example 3 have an enhanced ability to secrete the immunoregulatory factor PGE2.
  • Example 14 Comparative experiment of the ability of human umbilical cord mesenchymal stem cells to inhibit the proliferation of PBMC
  • the serum-free and heterologous mesenchymal stem cell medium of Example 3 was used as the test medium, and the serum-containing mesenchymal stem cell medium of Example 6 was used as the control serum-containing medium and Example 7
  • the serum-free and heterologous mesenchymal stem cell culture medium is used as the control serum-free culture medium.
  • human umbilical cord mesenchymal stem cell subculture expansion culture method in the present invention human umbilical cord mesenchymal stem cells are continuously cultured for 5 generations .
  • the human umbilical cord mesenchymal stem cells cultured in different media were tested for their ability to inhibit the proliferation of PBMC according to the following methods:
  • PHA (2.5 ⁇ g/mL) was added to the mesenchymal stem cell-PBMC co-culture well and one PBMC single culture well for activation stimulation.
  • Figure 6 shows the ability of continuous culture of human umbilical cord mesenchymal stem cells to inhibit the proliferation of PBMC using different media. It can be seen from the figure that under separate culture conditions, before and after PHA stimulation, the proportion of PBMC in the proliferation phase can rise from 1.23% to 56.94% (in the figure, P2 represents the proportion of non-proliferating cells, and P3 represents the proportion of proliferating cells). Co-cultivation with mesenchymal stem cells can effectively inhibit the proliferation of PBMC caused by PHA stimulation. Specifically, co-cultivation with mesenchymal stem cells cultured in a control serum-containing medium (Example 6) can reduce the proliferation ratio of PBMC from 56.94% to 22.60% (ie 34.34%).
  • Co-cultivation with mesenchymal stem cells cultured in the control serum-free medium can reduce the proliferation rate of PBMC from 56.94% to 39.31% (ie 17.63%).
  • Co-cultivation of mesenchymal stem cells cultured with the serum-free and heterologous mesenchymal stem cell medium of Example 3 can reduce the PBMC proliferation ratio from 56.94% to 10.69% (ie 46.25%).
  • the ratio of inhibiting PBMC proliferation of human umbilical cord mesenchymal stem cells cultured using the serum-free and heterologous mesenchymal stem cell medium of Example 3 under PHA stimulation is the control serum-containing medium and the control serum-free culture, respectively 1.35 times and 2.62 times of the base. The results show that the mesenchymal stem cells cultured in the serum-free and heterologous mesenchymal stem cell medium of Example 3 under PHA stimulation have the strongest ability to inhibit the proliferation of PBMC.
  • Example 15 Comparative experiment on the osteogenic differentiation ability of human umbilical cord mesenchymal stem cells
  • the serum-free and heterologous mesenchymal stem cell medium of Example 3 was used as the test medium, and the serum-free and heterologous mesenchymal stem cell medium of Example 7 was used as a control serum-free culture.
  • the human umbilical cord mesenchymal stem cells are continuously cultured for 5 generations.
  • the osteogenic differentiation ability of human umbilical cord mesenchymal stem cells cultured in different media was tested according to the following methods:
  • Fig. 7 The osteogenesis-directed differentiation of human umbilical cord mesenchymal stem cells cultured in different culture media is shown in Fig. 7. It can be seen from the figure that the serum-free and heterologous mesenchymal stem cell culture medium of Example 3 is continuously cultured in human umbilical cord mesenchymal stem cells for osteogenic differentiation and the positive area of Alizarin Red staining is the control of Example 7. 1.98 times of the serum medium group (p ⁇ 0.001). The results show that the serum-free and heterologous mesenchymal stem cell culture medium of Example 3 can improve the osteogenic differentiation ability of human umbilical cord mesenchymal stem cells.
  • Example 16 Comparative experiment on the ability of human umbilical cord mesenchymal stem cells to differentiate into cartilage
  • the serum-free and heterologous mesenchymal stem cell medium of Example 3 was used as the test medium, and the serum-free and heterologous mesenchymal stem cell medium of Example 7 was used as a control serum-free culture.
  • the human umbilical cord mesenchymal stem cells are continuously cultured for 5 generations.
  • the human umbilical cord mesenchymal stem cells cultured in different media were tested for their chondrogenic directional differentiation ability according to the following methods:
  • Fig. 8 shows the directional differentiation of human umbilical cord mesenchymal stem cells cultured in different culture media. It can be seen from the figure that the serum-free and heterologous mesenchymal stem cell medium of Example 3 continuously cultured human umbilical cord mesenchymal stem cells for chondrogenic differentiation and the positive area stained with Alcian Blue was the control of Example 7. The serum culture medium group was 2.63 times (p ⁇ 0.01). The results showed that the serum-free and heterologous mesenchymal stem cell culture medium of Example 3 can improve the ability of human umbilical cord mesenchymal stem cells to differentiate into cartilage.
  • Example 17 Comparative experiment of adipogenic differentiation ability of human umbilical cord mesenchymal stem cells
  • the serum-free and heterologous mesenchymal stem cell medium of Example 3 was used as the test medium, and the serum-free and heterologous mesenchymal stem cell medium of Example 7 was used as a control serum-free culture.
  • the human umbilical cord mesenchymal stem cells are continuously cultured for 5 generations.
  • Example 3 serum-free and heterologous mesenchymal stem cell culture medium continuously cultured human umbilical cord mesenchymal stem cells adipogenic differentiation oil red O staining positive area is the serum-free control of Example 7 3.25 times of the culture medium group (p ⁇ 0.001), the results show that the serum-free and heterologous mesenchymal stem cell culture medium of Example 3 can improve the adipogenic differentiation ability of human umbilical cord mesenchymal stem cells.

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Abstract

提供了一种无血清、无异源成分的间充质干细胞培养基及其应用。该培养基包含基础培养基和添加物，所述基础培养基为DMEM/F12培养基，所述添加物为人血小板裂解液和/或人脂质运载蛋白2。

Description

一种无血清、无异源成分的间充质干细胞培养基及其应用 技术领域
本发明涉及细胞培养技术领域。更具体地，涉及一种无血清、无异源成分的间充质干细胞培养基及其应用。
背景技术
间充质干细胞是一类具有自我复制更新和多向分化潜能的成体干细胞，广泛存在于胎儿和成人各组织中。除自我更新外，间充质干细胞具有独特的免疫调节和组织再生功能。一方面，间充质干细胞通过与免疫细胞间的相互作用以及旁分泌可溶性因子对免疫细胞进行调控，介导免疫反应平衡，帮助机体形成有利于病损组织细胞修复或再生的微环境。间充质干细胞的免疫调控包括抑制T淋巴细胞活化、降低B细胞活化增殖及抗体分泌、抑制自然杀伤细胞增殖和抑制树突状细胞成熟及抗原呈递。另一方面，间充质干细胞可分化为成骨细胞、软骨细胞、脂肪细胞、肝实质细胞、肌细胞以及神经元等多种功能细胞，参与损伤组织器官的重建。
目前，全球范围内在Clinical trails网站上注册的应用间充质干细胞移植治疗各种疾病的临床研究多达1000余项，包括间充质干细胞移植治疗移植物抗宿主病(graft versus host disease,GVHD)(ClinicalTrials.gov Identifier:NCT00366145)、股骨头坏死(osteonecrosis of femoral head,ONFH)(ClinicalTrials.gov Identifier:NCT01700920)、退行性关节炎(osteoarthritis,OA)(ClinicalTrials.gov Identifier:NCT01809769)、龙贝里综合征(Romberg's Disease)(ClinicalTrials.gov Identifier:NCT02494752)等，均取得了显著疗效。其治疗机制主要依赖于间充质干细胞的免疫调节和组织再生功能。例如，移植物抗宿主病的发生主要与活化的供体T细胞有关。活化的T细胞对抗原发生免疫应答后产生炎症因子IFNγ，而移植的间充质干细胞可响应IFNγ，通过分泌PGE2等免疫调节因子抑制T细胞的增殖，实现对移植物抗宿主病的治疗(Polchert D，Sobinsky J，Douglas G，Kidd M，Moadsiri A，Reina E，Genrich K，Mehrotra S，Setty S，Smith B et al:IFN-gamma activation of mesenchymal stem cells for treatment and prevention of graft versus host disease.European journal of immunology 2008，38(6):1745-1755.)。而将间充质干细胞应用于ONFH、OA、Romberg's Disease的治疗则一定程度上分别依赖于间充质干细胞成骨、成软骨、成脂分化能力。
随着间充质干细胞作为重要种子细胞在基础医学、再生医学、临床医学、组织工程等领域的广泛应用，符合产业转化和临床应用要求、能较好维持间充质干细胞免疫调节功能和多向分化潜能的高效体外扩增体系是支持间充质干细胞产业转化、推进间充质干细胞新药研发的关键因素。目前，常用的间充质干细胞培养基都含一定浓度的胎牛血清/动物蛋白。胎牛血清/动物蛋白属异种蛋白，在培养过程中可被间充质干细胞吞噬，间充质干细胞移植后在受者体内释放，极易引起免疫反应。其次，胎牛血清成分比较复杂，易受病毒、支原体和其他病原体的污染，在临床应用上存在潜在风险。研发无血清，无异源成分的间充质 干细胞培养基是产业发展的必然趋势。已有一些专利公开了无血清间充质干细胞培养基的成分，如专利CN109402050在基础培养基中加入了人血小板裂解液、青链霉素(Pen Strep)、谷氨酰胺、趋化因子XCL1、趋化因子CCL3、热休克蛋白(HSP70)、端粒酶抑制剂IFN-α 2b。也有一些公司开发了无血清，无异源成分的间充质干细胞培养基，如市场上广泛应用的美国Thermo Fisher公司的StemPro TM MSC SFM XenoFree(货号为A1067501)。但是，已开发的无血清间充质干细胞培养基都存在以下几个缺点:1)成分复杂。成分种类多达十种甚至更多，导致培养基批次间稳定性差、质控困难；2)不能较好维持间充质干细胞免疫调节功能。培养的间充质干细胞分泌免疫调节因子PGE2、抑制PBMC细胞增殖的能力弱；3)不能较好维持间充质干细胞多向分化潜能。培养的间充质干细胞定向分化为成骨细胞、软骨细胞、脂肪细胞的效率低。这些问题很大程度上限制了间充质干细胞的产业转化和临床应用。
因此，亟待提供一种无血清、无异源成分的间充质干细胞培养基，能解决上述问题中至少一个问题。
发明内容
本发明的一个目的在于提供一种间充质干细胞培养基，该培养基无血清和无异源成分，可显著提高间充质干细胞增殖能力的同时，也能很好地维持间充质干细胞的免疫调节功能和多向分化潜能。
本发明的另一个目的在于提供上述间充质干细胞培养基在培养间充质干细胞中的应用。
为达到上述目的，本发明首先提供了一种无血清、无异源成分的间充质干细胞培养基。
本发明所述间充质干细胞培养基包括基础培养基和添加物，所述基础培养基为DMEM/F12培养基，所述添加物为人血小板裂解液(human platelet lysate，hPL)和/或人脂质运载蛋白2(lipocalin 2或LCN2蛋白[Homo sapiens])。
本发明所述间充质干细胞培养基可以只由基础培养基和添加物组成，也可以包含其他组分；其中，所述添加物可以只由人血小板裂解液和人脂质运载蛋白2组成，也可以包含其他组分。
进一步，所述人血小板裂解液在所述间充质干细胞培养基的体积含量为1％-20％，所述人脂质运载蛋白2在所述间充质干细胞培养基的含量为10-200μg/mL。
在本发明优选的实施方式中，所述人血小板裂解液在所述间充质干细胞培养基的体积含量为2％-10％，所述人脂质运载蛋白2在所述间充质干细胞培养基的含量为20-100μg/mL。
在本发明更优选的实施方式中，所述人血小板裂解液在所述间充质干细胞培养基的体积含量为5％，所述人脂质运载蛋白2在所述间充质干细胞培养基的含量为50μg/mL。
进一步，所述间充质干细胞培养基的pH值为7.3-7.5，渗透压为300-350mOsm/kg。
为达到上述目的，本发明进一步提供了上述间充质干细胞培养基在培养间充质干细胞中的应用或在制备培养间充质干细胞的产品中的应用。
进一步，所述培养可以为原代分离培养和/或传代扩增培养。
上述间充质干细胞培养基在如下任一中的应用也在本发明的保护范围之内：
1)在提高间充质干细胞增殖能力中的应用；
2)在制备提高间充质干细胞增殖能力的产品中的应用。
3)在提高间充质干细胞免疫调节能力中的应用；
4)在制备提高间充质干细胞免疫调节能力的产品中的应用；
5)在提高间充质干细胞分化能力中的应用；
6)在制备提高间充质干细胞分化能力的产品中的应用。
在本发明优选的实施方式中，所述提高间充质干细胞免疫调节能力体现在利用本发明培养基对间充质干细胞进行培养可增强间充质干细胞分泌免疫调节因子PGE2和抑制PBMC细胞增殖的能力；所述提高间充质干细胞分化能力主要体现在利用本发明培养基对间充质干细胞进行培养可提高间充质干细胞定向分化成骨细胞、软骨细胞、脂肪细胞的效率。
本发明进一步还提供了人血小板裂解液和/或人脂质运载蛋白2在培养间充质干细胞中的应用或在制备培养间充质干细胞的产品中的应用。
进一步，所述培养可以为原代分离培养和/或传代扩增培养。
人血小板裂解液和/或人脂质运载蛋白2在如下任一中的应用也在本发明的保护范围之内：
1)在提高间充质干细胞增殖能力中的应用；
2)在制备提高间充质干细胞增殖能力的产品中的应用。
3)在提高间充质干细胞免疫调节能力中的应用；
4)在制备提高间充质干细胞免疫调节能力的产品中的应用；
5)在提高间充质干细胞分化能力中的应用；
6)在制备提高间充质干细胞分化能力的产品中的应用。
在本发明优选的实施方式中，所述提高间充质干细胞免疫调节能力体现在利用本发明培养基对间充质干细胞进行培养可增强间充质干细胞分泌免疫调节因子PGE2和抑制PBMC细胞增殖的能力；所述提高间充质干细胞分化能力主要体现在利用本发明培养基对间充质干细胞进行培养可提高间充质干细胞定向分化成骨细胞、软骨细胞、脂肪细胞的效率。
本发明中，所述间充质干细胞为人间充质干细胞，可以为人脐带、人骨髓、人脂肪来源的间充质干细胞，即人脐带间充质干细胞、人骨髓间充质干细胞和/或人脂肪间充质干细胞。
本发明中，所述产品可以为试剂或试剂盒。
本发明的有益效果如下：
1、本发明无血清和无异源成分的间充质干细胞培养基中，配方简单(仅含3个组分)、批次稳定、成本低廉，可大幅提高间充质干细胞的体外增殖能力，符合产业转化要求。
2、本发明无血清和无异源成分的间充质干细胞培养基不含异源物质，不存在引入动物源性病原微生物的风险，符合临床应用的安全性要求。
3、本发明无血清和无异源成分的间充质干细胞培养基能增强间充质干细胞的生物学效应，包括提高与适应症匹配的免疫调节能力和多向分化功能，符合临床应用的有效性 要求。
附图说明
下面结合附图对本发明的具体实施方式作进一步详细的说明。
图1为分别利用实施例1-5中无血清、无异源成分的间充质干细胞培养基、实施例6的对照含血清培养基和实施例7的对照无血清培养基对人脐带间充质干细胞连续培养10代的累积细胞群体倍增数图。
图2为分别利用实施例3中无血清、无异源成分的间充质干细胞培养基、实施例6的对照含血清培养基和实施例7的对照无血清培养基对人骨髓间充质干细胞连续培养5代的累积细胞群体倍增数图。
图3为分别利用实施例3中无血清、无异源成分的间充质干细胞培养基、实施例6的对照含血清培养基和实施例7的对照无血清培养基对人脂肪间充质干细胞连续培养4代的累积细胞群体倍增数图。
图4为分别利用实施例3中无血清、无异源成分的间充质干细胞培养基、实施例6的对照含血清培养基和实施例7的对照无血清培养基连续培养人脐带间充质干细胞、人骨髓间充质干细胞、人脂肪间充质干细胞的细胞形态图，标尺：100μm。
图5为分别利用实施例3中无血清、无异源成分的间充质干细胞培养基和实施例7的对照无血清培养基连续培养人脐带间充质干细胞，炎症因子刺激前后人脐带间充质干细胞PGE2分泌图，*p<0.05，***p<0.001。
图6为分别利用实施例3中无血清、无异源成分的间充质干细胞培养基和实施例6的对照含血清培养基，实施例7的对照无血清培养基连续培养人脐带间充质干细胞对PBMC增殖抑制效率图，图中P2代表不增殖细胞比例，P3代表增殖细胞比例。
图7为分别利用实施例3中无血清、无异源成分的间充质干细胞培养基和实施例7的对照无血清培养基连续培养人脐带间充质干细胞成骨定向分化茜素红染色图，标尺：100μm，***p<0.001。
图8为分别利用实施例3中无血清、无异源成分的间充质干细胞培养基和实施例7的对照无血清培养基连续培养人脐带间充质干细胞成软骨定向分化阿利新蓝染色图，标尺：100μm，**p<0.01。
图9为分别利用实施例3中无血清、无异源成分的间充质干细胞培养基和实施例7的对照无血清培养基连续培养人脐带间充质干细胞成脂定向分化油红O染色图，标尺：100μm，***p<0.001。
具体实施方式
为了更清楚地说明本发明，下面结合优选实施例和附图对本发明做进一步的说明。附图中相似的部件以相同的附图标记进行表示。本领域技术人员应当理解，下面所具体描述的内容是说明性的而非限制性的，不应以此限制本发明的保护范围。
针对现有技术中的含血清间充质干细胞培养基中动物血清或动物源性成分的使用风险、培养的间充质干细胞体外扩增能力和免疫调节功能的维持能力有限等问题，及无血清间充质干细胞培养基的成分复杂和不能较好维持间充质干细胞体外扩增能力、免疫 调节功能和多向分化潜能，本发明提供了一种无血清、无异源成分的间充质干细胞培养基。
本发明无血清、无异源成分的间充质干细胞培养基包括基础培养基和添加物。所述基础培养基为DMEM/F12培养基，其含有糖类、氨基酸类、维生素和无机盐等基础物质，为间充质干细胞的新陈代谢提供能量来源。所述添加物为人血小板裂解液和人脂质运载蛋白2，其中，人血小板裂解液中富含各类生长因子，包括PDGF，VEGF，EGF等，可以替代胎牛血清支持间充质干细胞的生长；人脂质运载蛋白2具有抗氧化，促进细胞增殖，抑制干细胞衰老等多种功能，但是人脂质运载蛋白2尚未应用于间充质干细胞的培养，对其在体外培养中调控间充质干细胞增殖、维持免疫调节功能及多向分化潜能的作用尚未有明确的结论。本发明发明人研究发现了以DMEM/F12培养基、人血小板裂解液和人脂质运载蛋白2为配方的间充质干细胞培养基对间充质干细胞有促进细胞增殖、高效维持细胞免疫调节功能及多向分化潜能的作用，特定浓度下效果尤其显著。例如，所述人血小板裂解液在所述间充质干细胞培养基的体积含量为1-20％，所述人脂质运载蛋白2在所述间充质干细胞培养基的含量为10-200μg/mL。具体地，人血小板裂解液在所述间充质干细胞培养基的体积含量可以为1％、5％、10％和20％等等或体积含量之间的任意范围，人脂质运载蛋白2在所述间充质干细胞培养基的含量为10μg/mL、50μg/mL、100μg/mL和200μg/mL等等或含量之间的任意范围。
本发明无血清和无异源成分的间充质干细胞培养基，成分简单，应用于间充质干细胞的培养，具有如下优势：第一，能提高间充质干细胞的细胞增殖能力，具体体现在：使用本发明培养基培养的人脐带间充质干细胞、骨髓间充质干细胞、脂肪间充质干细胞的累积细胞群体倍增数分别是对照含血清培养基和对照无血清培养基的1.82、1.18、1.66和1.67、1.54、1.71倍；第二，能提高间充质干细胞的免疫调节能力，分泌免疫调节因子PGE2和抑制PBMC细胞增殖的能力强，具体体现在：在TNFα和IFNγ的共同刺激下利用本发明培养基培养的人脐带间充质干细胞的PGE2分泌量是对照无血清培养基的14.79倍；在PHA刺激下使用本发明培养基培养的人脐带间充质干细胞抑制PBMC增殖分别是对照含血清培养基和对照无血清培养基的1.35倍和2.62倍；第三，能提高间充质干细胞的多向分化能力，定向分化成骨细胞、软骨细胞、脂肪细胞的效率显著提高，具体体现在：使用本发明培养基培养的人脐带间充质干细胞成骨(茜素红)、成软骨(阿利新蓝)和成脂(油红O)分化的阳性染色面积分别是对照无血清培养基的1.98、2.63、3.25倍。
下面通过具体实施例对本发明作进一步的说明。
下述实施例中的材料，如无特殊说明，均为本领域常用材料，均可从商业途径得到。下述实施例中的细胞培养条件如无特殊说明，均为37℃，5％CO 2。下述实施例中的实验数据以平均值±标准差表示，用GraghPad Prism 5统计软件分析，*p<0.05，**p<0.01，***p<0.001。
本发明的实施例中所使用的试剂、细胞及抗体来源如下：
DMEM/F12培养基，Thermo Fisher公司，货号为12400024。具体配方见表1。
表1 DMEM/F12培养基配方表
组分(英文名称+中文) 分子量 浓度(mg/L)
Glycine(甘氨酸) 75.0 18.75
L-Alanine(L-丙氨酸) 89.0 4.45
L-Arginine hydrochloride(L-精氨酸盐酸盐) 211.0 147.5
L-Asparagine-H 2O(L-天冬酰胺一水合物) 150.0 7.5
L-Aspartic acid(L-天冬氨酸) 133.0 6.65
L-Cysteine hydrochloride-H 2O(L-半胱氨酸盐酸盐一水合物) 176.0 17.56
L-Cystine 2HCl(L-胱氨酸二盐酸盐) 313.0 31.29
L-Glutamic Acid(L-谷氨酸) 147.0 7.35
L-Glutamine(L-谷氨酰胺) 146.0 365.0
L-Histidine hydrochloride-H 2O(L-组氨酸单盐酸盐一水合物) 210.0 31.48
L-Isoleucine(L-异亮氨酸) 131.0 54.47
L-Leucine(L-亮氨酸) 131.0 59.05
L-Lysine hydrochloride(L-赖氨酸盐酸盐) 183.0 91.25
L-Methionine(L-蛋氨酸) 149.0 17.24
L-Phenylalanine(L-苯丙氨酸) 165.0 35.48
L-Proline(L-脯氨酸) 115.0 17.25
L-Serine(L-丝氨酸) 105.0 26.25
L-Threonine(L-苏氨酸) 119.0 53.45
L-Tryptophan(L-色氨酸) 204.0 9.02
L-Tyrosine disodium salt dihydrate(L-酪氨酸二钠盐二水合物) 261.0 55.79
L-Valine(L-缬氨酸) 117.0 52.85
Biotin(生物素) 244.0 0.0035
Choline chloride(氯化胆碱) 140.0 8.98
D-Calcium pantothenate(D-泛酸钙) 477.0 2.24
Folic Acid(叶酸) 441.0 2.65
Niacinamide(烟酰胺) 122.0 2.02
Pyridoxine hydrochloride(盐酸吡哆素) 206.0 2.031
Riboflavin(核黄素) 376.0 0.219
Thiamine hydrochloride(盐酸硫胺素) 337.0 2.17
Vitamin B12(维生素B12) 1355.0 0.68
i-Inositol(肌醇) 180.0 12.6
Calcium Chloride(CaCl 2)anhydrous(氯化钙) 111.0 116.6
Cupric sulfate(CuSO 4-5H 2O)(硫酸铜五水合物) 250.0 0.0013
Ferric Nitrate(Fe(NO 3) 3-9H 2O)(硝酸铁九水合物) 404.0 0.05
Ferric sulfate(FeSO 4-7H 2O)(硫酸亚铁七水合物) 278.0 0.417
Magnesium Chloride(MgCl 2)anhydrous(氯化镁) 95.0 28.64
Magnesium Sulfate(MgSO 4)anhydrous(硫酸镁) 120.0 48.84
Potassium Chloride(KCl)(氯化钾) 75.0 311.8
Sodium Chloride(NaCl)(氯化钠) 58.0 6995.5
Sodium Phosphate dibasic(Na 2HPO 4)anhydrous(磷酸氢二钠) 142.0 71.02
Sodium Phosphate monobasic(NaH 2PO 4-H 2O)(磷酸二氢钠二水合物) 138.0 62.5
Zinc sulfate(ZnSO 4-7H 2O)(硫酸锌七水合物) 288.0 0.432
D-Glucose(D-(+)-葡萄糖) 180.0 3151.0
HEPES(4-(2-羟乙基)哌嗪-1-乙磺酸) 238.0 3574.5
Hypoxanthine Na(次黄嘌呤单钠盐) 159.0 2.39
Linoleic Acid(亚油酸) 280.0 0.042
Lipoic Acid(α-硫辛酸) 206.0 0.105
Phenol Red(酚红) 376.4 8.1
Putrescine 2HCl(腐胺二盐酸盐) 161.0 0.081
Thymidine(胸苷) 242.0 0.365
StemPro TM MSC SFM XenoFree，Thermo Fisher公司，货号为A1067501。该培养基包含：StemPro TM MSC SFM基础培养基(货号为A13829-01)和StemPro TM MSC SFM XenoFree添加剂(货号为A11577-01)。具体配方参见其官网： https://www.thermofisher.com/order/catalog/product/A1067501#/A1067501。
人血小板裂解液，达科为生物技术集团，货号为EPA-500。
人脂质运载蛋白2，Abcam公司，货号为ab95007。人脂质运载蛋白2为Genbank号为AAH33089.1(更新日期为09-JUN-2008)的DNA序列所编码的蛋白质，由198个氨基酸残基组成。
胎牛血清，Thermo Fisher公司，货号为16140071。
Human Fibronectin Protein，R&D systems公司，货号为1918-FN。
Human TNFαProtein，R&D systems公司，货号为210-TA-020/CF。
Human IFNγProtein，R&D systems公司，货号为285-IF/CF。
Prostaglandin E2(PGE2)ELISA检测试剂盒，Abcam公司，货号为ab133055。
人间质干细胞成骨诱导分化试剂盒，赛业生物科技集团，货号为HUXUC-90021。
人间质干细胞成软骨诱导分化试剂盒，赛业生物科技集团，货号为HUXUC-90041。
人间质干细胞成脂诱导分化试剂盒，赛业生物科技集团，货号为HUXUC-90031。
人脐带间充质干细胞，北京全式金生物技术有限公司，货号为MC401。
人骨髓间充质干细胞，广州赛莱拉干细胞科技股份有限公司，货号为S-08-001。
人脂肪间充质干细胞，广州赛莱拉干细胞科技股份有限公司，货号为S-08-002。
人外周血单核细胞，澳赛尔斯生物技术(上海)有限公司，货号为PB006。
CD90-FITC抗体，Thermo Fisher公司，货号为11-0909-41。
CD73-FITC抗体，Thermo Fisher公司，货号为11-0739-41。
CD105-PE抗体，Thermo Fisher公司，货号为12-1057-41。
CD34-FITC抗体，Thermo Fisher公司，货号为11-0349-41。
CD45-PE抗体，Biolegend公司，货号为304007。
HLA-DR-PE抗体，Biolegend公司，货号为307605。
PE Mouse IgG1,κIsotype Ctrl抗体，Biolegend公司，货号为400211。
FITC Mouse IgG1,κIsotype Ctrl抗体，Biolegend公司，货号为400107。
下述实施例中用于人脐带间充质干细胞的原代分离培养方法，包括以下步骤：
1)用1×PBS漂洗脐带标本若干次，将脐带血管腔的血液彻底挤出。
2)将脐带剪成2cm长若干段。
3)固定好脐带组织，纵向剖开脐带，剥离脐带外皮。
4)露出2条脐动脉和1条脐静脉，将静脉和动脉拉出丢弃。
5)血管和外皮清理干净后，留下的部分则为内膜，即华通氏胶(Wharton's jelly)。
6)将华通氏胶用1×PBS洗涤2-3次。
7)将华通氏胶剪碎至1mm左右大小后，均匀铺于10cm培养皿中，室温静置5-10分钟，至组织块充分贴附到皿底。
8)向贴附好组织块的培养皿中缓慢加入4mL本发明下述实施例中的培养基，轻微晃动培养皿，使培养基浸泡到组织块，放入培养箱中(37℃，5％CO 2)培养。
9)培养1-2天后，向培养皿中添加2-3mL本发明下述实施例中的培养基。
10)培养7天后，观察细胞爬出情况，此时组织块边缘爬出的细胞即为原代脐带间充质干细胞。
下述实施例中人脐带间充质干细胞、人骨髓间充质干细胞、人脂肪间充质干细胞传代扩增培养方法，包括以下步骤：
1)用1×PBS稀释Fibronectin至10μg/mL，加入到待包被的培养皿中(加入量请参考下表)。37℃孵育1小时或者2-8℃孵育过夜，得到预先包被Fibronectin的培养皿。在孵育过程中避免基质干掉，使用时弃掉基质接种细胞即可。
注意：稀释后的Fibronectin不可长期保存。请用前稀释。
细胞培养板(皿) 面积 单孔包被量
6-well 10cm 2 1mL
12-well 4cm 2 0.5mL
24-well 2cm 2 0.25mL
35mm 10cm 2 1mL
60mm 20cm 2 2mL
100mm 60cm 2 6mL
2)按照接种密度为5×10 3-2×10 4个活细胞/cm 2接种间充质干细胞到预先包被Fibronectin的培养皿中。
3)放入培养箱(37℃，5％CO 2)中进行培养，24小时后换等量本发明下述实施例中的培养基。以后隔天换等量本发明下述实施例中的培养基。
4)显微镜下观察，细胞汇合度达到80％-90％时进行传代扩增。
5)吸弃旧的培养基，加入等量1×PBS润洗一次，吸弃。
6)每6孔板孔中加入500μL预热的TrypLE TM Express Enzyme，37℃消化5分钟，待细胞完全脱落。
7)加入2mL 37℃预热的本发明下述实施例中的培养基，轻柔吹吸成单细胞悬液。
8)300×g离心5分钟，弃上清。再加入适量37℃预热的本发明下述实施例中的培养基重悬细胞，进行活细胞计数，推荐5×10 3-2×10 4个活细胞/cm 2(例如1×10 4活细胞/cm 2)的密度接种至预先包被Fibronectin的培养皿中。
9)放入培养箱(37℃，5％CO 2)中进行培养，24小时后换等量本发明下述实施例中的培养基。以后隔天换等量本发明下述实施例中的培养基。
实施例1 一种无血清、无异源成分的间充质干细胞培养基
一种无血清、无异源成分的间充质干细胞培养基，所述培养基由DMEM/F12培养基(1×)、人血小板裂解液和人脂质运载蛋白2组成，所述人血小板裂解液在所述间充质干 细胞培养基的体积含量为20％，所述人脂质运载蛋白2在所述间充质干细胞培养基的含量为200μg/mL。
该间充质干细胞培养基的pH值为7.38，渗透压为341mOsm/kg。
实施例2 一种无血清、无异源成分的间充质干细胞培养基
一种无血清、无异源成分的间充质干细胞培养基，所述培养基由DMEM/F12培养基(1×)、人血小板裂解液和人脂质运载蛋白2组成，所述人血小板裂解液在所述间充质干细胞培养基的体积含量为10％，所述人脂质运载蛋白2在所述间充质干细胞培养基的含量为100μg/mL。
该间充质干细胞培养基的pH值为7.35，渗透压为335mOsm/kg。
实施例3 一种无血清、无异源成分的间充质干细胞培养基
一种无血清、无异源成分的间充质干细胞培养基，所述培养基由DMEM/F12培养基(1×)、人血小板裂解液和人脂质运载蛋白2组成，所述人血小板裂解液在所述间充质干细胞培养基的体积含量为5％，所述人脂质运载蛋白2在所述间充质干细胞培养基的含量为50μg/mL。
该间充质干细胞培养基的pH值为7.42，渗透压为346mOsm/kg。
实施例4 一种无血清、无异源成分的间充质干细胞培养基
一种无血清、无异源成分的间充质干细胞培养基，所述培养基由DMEM/F12培养基(1×)、人血小板裂解液和人脂质运载蛋白2组成，所述人血小板裂解液在所述间充质干细胞培养基的体积含量为5％，所述人脂质运载蛋白2在所述间充质干细胞培养基的含量为10μg/mL。
该间充质干细胞培养基的pH值为7.46，渗透压为337mOsm/kg。
实施例5 一种无血清、无异源成分的间充质干细胞培养基
一种无血清、无异源成分的间充质干细胞培养基，所述培养基由DMEM/F12培养基(1×)、人血小板裂解液和人脂质运载蛋白2组成，所述人血小板裂解液在所述间充质干细胞培养基的体积含量为1％，所述人脂质运载蛋白2在所述间充质干细胞培养基的含量为10μg/mL。
该间充质干细胞培养基的pH值为7.37，渗透压为329mOsm/kg。
实施例6 一种含血清的间充质干细胞培养基(对照含血清培养基)
一种含血清的间充质干细胞培养基，所述培养基由DMEM/F12培养基(1×)和胎牛血清组成，所述胎牛血清在所述间充质干细胞培养基的体积含量为10％。
该间充质干细胞培养基的pH值为7.44，渗透压为349mOsm/kg。
实施例7 一种无血清、无异源成分的间充质干细胞培养基(对照无血清培养基)
一种无血清、无异源成分的间充质干细胞培养基，所述培养基为StemPro TM MSC SFM  XenoFree。所述培养基由StemPro TM MSC SFM基础培养基和StemPro TM MSC SFM XenoFree添加剂组成，所述StemPro TM MSC SFM基础培养基在所述间充质干细胞培养基的体积含量为99％，所述StemPro TM MSC SFM XenoFree添加剂在所述间充质干细胞培养基的体积含量为1％。
该间充质干细胞培养基的pH值为7.35，渗透压为323mOsm/kg。
实施例8 人脐带间充质干细胞的体外扩增能力对比试验
以人脐带间充质干细胞为受试细胞，以实施例1、实施例2、实施例3、实施例4和实施例5的无血清、无异源成分的间充质干细胞培养基为受试培养基，同时以实施例6的含血清的间充质干细胞培养基作为对照含血清培养基和实施例7的无血清、无异源成分的间充质干细胞培养基作为对照无血清培养基，按照本发明中上述人脐带间充质干细胞传代扩增培养方法对人脐带间充质干细胞连续培养10代，记录利用不同培养基培养人脐带间充质干细胞时每代活细胞计数数据，并计算累积细胞群体倍增数(cumulative population doubling level，CPDL)以比较不同培养基对人脐带间充质干细胞的体外增殖能力。
使用不同培养基对人脐带间充质干细胞连续培养10代的累积细胞群体倍增数结果如图1所示。由图可以看出，相比于实施例1-5的无血清、无异源成分的间充质干细胞培养基，人脐带间充质干细胞在实施例6的对照含血清培养基和实施例7的对照无血清培养基中的体外增殖能力较弱；另外，实施例3的无血清、无异源成分的间充质干细胞培养基相对于其他培养基对于人脐带间充质干细胞的体外增殖能力最强。使用实施例3培养基培养的人脐带间充质干细胞的累积细胞群体倍增数分别是对照含血清培养基和对照无血清培养基的1.82和1.67倍。
实施例9 人骨髓间充质干细胞的体外扩增能力对比实验
以人骨髓间充质干细胞为受试细胞，以实施例3的无血清、无异源成分的间充质干细胞培养基为受试培养基，同时以实施例6的含血清的间充质干细胞培养基作为对照含血清培养基和实施例7的无血清、无异源成分的间充质干细胞培养基作为对照无血清培养基，按照本发明中上述人骨髓间充质干细胞传代扩增培养方法对人骨髓间充质干细胞连续培养5代，记录利用不同培养基培养人骨髓间充质干细胞时每代活细胞计数数据，并计算累积细胞群体倍增数以比较不同培养基对人骨髓间充质干细胞的体外增殖能力。
使用不同培养基对人骨髓间充质干细胞连续培养5代的累积细胞群体倍增数结果如图2所示，由图可以看出，人骨髓间充质干细胞培养在实施例3的无血清、无异源成分的间充质干细胞培养基中的体外增殖能力最强，在实施例6的对照血清培养基中的体外增殖能力一般，在实施例7的对照无血清培养基中的体外增殖能力最弱。使用实施例3培养基培养的人骨髓间充质干细胞的累积细胞群体倍增数分别是对照含血清培养基和对照无血清培养基的1.18和1.54倍。
实施例10 人脂肪间充质干细胞的体外扩增能力对比实验
以人脂肪间充质干细胞为受试细胞，以实施例3的无血清、无异源成分的间充质干细胞培养基为受试培养基，同时以实施例6的含血清的间充质干细胞培养基作为对照含血清 培养基和实施例7的无血清、无异源成分的间充质干细胞培养基作为对照无血清培养基，按照本发明中上述人脂肪间充质干细胞代扩增培养方法对人脂肪间充质干细胞连续培养4代，记录利用不同培养基培养人脂肪间充质干细胞时每代活细胞计数数据，计算累积细胞群体倍增数以比较不同培养基对人脂肪间充质干细胞的体外增殖能力。
使用不同培养基对人脂肪间充质干细胞连续培养4代的累积细胞群体倍增数结果如图3所示，由图可以看出，人脂肪间充质干细胞培养在实施例3的无血清、无异源成分的间充质干细胞培养基中的体外增殖能力最强，在实施例6的对照血清培养基和实施例7的对照无血清培养基中的体外增殖能力一般。使用实施例3培养基培养的人脂肪间充质干细胞的累积细胞群体倍增数分别是对照含血清培养基和对照无血清培养基的1.66和1.71倍。
实施例11 培养人脐带间充质干细胞、人骨髓间充质干细胞、人脂肪间充质干细胞的细胞形态对比实验
以实施例3的无血清、无异源成分的间充质干细胞培养基为受试培养基，同时以实施例6的含血清的间充质干细胞培养基作为对照含血清培养基和实施例7的无血清、无异源成分的间充质干细胞培养基作为对照无血清培养基，按照本发明中上述人脐带间充质干细胞、人骨髓间充质干细胞、人脂肪间充质干细胞传代扩增培养方法对人脐带间充质干细胞、人骨髓间充质干细胞、人脂肪间充质干细胞连续培养5代，分别在第6代第3天(P6，D3)、第7代第4天(P7，D4)、第7代第5天(P7，D5)时在显微镜下采集人脐带间充质干细胞、人骨髓间充质干细胞、人脂肪间充质干细胞的图片。
使用实施例3的无血清、无异源成分的间充质干细胞培养基、实施例6的对照无血清培养基和实施例7的对照含血清培养基连续培养人脐带间充质干细胞、人骨髓间充质干细胞、人脂肪间充质干细胞的细胞形态图如图4所示，由图可以看出，与实施例6的对照含血清培养基和实施例7的对照无血清培养基相比，在实施例3中无血清、无异源成分的间充质干细胞培养基的连续培养的人脐带间充质干细胞、人骨髓间充质干细胞、人脂肪间充质干细胞的细胞形态呈梭形，体积更小且均等、呈旋涡状生长。
实施例12 流式细胞术鉴定人脐带间充质干细胞表面标志物的对比实验
以实施例3的无血清、无异源成分的间充质干细胞培养基为受试培养基，同时以实施例6的含血清的间充质干细胞培养基作为对照含血清培养基和实施例7的无血清、无异源成分的间充质干细胞培养基作为对照无血清培养基，按照本发明中上述人脐带间充质干细胞传代扩增培养方法，对人脐带间充质干细胞连续培养9代。
按照下述方法对不同培养基培养的第10代的人脐带间充质干细胞进行纯度分析：
1)显微镜下观察，细胞汇合度达到80％-90％时，吸弃旧培养基。
2)用1×PBS润洗细胞一次。
3)每6孔板孔中加入500μL预热的TrypLE TM Express Enzyme，37℃消化3-5分钟，待细胞完全脱落。一次检测需要2孔细胞量。
4)每6孔板孔加入2mL预热的相同培养基，轻柔吹吸成单细胞悬液。
5)细胞计数后分装成8管，每管1×10 5-2×10 5个细胞，300×g离心5分钟，弃上清。
6)按下表在每管中加入相应抗体,充分混匀。
序号 10％FBS in 1×PBS 抗体
#1 100μL 0.1μL FITC Mouse IgG1,κIsotype Ctrl
#2 100μL 1μL PE Mouse IgG1,κIsotype Ctrl
#3 100μL 1μL CD90-FITC
#4 100μL 1μL CD73-FITC
#5 100μL 1μL CD105-PE
#6 100μL 1μL CD34-FITC
#7 100μL 1μL CD45-PE
#8 100μL 1μL HLA-DR-PE
7)避光室温孵育1h，每隔15min摇晃一次，避免细胞沉降。
8)每管加1mL 1×PBS洗细胞2-3遍。
9)最后每管用200μL 1×PBS重悬。
10)将样品管置于冰上进行流式细胞仪检测分析。
使用不同培养基连续培养人脐带间充质干细胞9代后细胞表面标志物表达数据如表2所示，由表可以看出，实施例6的对照含血清培养基培养的人脐带间充质干细胞表达CD90、CD73、CD105、CD34、CD45和HLA-DR的细胞纯度为99.90±0.18％、96.53±3.75％、99.35±1.08％、0.37±0.07％、0.98±0.67％和1.17±0.02％(n＝3)，实施例7的对照无血清培养基培养的人脐带间充质干细胞表达CD90、CD73、CD105、CD34、CD45和HLA-DR的细胞纯度为99.99±0.02％、97.08±2.72％、99.89±0.05％、0.65±0.14％、0.43±0.34％和0.31±0.11％(n＝3)，实施例3的无血清、无异源成分的培养基培养的人脐带间充质干细胞表达CD90、CD73、CD105、CD34、CD45和HLA-DR的细胞纯度为99.86±0.14％、98.98±0.41％、99.86±0.10％、0.36±0.29％、0.04±0.02％和0.05±0.04％(n＝3)。结果表明使用不同培养基连续培养得到的人脐带间充质干细胞纯度均较高。
表2 使用不同培养基连续培养后，第10代人脐带间充质干细胞表面标志物表达情况(n＝3)
Figure PCTCN2021080728-appb-000001
实施例13 炎症因子刺激条件下人脐带间充质干细胞PGE2分泌能力的对比实验
以实施例3的无血清、无异源成分的间充质干细胞培养基为受试培养基，同时以实施 例7的无血清、无异源成分的间充质干细胞培养基作为对照无血清培养基，按照本发明中上述人脐带间充质干细胞传代扩增培养方法，对人脐带间充质干细胞连续培养5代。
按照下述方法对不同培养基培养的人脐带间充质干细胞进行炎症因子刺激条件下PGE2分泌能力检测：
1)按照1×10 5活细胞/6孔板孔的密度，接种间充质干细胞到2个6孔板孔中，于37℃，CO 2培养箱中培养。
2)接种后第2天(48h)换液。2孔细胞中1孔作为空白对照(不处理，仅换相同培养基)，另1孔用TNFα和IFNγ处理(换相同培养基+20ng/mL TNFα+20ng/mL IFNγ)。
3)处理24h后分别4℃，5000rpm离心15min，收集细胞上清，分装(100μL/管)，作为空白对照和TNFα+IFNγ处理的待测样品进行ELISA检测，或冻于-80℃保存待检。
4)按照PGE2 ELISA试剂盒的说明书，取适量去离子水将20×Wash Buffer稀释成1×Wash Buffer。
5)按照PGE2 ELISA试剂盒的说明书，用相同细胞培养基将试剂盒中标准品进行梯度稀释，制成不同浓度标准品溶液。
6)将空白对照和TNFα+IFNγ处理的待测样品平衡至室温后，用相同细胞培养基稀释40倍。
7)按照PGE2 ELISA试剂盒的说明书，向96孔Elisa板孔中加入不同浓度标准品溶液和待测样品。
8)除试剂盒阳性对照孔(TA)和试剂盒阴性对照孔(Blank)外，每个孔中加入50μL PGE2-AP conjugate。
9)除试剂盒阳性对照孔(TA)、试剂盒阴性对照孔(Blank)和试剂盒非特异结合对照孔(NSB)外，每个孔中加入50μL Anti-PGE2 Antibody。
10)将96孔Elisa板放在平板摇床上，500rpm，室温孵育2小时。
11)弃掉溶液，每孔加入200μL 1×Wash Buffer，平板摇床上，100rpm润洗5min。重复3遍。
12)洗3遍后，将板子彻底晾干。
13)在TA孔中加入5μL PGE2-AP Conjugate。
14)在每个孔中加入200μL pNpp Substrate Solution。37℃培养箱中孵育1h。
15)在每个孔中加入50μL Stop Solution。晃匀后，立即用多功能微孔板检测仪在405nm波长下读取OD值。
使用不同培养基连续培养人脐带间充质干细胞，炎症因子TNFα和IFNγ的共同刺激后人脐带间充质干细胞PGE2分泌图如图5。由图可以看出，实施例7的对照无血清培养基培养的人脐带间充质干细胞在静息状态(Basal)和炎症因子刺激状态(TNFα+IFNγ)下，PGE2分泌量为0.68ng/mL和1.42ng/mL(p<0.05，n＝3)。实施例3的无血清、无异源成分的间充质干细胞培养基培养的人脐带间充质干细胞在静息状态(Basal)和炎症因子刺激状态(TNFα+IFNγ)下，PGE2分泌量为0.91ng/mL和21.00ng/mL(p<0.001，n＝3)。在炎症因子刺激状态下，利用实施例3的无血清、无异源成分的间充质干细胞培养基培养的人脐带间充质干细胞的PGE2分泌量是对照无血清培养基的14.79倍。结果表明实施例3的无血清、无异源成分的间充质干细胞培养基培养的人脐带间充质干细胞的分泌免疫调节 因子PGE2的能力增强。
实施例14 人脐带间充质干细胞对PBMC增殖抑制能力的对比实验
以实施例3的无血清、无异源成分的间充质干细胞培养基为受试培养基，同时以实施例6的含血清的间充质干细胞培养基作为对照含血清培养基和实施例7的无血清、无异源成分的间充质干细胞培养基作为对照无血清培养基，按照本发明中上述人脐带间充质干细胞传代扩增培养方法，对人脐带间充质干细胞连续培养5代。
按照下述方法对不同培养基培养的人脐带间充质干细胞进行对PBMC增殖抑制能力检测：
1)按照1×10 5活细胞/孔的密度，接种间充质干细胞到1个6孔板孔中，于37℃，CO 2培养箱中培养。
2)取5×10 6个活PBMC,用5mL 1×CFDA SE染色液重悬，轻轻混匀。
3)于37℃培养箱中，避光孵育30分钟。
4)500×g离心5分钟，离心后去上清，加入5mL 1×PBS润洗。
5)重复上述操作一次。
6)加入500uL PBMC细胞培养基制成PBMC细胞悬液。
7)分别取100uL PBMC悬液与不同培养条件的间充质干细胞共培养，另外200uL PBMC悬液分别接种到2个空白孔中。
8)向间充质干细胞-PBMC共培养孔和1个PBMC单独培养孔中添加PHA(2.5μg/mL)进行活化刺激。
9)72h后收集5个孔中悬浮的PBMC进行流式检测，选用FITC通道。
使用不同培养基连续培养人脐带间充质干细胞对PBMC增殖抑制能力图如图6所示。由图可以看出，在单独培养条件下，受PHA刺激前后，处于增殖期的PBMC比例可从1.23％上升至56.94％(图中P2代表不增殖细胞比例，P3代表增殖细胞比例)。与间充质干细胞共同培养，可有效抑制由PHA刺激引起的PBMC增殖。具体地，与对照含血清培养基(实施例6)培养的间充质干细胞共培养，可将PBMC增殖比率从56.94％降低到22.60％(即34.34％)。与对照无血清培养基(实施例7)培养的间充质干细胞共培养，可将PBMC增殖比率从56.94％降低到39.31％(即17.63％)。与实施例3的无血清、无异源成分的间充质干细胞培养基培养的间充质干细胞共培养，可将PBMC增殖比率从56.94％降低到10.69％(即46.25％)。经计算，在PHA刺激下使用实施例3的无血清、无异源成分的间充质干细胞培养基培养的人脐带间充质干细胞抑制PBMC增殖比率分别是对照含血清培养基和对照无血清培养基的1.35倍和2.62倍。结果表明，在PHA刺激下实施例3的无血清、无异源成分的间充质干细胞培养基培养的间充质干细胞对PBMC增殖抑制能力最强。
实施例15 人脐带间充质干细胞成骨定向分化能力的对比实验
以实施例3的无血清、无异源成分的间充质干细胞培养基为受试培养基，同时以实施例7的无血清、无异源成分的间充质干细胞培养基作为对照无血清培养基，按照本发明中上述人脐带间充质干细胞传代扩增培养方法，对人脐带间充质干细胞连续培养5代。
按照下述方法对不同培养基培养的人脐带间充质干细胞进行成骨定向分化能力检测：
1)加500μL 0.1％明胶到一个12孔板孔中，摇匀，使明胶覆盖整个底面。在超净台内孵育至少30min。
2)弃明胶，待12孔板孔晒干后，可用于接种细胞。
3)按照2×10 4活细胞/cm 2的密度，接种人脐带间充质干细胞至0.1％明胶包被过的12孔板孔中。
4)细胞汇合度达到60％-70％时，吸走旧培养基，加入1mL间充质干细胞成骨分化培养基。
5)每隔3天，吸走旧培养基，加入等量间充质干细胞成骨分化培养基。
6)诱导21天后，可观察到明显的钙结节。成骨定向分化结束。
7)进行茜素红染色。吸弃培养基，每孔加入2mL 1×PBS润洗1-2次。
8)每孔加入2mL 4％多聚甲醛溶液，室温固定30min。
9)吸弃多聚甲醛溶液，每孔加入2mL 1×PBS润洗2次。
10)每孔加入1mL茜素红染液，染3-5min。
11)吸弃茜素红染液，每孔加入2mL 1×PBS润洗3次。
12)将培养板置于显微镜下观察成骨染色结果。
使用不同培养基连续培养的人脐带间充质干细胞成骨定向分化茜素红染色图如图7。由图可以看出，实施例3的无血清、无异源成分的间充质干细胞培养基连续培养的人脐带间充质干细胞成骨定向分化茜素红染色阳性面积是实施例7的对照无血清培养基组的1.98倍(p<0.001)。结果表明实施例3的无血清、无异源成分的间充质干细胞培养基能提高人脐带间充质干细胞成骨定向分化能力。
实施例16 人脐带间充质干细胞成软骨定向分化能力的对比实验
以实施例3的无血清、无异源成分的间充质干细胞培养基为受试培养基，同时以实施例7的无血清、无异源成分的间充质干细胞培养基作为对照无血清培养基，按照本发明中上述人脐带间充质干细胞传代扩增培养方法，对人脐带间充质干细胞连续培养5代。
按照下述方法对不同培养基培养的人脐带间充质干细胞进行成软骨定向分化能力检测：
1)根据活细胞计数结果，吸取3×10 5-4×10 5个人脐带间充质干细胞活细胞到15mL离心管中，250×g离心4min。
2)离心后吸弃上清。加入0.5mL间充质干细胞成软骨分化培养基重悬细胞沉淀，以清洗脐带间质干细胞。
3)室温下150×g离心5min。离心后吸弃上清，以再次清洗脐带间质干细胞。
4)加入0.5mL间充质干细胞成软骨分化培养基重悬细胞沉淀，室温下150×g离心5min。
5)拧松离心管盖以便于气体交换，将其放置于37℃，5％CO 2的培养箱中培养。24小时之内不要摇动离心管。
6)24h-48h后，细胞聚团。轻弹离心管底部使细胞团脱离管底悬浮在液体中。
7)避开细胞团，小心吸出旧培养基，加入0.5mL间充质干细胞成软骨分化培养基。拧松离心管盖，放置于37℃，5％CO 2的培养箱中培养。
8)每隔3天吸弃旧培养基，加入等量间充质干细胞成软骨分化培养基。
9)诱导28天后，吸弃培养基，加入3mL 1×PBS润洗2次。
10)加入2mL 4％多聚甲醛溶液，室温固定30min。
11)按常规方法进行石蜡包埋切片。
12)按常规方法将样片脱蜡，通过梯度乙醇后入蒸馏水再水化。
13)将样片放入阿利新蓝酸化液浸泡3min。
14)将样片放入阿利新蓝染液染色30min。
15)流水冲洗样片5min。
16)将样片过梯度乙醇脱水、二甲苯透明后，用中性树胶封片。
17)在显微镜下观察成软骨染色结果。
使用不同培养基连续培养的人脐带间充质干细胞成软骨定向分化阿利新蓝染色图如图8所示。由图可以看出，实施例3的无血清、无异源成分的间充质干细胞培养基连续培养的人脐带间充质干细胞成软骨定向分化阿利新蓝染色阳性面积是实施例7的对照无血清培养基组的2.63倍(p<0.01)，结果表明实施例3的无血清、无异源成分的间充质干细胞培养基能提高人脐带间充质干细胞成软骨定向分化能力。
实施例17 人脐带间充质干细胞成脂定向分化能力的对比实验
以实施例3的无血清、无异源成分的间充质干细胞培养基为受试培养基，同时以实施例7的无血清、无异源成分的间充质干细胞培养基作为对照无血清培养基，按照本发明中上述人脐带间充质干细胞传代扩增培养方法，对人脐带间充质干细胞连续培养5代。
按照下述方法对不同培养基培养的人脐带间充质干细胞进行成脂定向分化能力检测：
1)按照2×10 4活细胞/cm 2的密度，接种人脐带间充质干细胞至12孔板孔中，添加1mL相应间充质干细胞培养基，放置于37℃，5％CO 2的培养箱中培养。
2)当细胞汇合度达到100％时，吸弃培养基，加入1mL间充质干细胞成脂分化培养基。
3)每隔3天，吸弃旧培养基，加入等量间充质干细胞成脂分化培养基。
4)诱导21天后，观察脂滴形成情况，进行油红O染色。
5)吸弃间质干细胞成脂分化培养基，加入1mL 1×PBS润洗1-2次。
6)吸弃1×PBS，加入2mL 4％多聚甲醛溶液，固定30min。
7)吸弃多聚甲醛溶液，加入1mL 1×PBS润洗2次。
8)加入1mL油红O染液，室温染色30min。
9)吸弃油红O染液，加入1mL 1×PBS润洗3次。
10)在显微镜下观察成脂分化油红O染色结果。
使用不同培养基连续培养的人脐带间充质干细胞成脂定向分化油红O染色图如图9所示。由图可以看出，实施例3无血清、无异源成分的间充质干细胞培养基连续培养的人脐带间充质干细胞成脂定向分化油红O染色阳性面积是实施例7的对照无血清培养基组的3.25倍(p<0.001)，结果表明实施例3的无血清、无异源成分的间充质干细胞培养基能提高人脐带间充质干细胞成脂定向分化能力。
显然，本发明的上述实施例仅仅是为清楚地说明本发明所作的举例，而并非是对本发明的实施方式的限定，对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动，这里无法对所有的实施方式予以穷举，凡是属于本发明的技术方案所引伸出的显而易见的变化或变动仍处于本发明的保护范围之列。

Claims (10)

  1. 一种无血清、无异源成分的间充质干细胞培养基，其特征在于：所述间充质干细胞培养基包括基础培养基和添加物；
    所述基础培养基为DMEM/F12培养基，所述添加物为人血小板裂解液和/或人脂质运载蛋白2。
  2. 根据权利要求1所述的间充质干细胞培养基，其特征在于：所述人血小板裂解液在所述间充质干细胞培养基的体积含量为1％-20％，所述人脂质运载蛋白2在所述间充质干细胞培养基的含量为10-200μg/mL；优选的，所述人血小板裂解液在所述间充质干细胞培养基的体积含量为2％-10％，所述人脂质运载蛋白2在所述间充质干细胞培养基的含量为20-100μg/mL。
  3. 根据权利要求2所述的间充质干细胞培养基，其特征在于：所述人血小板裂解液在所述间充质干细胞培养基的体积含量为5％，所述人脂质运载蛋白2在所述间充质干细胞培养基的含量为50μg/mL。
  4. 根据权利要求1-3任一所述的间充质干细胞培养基，其特征在于：所述间充质干细胞培养基的pH值为7.3-7.5，渗透压为300-350mOsm/kg。
  5. 权利要求1-4任一所述的间充质干细胞培养基在培养间充质干细胞中的应用或在制备培养间充质干细胞的产品中的应用。
  6. 权利要求1-4任一所述的间充质干细胞培养基在如下任一中的应用：
    1)在提高间充质干细胞增殖能力中的应用；
    2)在制备提高间充质干细胞增殖能力的产品中的应用。
    3)在提高间充质干细胞免疫调节能力中的应用；
    4)在制备提高间充质干细胞免疫调节能力的产品中的应用；
    5)在提高间充质干细胞分化能力中的应用；
    6)在制备提高间充质干细胞分化能力的产品中的应用。
  7. 人血小板裂解液和/或人脂质运载蛋白2在如下任一中的应用：
    1)在培养间充质干细胞中的应用；
    2)在制备培养间充质干细胞的产品中的应用；
    3)在提高间充质干细胞增殖能力的应用；
    4)在制备提高间充质干细胞增殖能力的产品中的应用
    5)在提高间充质干细胞免疫调节能力中的应用；
    6)在制备提高间充质干细胞免疫调节能力的产品中的应用；
    7)在提高间充质干细胞分化能力中的应用；
    8)在制备提高间充质干细胞分化能力的产品中的应用。
  8. 根据权利要求5-7任一所述的应用，其特征在于：所述间充质干细胞为人间充质干细胞；优选的，所述人间充质干细胞为人脐带间充质干细胞、人骨髓间充质干细胞和/或人脂肪间充质干细胞。
  9. 根据权利要求5或7所述的应用，其特征在于：所述培养为原代分离培养和/或 传代扩增培养。
  10. 根据权利要求5-8任一所述的应用，其特征在于：所述产品为试剂或试剂盒。
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WO2022116001A1 (zh) * 2020-12-01 2022-06-09 深圳先进技术研究院 一种调控间充质干细胞分化和旁分泌功能的方法
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