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WO2017096617A1 - 一种用于分步培养hUC-MSC的试剂盒及采用所述试剂盒获得的hUC-MSC - Google Patents

一种用于分步培养hUC-MSC的试剂盒及采用所述试剂盒获得的hUC-MSC Download PDF

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WO2017096617A1
WO2017096617A1 PCT/CN2015/097150 CN2015097150W WO2017096617A1 WO 2017096617 A1 WO2017096617 A1 WO 2017096617A1 CN 2015097150 W CN2015097150 W CN 2015097150W WO 2017096617 A1 WO2017096617 A1 WO 2017096617A1
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cells
medium
huc
volume
msc
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郭镭
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郭镭
里程
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Priority to US16/061,272 priority Critical patent/US11091739B2/en
Priority to PCT/CN2015/097150 priority patent/WO2017096617A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/44—Thiols, e.g. mercaptoethanol
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/46—Amines, e.g. putrescine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
    • C12N2506/1369—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from blood-borne mesenchymal stem cells, e.g. MSC from umbilical blood

Definitions

  • the invention relates to the research field of stem cells, in particular to a novel and high-efficiency serum-free stepwise culture kit for hUC-MSC.
  • Mesenchymal stem cells are ubiquitous in various tissues and organs of human body, and have multi-directional differentiation potential. They have functions of stimulating tissue regeneration and regulating immunity, and have broad application prospects in the field of cell therapy.
  • hUC-MSC Human Umbilical Cord mesenchymal stem cells derived from human umbilical cord express unique markers of various embryonic stem cells, have large differentiation potential, strong proliferative ability, low immunogenicity, and convenient materials. , no restrictions on ethical issues, easy to industrialize and other characteristics, and studies have confirmed that hUC-MSC has good therapeutic effects in animal models and clinical studies of diseases such as neurological diseases, immune system, endocrine system, cancer, heart disease, etc. It may become the most versatile stem cell for clinical application.
  • hUC-MSC In order to further apply hUC-MSC to clinical practice, the most important thing is that the large-scale expansion of hUC-MSC in vitro reaches an effective clinical therapeutic dose. Therefore, in vitro culture of hUC-MSC has become one of the most basic and most important technologies.
  • Existing hUC-MSC culture methods mostly use FBS and streptomycin in the basal medium, but the non-human serum components are complex, which makes hUC-MSCs easy to differentiate during long-term culture, and there is no non-existing pathogens. Danger.
  • Another object of the present invention is to provide hUC-MSC obtained by the medium of the present invention.
  • the present invention provides a novel kit culture protocol for serum-free stepwise culture of hUC-MSC, the kit comprising at least two culture media of different compositions, and the medium in the kit can be used in serum-free
  • the stepwise culture and long-term expansion culture of hUC-MSC were carried out under the environment, and at the same time, it was ensured that it maintained pluripotency and strong proliferative ability under long-term culture.
  • the invention provides a kit for stepwise culturing hUC-MSC, the kit comprising separately placed TME medium and TMD medium. Both media were serum free and used for stepwise culture of hUC-MSCs.
  • the hUC-MSC is a human umbilical cord mesenchymal stem cell isolated from a healthy neonatal umbilical cord tissue that is naturally delivered or cesarean section.
  • a human umbilical cord mesenchymal stem cell isolated from a healthy neonatal umbilical cord tissue that is naturally delivered or cesarean section.
  • the TME medium comprises a-MEM, ⁇ -mercaptoethanol, and non-essential amino acids.
  • the TME medium contains 0.05-0.2 parts by volume of ⁇ -mercaptoethanol, 0.5-2 parts by volume of an aqueous solution of non-essential amino acids, and 95-100 parts by volume of a-MEM, wherein the non-essential amino acid aqueous solution comprises The concentration is 8-12 mM each of glycine, alanine, L-tianmenamide, L-aspartic acid, glutamic acid, proline and serine; more preferably, the TME medium contains 0.1 part by volume of ⁇ - mercaptoethanol, 1 part by volume of a non-essential amino acid aqueous solution and 99 parts by volume of a-MEM; further preferably, the TME medium consists of the a-MEM, ?-mercaptoethanol and a non-essential amino acid aqueous solution.
  • the TMD medium contains a-MEM/DMEM-F12, ⁇ -mercaptoethanol, non-essential amino acids, recombinant human basic fibroblast growth factor (b-FGF), and serum substitute.
  • the TMD medium comprises 0.05-0.2 parts by volume of ⁇ -mercaptoethanol, 0.5-2 a volume of aqueous solution of non-essential amino acids, 8-12 parts by volume of serum replacement, 85-95 parts by volume of a-MEM/DMEM-F12, and recombinant human basic fibroblast growth factor at a final concentration of 5-15 ng/ml
  • the non-essential amino acid aqueous solution comprises glycine, alanine, L-aspartamide, L-aspartic acid, glutamic acid, proline and serine at a concentration of 8-12 mM each; more preferably, said The TMD medium contains 0.1 parts by volume of ⁇ -mercaptoethanol, 1 part by volume of an aqueous solution of non-essential amino acids, 10 parts by volume of serum substitute, 89 parts by volume of a-MEM/DMEM-F12, and a final concentration of 10 ng/ml.
  • the TMD medium consists of the a-MEM/DMEM-F12, ⁇ -mercaptoethanol, an aqueous solution of a non-essential amino acid, a recombinant human basic fibroblast growth factor, and a serum substitute.
  • the non-essential amino acid aqueous solution may be a product of Gibco Corporation No. 11140.
  • the serum replacement may be used KnockOut TM Serum Replacement (Gibco Products, Catalog No. 10828-010).
  • the hUC-MSC can be cultured stepwise using the kit of the present invention, and the culture method comprises first culturing hUC-MSC using the TME medium in the kit, and then culturing using the TMD medium in the kit. That is, two culture media are used for culture;
  • the culture method comprises the following steps:
  • the hUC-MSC cells collected in step (3) are taken to detect one or more of the following items: differentiation ability, cell activity, cell purity, cell contamination, and proliferation characteristics.
  • the culture method comprises the following steps:
  • the hUC-MSC cells collected in step (3) were taken to detect all of the following items: differentiation ability, cell activity, cell purity, cell contamination, and proliferation characteristics.
  • the serum-free stepwise culture method for hUC-MSC comprises the following steps:
  • the following items were examined: differentiation ability, cell activity, cell purity, cell contamination, and proliferation characteristics.
  • the present invention also provides hUC-MSC obtained by the above method.
  • the hUC-MSC has the following characteristics:
  • the present invention also provides a TME medium and/or a TMD medium used in the above culture method.
  • the TME medium and the TMD medium in the kit of the present invention are serum-free components, and the composition is clear, thereby avoiding cells caused by serum batch differences in the culture of the cultured cells.
  • the unstable growth process also precludes the possibility of spreading heterologous pathogens.
  • the TME medium was first cultured to promote hUC-MSC adherence, and then replaced with TMD medium for rapid amplification, which solved the conventional serum-free culture.
  • the cells have poor adherence ability and slow proliferation, and the cells can maintain good proliferation ability and multi-directional differentiation potential during long-term culture, which provides an efficient solution for in vitro culture of animal cells.
  • kit of the present invention is simple to operate and shortens the primary culture time.
  • the mesenchymal stem cells obtained by the method of the invention have high activity, high purity and strong differentiation ability, and the established cell bank can be directly used for Scientific research and clinical adjuvant therapy.
  • Fig. 1 is a diagram showing the culture of hUC-MSCs using a serum-containing medium, wherein Fig. 1A shows the morphology of cells after inoculation for 2 hours, Fig. 1B shows the morphology of cells after 24 hours of inoculation, and Fig. 1C shows the morphology of cells after 48 hours of inoculation.
  • Figure 2 is a cell diagram during the screening of the medium composition, wherein Figure 2A shows the cell morphology of the high-concentration ⁇ -mercaptoethanol medium after 4 hours of cell inoculation, and Figure 2B shows the cells after 48 hours of inoculation with the low-concentration serum replacement medium. Morphology, Fig. 2C shows the morphology of the cells after inoculation for 24 hours in the high-concentration serum replacement medium, Fig. 2D shows the morphology of the cells after inoculation for 24 hours in the low-concentration bFGF medium, and Fig. 2E shows the morphology of the cells in the high-concentration bFGF medium after passage.
  • Figure 3 is a diagram showing the culture of hUC-MSCs using the TME medium in the kit, wherein Figure 3A shows the cell morphology after 2 hours of inoculation, Figure 3B shows the cell morphology after 24 hours of inoculation, and Figure 3C shows the cells after 48 hours of inoculation. form.
  • Figure 4 is a diagram showing the culture of hUC-MSCs using the TMD medium in the kit, wherein Figure 4A shows the cell morphology after 2 hours of inoculation, Figure 4B shows the cell morphology after 24 hours of inoculation, and Figure 4C shows the cells after 48 hours of inoculation. form.
  • Figure 5 is a diagram showing the stepwise culture of hUC-MSC using the kit of the present invention, wherein Figure 5A shows the cell morphology after inoculation with TME medium in the kit for 2 hours, and Figure 5B shows the reagent used.
  • the TME medium in the cassette was replaced with TMD medium for 4 hours and then cultured for 24 hours.
  • Figure 5C was inoculated with TME medium in the kit for 4 hours and then changed to TMD medium for further 48 hours. Cell morphology.
  • Figure 6 shows the results of analyzing cell surface molecules by flow cytometry of hUC-MSC obtained by serum-free stepwise culture in the kit of the present invention, showing that the hUC-MSC expresses CD29, CD44, The positive proportion of CD73, CD90, CD105 and HLA-ABC was greater than 99%; the positive proportion of CD45, CD34 and HLA-DR was less than 1%.
  • Fig. 7 is a result of analyzing the cell viability and growth characteristics of the obtained hUC-MSC by Vi-Cell cell viability analyzer, wherein Fig. 7A is a diameter distribution map of hUC-MSC, and Fig. 7B is a growth curve of hUC-MSC, and Fig. 7C is a growth curve of hUC-MSC.
  • the real-time viability analysis of hUC-MSC showed that the hUC-MSC activity was above 99.7%, the cell diameter was around 13 ⁇ m, and it had the proliferation characteristics of latency, logarithmic growth and plateau.
  • Figure 8 is a result of the induced differentiation of hUC-MSCs obtained into osteoblasts and osteoblasts, wherein Fig. 8A shows a dark red compound produced by the color reaction of alizarin red with the calcium nodules of the osteogenesis process. 8B shows the fat bubble-specific coloration of oil red O to adipocytes.
  • Figure 9 shows the hUC-MSC pluripotency-specific protein obtained by immunofluorescence staining, from left to right, from top to bottom, SSEA-4, SOX-2, OCT-4, and NANOG.
  • Test medium 89 parts by volume of ⁇ -MEM, 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 U/ml streptomycin, 0.1 part by volume of ⁇ -mercaptoethanol, 10 ng/ml of b-FGF 1 part by volume of an aqueous solution of a non-essential amino acid (11140, Gibco).
  • FBS fetal bovine serum
  • penicillin 100 U/ml
  • streptomycin 100 U/ml streptomycin
  • 0.1 part by volume of ⁇ -mercaptoethanol 10 ng/ml of b-FGF 1 part by volume of an aqueous solution of a non-essential amino acid (11140, Gibco).
  • the 3rd generation hUC-MSC isolated from the natural umbilical cord of the newborn baby was inoculated into the T75 cell culture flask at a density of 2 ⁇ 10 4 cells/cm 2 and 15 mL was added.
  • the medium was transferred to a 37 ° C incubator with a CO 2 concentration of 5%. After 2 hours of inoculation, the adherence of the cells was observed. A large number of cells of hUC-MSC adhered to the cells and extended tentacles; after 48 hours, hUC-MSCs reached 90% confluence; the cells were stretched and bright.
  • Example 1 cells were inoculated at the same cell source and at the same density, and 15 mL of a commercially available serum-free medium (product of Saiye Co., Ltd., product number HUXUC-90061) was added to culture the cells. After inoculation for 2 hours, the cells were attached, the cells were bright, mostly round, and the antennae were stretched. After 24 hours of inoculation, the cells were observed.
  • a commercially available serum-free medium product of Saiye Co., Ltd., product number HUXUC-90061
  • the hUC-MSC was bright under the microscope, and the antenna extended, and the amplification was not obvious; After that, the cell confluence rate was about 50%; after 72 hours of inoculation, the cells were observed, and the hUC-MSC cells were bright, reaching more than 90% confluence, and the cells were cryopreserved by trypsin digestion.
  • the cells After the cells reach 100% confluence, after continuing the culture, the cells begin to curl off from the edge of the culture flask. From this, it can be seen that when the serum component is lacking, the cells are easily detached, and it is difficult to maintain a good adherence state.
  • Test medium 0.01, 0.02, 0.05, 0.1, 0.15, 0.2, 0.3 or 0.5 parts by volume of ⁇ -mercaptoethanol, 1 part by volume of aqueous solution of non-essential amino acids (11140, Gibco), 99 parts by volume of a-MEM .
  • Example 1 cells were seeded at the same density with the same cell source, and 15 mL of the test medium was added to culture the cells. Observe the cell adherence.
  • TME medium 0.1 part by volume of ⁇ -mercaptoethanol, 1 part by volume of an aqueous solution of non-essential amino acids (11140, Gibco), 99 parts by volume of a-MEM.
  • Test medium 0.1 part by volume of ⁇ -mercaptoethanol, 10 ng/ml of recombinant human basic fibroblast growth factor (b-FGF, Peprotech), 1 part by volume of aqueous solution of non-essential amino acids (11140, Gibco), 1, 2, 5, 8, 10, 12, 15 or 20 parts by volume of Knockout FBS serum replacement (10828-028, Gibco), 89 parts by volume of a-MEM.
  • Example 1 cells were seeded at the same density with the same cell source, and 15 mL of TME medium was added. Two hours after the inoculation, the cells were observed to adhere to the cells, and the culture was continued. After about 4 hours of inoculation, the cells were completely adhered, and 15 mL of the test medium was replaced. Observe the growth of the cells.
  • RESULTS In the three concentration groups containing 1, 2, and 5 volumes of serum substitutes in the medium, the cells proliferated slowly. After 24 hours of inoculation, the cells were observed. Some cells of hUC-MSC aggregated, the cells were flat, and the refractive index was poor. The confluence reached about 20%. After 48 hours of inoculation, the cells were observed. The hUC-MSC cells were bright, and after about 60% confluence, the proliferation was basically stopped (see Figure 2B); in the medium, there were 8, 10, and 12 volumes, respectively. In the three concentration groups of serum substitutes, the cells grew well. After 24 hours of inoculation, the cells were observed.
  • the hUC-MSCs were spindle-shaped and vortex-like, with high elongation and bright cells, and the confluence reached 40-60%. After the hour, the cells were observed, hUC-MSC cells were bright, reaching more than 90% confluence; in the two concentration groups containing 15 and 20 volumes of serum substitutes in the medium, the same condition as in the low concentration group occurred, and the cells grew slowly. The cells are flattened and the outline is clear (see Figure 2C).
  • TME medium 0.1 part by volume of ⁇ -mercaptoethanol, 1 part by volume of an aqueous solution of non-essential amino acids (11140, Gibco), 99 parts by volume of a-MEM.
  • Test medium 0.1 part by volume of ⁇ -mercaptoethanol, 1, 2, 5, 8, 10, 12, 15, 18 or 20 ng/ml of recombinant human basic fibroblast growth factor (b-FGF, Peprotech) , 1 part by volume of a non-essential amino acid aqueous solution (11140, Gibco), 10 parts by volume of Knockout FBS serum replacement (10828-028, Gibco), 89 parts by volume of a-MEM.
  • b-FGF human basic fibroblast growth factor
  • Example 1 cells were seeded at the same density with the same cell source, and 15 mL of TME medium was added. Two hours after the inoculation, the cells were observed to adhere to the cells, and the culture was continued. After about 4 hours of inoculation, the cells were completely adhered, and 15 mL of the test medium was replaced. Observe the growth of the cells.
  • TME medium 0.1 part by volume of ⁇ -mercaptoethanol, 1 part by volume of an aqueous solution of non-essential amino acids (11140, Gibco), 99 parts by volume of a-MEM.
  • the third generation of hUC-MSC isolated from the natural umbilical cord of the newborn baby was inoculated into the T75 cell culture flask at a density of 2 ⁇ 10 4 cells/cm 2 and cultured with 15 mL of TME.
  • the base was transferred to a 37 ° C incubator with a CO 2 concentration of 5%.
  • Two hours after the inoculation the cells were attached to the antennae, and the cells were removed from the incubator at 24 hours and 48 hours. The cells were in good condition, but a large number of floating dead cells appeared.
  • the proliferation was not obvious; on the third day after inoculation, the fresh ME medium was replaced and the culture was continued, and the cells gradually fell off from the bottom of the bottle and the proliferation was not obvious.
  • TMD medium 0.1 part by volume of ⁇ -mercaptoethanol, 10 ng/ml of recombinant human basic fibroblast growth factor (b-FGF, Peprotech), 1 part by volume of aqueous solution of non-essential amino acids (11140, Gibco), 10 Volume fraction of Knockout FBS serum replacement (10828-028, Gibco), 89 parts by volume of a-MEM
  • Example 4 cells were seeded at the same density with the same cell source, and 15 mL of TMD medium was added. Similarly, after 2 hours of inoculation, cell adhesion was observed, and hUC-MSC still remained. A large number of cells floated unattached; cells were observed after 24 hours, cell adherence was uneven, local aggregation; after 48 hours, hUC-MSC cells adhered to a larger area of aggregation; on the third day after inoculation, fresh TMD culture was replaced. The cells continued to culture, some of the cells fell off during the liquid exchange process, and a small number of cells died of local aging. After 96 hours of culture, the cell confluence was about 90%.
  • Example 6 Serum-free stepwise culture of hUC-MSC using the kit of the present invention
  • TME medium 0.1 part by volume of ⁇ -mercaptoethanol, 1 part by volume of an aqueous solution of non-essential amino acids (11140, Gibco), 99 parts by volume of a-MEM;
  • TMD medium 0.1 part by volume of ⁇ -mercaptoethanol, 10 ng/ml of recombinant human basic fibroblast growth factor (b-FGF, Peprotech), 1 part by volume of aqueous solution of non-essential amino acids (11140, Gibco), 10 Volume fraction of Knockout FBS serum replacement (10828-028, Gibco), 89 parts by volume of a-MEM.
  • Example 4 cells were seeded at the same density with the same cell source, and 15 mL of TME medium was added. After 2 hours of inoculation, the cells were observed to adhere to the culture, and the culture was continued. After about 4 hours of inoculation, the cells were completely adherent, and the fresh TMD medium was replaced. After 24 hours of inoculation, the cells were observed, and hUC-MSCs were fusiformly vortex-likely aggregated. High, cells are bright, confluence is 40-60%; cells are observed 48 hours after inoculation, hUC-MSC cells are bright, more than 90% confluence, and cells are cryopreserved by trypsin digestion.
  • the cells were not rolled up and left to maintain a good adherence for a long time.
  • Example 6 Comparing Example 6 with Example 1, it can be seen that the present invention adopts TME medium and TMD medium for serum-free stepwise culture, and achieves the same results as conventional serum culture, but at the same time avoids introduction in cultured cells. Serum causes the risk of spreading heterologous pathogens, and also avoids the instability of cell growth due to differences in serum batches during culture.
  • Example 6 The passage 3 cells cultured in Example 6 were taken, and after the cells were grown to 90% confluence, 2 mL of 0.125% trypsin was digested, and then centrifuged at 1200 rpm for 6 minutes at 4 ° C, the supernatant was discarded, and the cells were washed twice, and the cells were washed twice.
  • Example 8 Cell viability analyzer for analyzing cell viability and growth characteristics of hUC-MSC
  • Example 6 The passage 3 cells cultured in Example 6 were inoculated into a T25 flask, and after the cells reached 95%-100% confluence, 0.125% trypsinization was performed, and the collected cells were seeded at a density of 1 ⁇ 10 5 /well in two 6 Orifice plate. After the cells were all adherent and partially grown for 10 hours, two wells of cells were collected and 500 ⁇ L of PBS was used to prepare a cell suspension, which was analyzed by a cell analysis (cell viability analyzer Vi-Cell XR, Beckman). Samples were taken every 12 hours thereafter and growth curves were plotted.
  • a cell analysis cell viability analyzer Vi-Cell XR, Beckman
  • the 3rd generation hUC-MSC cultured in Example 6 was inoculated to a 6-well cell culture plate at 3 ⁇ 10 4 cells/cm 2 , and 24 hours later, freshly prepared human UC MSC osteogenic differentiation medium was added per well ( HUXUC-90021, Saiye product) 2mL, after which every 3 days to replace the fresh osteogenic differentiation induction medium, 2 weeks after the paraformaldehyde fixation, alizarin red staining 3-5min.
  • Fig. 8A shows that after two weeks of osteogenic induction, the hUC-MSC obtained by the method of the present invention has a deep red color reaction with the calcium nodules of the alizarin red and the osteogenesis process.
  • the 3rd generation hUC-MSC cultured in Example 6 was inoculated into a 6-well cell culture plate at 2 ⁇ 10 4 cells/cm 2 , and after the cells reached 100% confluence, the adipogenic differentiation medium A was added to each well ( HUXUC-90031, the Saiye product) began to induce, and after 3 days, it was replaced with the adipogenic differentiation medium B for 24 hours, and thus circulated.
  • Fig. 8B shows that the hUC-MSC obtained by the method of the present invention stains the adipogenic cells significantly after two weeks of adipogenic induction.
  • the 5th generation hUC-MSC cultured in Example 6 was inoculated into a 24-well cell culture plate at a density of 5 ⁇ 10 3 cells per well, and the cells were grown to 30% to 50% confluence and fixed with 4% paraformaldehyde. After minute, it was perforated with 0.25% TritonX-100 for 20 minutes, and the goat serum was blocked and then diluted with anti-human primary antibody (anti-SOX2 antibody, anti-OCT4 antibody, anti-NANOG antibody and anti-NANOG antibody).

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Abstract

一种人脐带间充质干细胞(human umbilical cord mesenchymal stem cells，hUC-MSC)无血清培养方法，该方法采用分步法培养hUC-MSC：先使用TME培养基培养3-4小时促进hUC-MSC贴壁，然后更换为TMD培养基进行快速扩增。

Description

一种用于分步培养hUC-MSC的试剂盒及采用所述试剂盒获得的hUC-MSC 技术领域
本发明涉及干细胞的研究领域，特别是涉及一种hUC-MSC的新型、高效的无血清分步培养试剂盒。
背景技术
间充质干细胞普遍存在于人体多种组织和器官中，并具有多向分化潜能，具有刺激组织再生、调节免疫等功能，在细胞治疗领域有着广阔的应用前景。
骨髓间充质干细胞已经在临床上得到了广泛应用，而目前的研究表明，脐带来源的间充质干细胞不但能够成为骨髓间充质干细胞的理想替代物，而且具有更大的应用潜能。其中，源于人脐带的人脐带间充质干细胞(human Umbilical Cord mesenchymal stem cells，hUC-MSC)表达多种胚胎干细胞的特有标志，具有分化潜力大、增殖能力强、免疫原性低、取材方便、无道德伦理问题的限制、易于工业化制备等特征，而且研究证实hUC-MSC在神经疾病、免疫系统、内分泌系统以及癌症、心脏病等疾病的动物模型和临床研究中有良好治疗效果，因此有可能成为最具临床应用前景的多能干细胞。
若要将hUC-MSC进一步应用于临床，最重要的就是hUC-MSC的体外大量扩增达到有效的临床治疗剂量，因此hUC-MSC的体外培养成为了最基础同时也是最重要的技术之一。现有的hUC-MSC培养方法多采用在基础培养基中添加FBS、青链霉素，但非人血清成分复杂，使hUC-MSC在长期的培养过程中易分化，且非存在传播异种病原体的危险。
此外，虽然已有科研工作者开发出多种类型的血清替代物，但目前市场可购买的供hUC-MSC培养的血清替代物以及完全培养基的培养效果仍不理想，尤其对于干细胞的贴壁、增殖、以及细胞长期培养后的稳定性的 维持等特性均无法达到预期效果。
发明内容
因此本发明的目的是针对本领域的需要，提供一种用于培养hUC-MSC的培养基，从而获得贴壁能力好、增殖快速、易分化的hUC-MSC。
本发明的另一目的是提供通过本发明的培养基获得的hUC-MSC。
具体而言，本发明提供一种新型的无血清分步培养hUC-MSC的试剂盒培养方案，该试剂盒至少包括不同组成的两种培养基，利用该试剂盒中的培养基可在无血清环境下进行hUC-MSC的分步培养和长期扩增培养，同时确保其在长期培养情况下依然保持多潜能性及较强的增殖能力。
本发明的技术方案如下。
一方面，本发明提供了一种用于分步培养hUC-MSC的试剂盒，所述试剂盒包括分开放置的TME培养基和TMD培养基。该两种培养基均无血清成分，且用于分步培养hUC-MSC。
其中，所述hUC-MSC为从自然分娩或剖宫产的健康新生儿脐带组织分离出的人脐带间充质干细胞。目前本领域已有多种公知的人脐带间充质干细胞分离方法。
本发明提供的试剂盒中，所述TME培养基包含a-MEM、β-巯基乙醇和非必需氨基酸。优选地，所述TME培养基含有0.05-0.2体积份的β-巯基乙醇、0.5-2体积份的非必需氨基酸水溶液和95-100体积份的a-MEM，其中，所述非必需氨基酸水溶液包含浓度各为8-12mM的甘氨酸、丙氨酸、L-天门酰胺、L-天冬氨酸、谷氨酸、脯氨酸和丝氨酸；更优选地，所述TME培养基含有0.1体积份的β-巯基乙醇、1体积份的非必需氨基酸水溶液和99体积份的a-MEM；进一步优选地，所述TME培养基由所述a-MEM、β-巯基乙醇和非必需氨基酸水溶液组成。
本发明提供的试剂盒中，所述TMD培养基含有a-MEM/DMEM-F12、β-巯基乙醇、非必需氨基酸、重组人碱性成纤维生长因子(b-FGF)和血清替代物。
优选地，所述TMD培养基包含0.05-0.2体积份的β-巯基乙醇、0.5-2 体积份的非必需氨基酸水溶液、8-12体积份的血清替代物、85-95体积份的a-MEM/DMEM-F12和终浓度为5-15ng/ml的重组人碱性成纤维生长因子，其中，所述非必需氨基酸水溶液包含浓度各为8-12mM的甘氨酸、丙氨酸、L-天门酰胺、L-天冬氨酸、谷氨酸、脯氨酸和丝氨酸；更优选地，所述TMD培养基包含0.1体积份的β-巯基乙醇、1体积份的非必需氨基酸水溶液、10体积份的血清替代物、89体积份的a-MEM/DMEM-F12和终浓度为10ng/ml的重组人碱性成纤维生长因子。优选地，所述TMD培养基由所述a-MEM/DMEM-F12、β-巯基乙醇、非必需氨基酸水溶液、重组人碱性成纤维生长因子和血清替代物组成。
根据本申请的具体实施方式，所述非必需氨基酸水溶液可以采用Gibco公司货号为11140的产品。
根据本申请的具体实施方式，所述血清替代物可以采用KnockOutTM Serum Replacement(Gibco公司产品，货号10828-010)。
使用本发明的试剂盒可以分步培养hUC-MSC，培养方法包括：先使用所述试剂盒中的TME培养基培养hUC-MSC，然后使用所述试剂盒中的TMD培养基进行培养。即先后采用两种培养基进行培养；
进一步地，所述培养方法包括以下步骤：
(1)将hUC-MSC以0.5-4×104个细胞/cm2的密度接种于所述试剂盒中的TME培养基中培养3-6小时；
(2)弃去TME培养基，PBS清洗，更换为所述试剂盒中的TMD培养基，每3-5天更换新鲜的TMD培养基；
(3)待细胞达70-90％汇合后，收集细胞备用、冻存或重复步骤(1)和(2)传代培养；
可选择地，取步骤(3)收集的hUC-MSC细胞，检测以下项目中的一项或多项：分化能力、细胞活性、细胞纯度、细胞污染和增殖特性。
优选地，所述培养方法包括以下步骤：
(1)将hUC-MSC以2×104个细胞/cm2的密度接种于所述试剂盒中的TME培养基中培养3-4小时；
(2)弃去TME培养基，PBS清洗1次，更换为预先37℃孵育的所述 试剂盒中的TMD培养基，每3天更换新鲜的TMD培养基；
(3)待细胞达90％汇合后，收集细胞备用、冻存或重复步骤(1)和(2)传代培养；
可选择地，取步骤(3)收集的hUC-MSC细胞，检测以下项目中的全部：分化能力、细胞活性、细胞纯度、细胞污染和增殖特性。
根据本发明的具体实施方式，所述用于hUC-MSC的无血清分步培养方法包括以下步骤：
(1)使用TME培养基以一定密度接种hUC-MSC于六孔板或T75培养瓶内进行贴壁培养；优选地，hUC-MSC的接种密度为约2×104个细胞/cm2；
(2)接种4小时后，用移液器小心吸弃旧TME培养基，用PBS洗一遍，更换为同样预先37℃孵育的TMD培养基；
(3)待细胞达90％汇合后，利用上述方案进行传代培养或冻存细胞。
选择性地，针对步骤(3)所得的hUC-MSC，检测以下项目：分化能力、细胞活性、细胞纯度、细胞污染和增殖特性。
另一方面，本发明还提供通过上述方法获得的hUC-MSC。
优选地，所述hUC-MSC具有以下特征：
(1)粘附于塑料培养器皿成梭形旋涡状生长；
(2)表达CD29、CD44、CD73、CD90、CD105和HLA-ABC的阳性比例大于99.0％；表达CD45、CD34和HLA-DR的阳性比例小于1.0％；
(3)体外可诱导分化为成骨细胞和成脂细胞；
(4)活细胞检测比率达99％以上；
(5)呈典型的“S型”生长曲线特性；
(6)多能性基因表达，所述多能性基因为选自SSEA-4、OCT-4、NANOG和SOX-2中的一种或多种。
又一方面，本发明还提供在上述培养方法中采用的TME培养基和/或TMD培养基。
本发明中所述试剂盒中的TME培养基和TMD培养基均为无血清组分，且组成明晰，避免了在培养细胞培养过程中因血清批次差异导致细胞 生长过程不稳定的情况，也排除了传播异种病原体危险的可能。
此外，利用试剂盒中的两种培养基进行分步培养，先使用TME培养基培养促进hUC-MSC贴壁，然后更换为TMD培养基进行快速扩增，很好地解决了常规无血清培养中细胞贴壁能力差，增殖缓慢的缺点，且在长期培养过程中，细胞仍能保持良好的增殖能力和多向分化潜能，为动物细胞的体外培养提供了一种高效的解决方案。
并且，本发明的试剂盒操作简单易行，缩短了原代培养时间。
经流式细胞仪测定，经活力检测、分化能力鉴定以及多能性基因分析，通过本发明方法获得的间充质干细胞活率高，纯度高，分化能力强，建立的细胞库可直接用于科学研究和临床辅助治疗。
附图说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1是使用含血清培养基培养hUC-MSC的图，其中图1A为接种2小时后的细胞形态，图1B为接种24小时后的细胞形态，图1C为接种48小时后的细胞形态。
图2是在培养基组成筛选过程中的细胞图，其中图2A为高浓度β-巯基乙醇培养基在细胞接种4小时后细胞形态，图2B为低浓度血清替代物培养基接种48小时后细胞形态，图2C为高浓度血清替代物培养基接种24小时后细胞形态，图2D为低浓度bFGF培养基接种24小时后细胞形态，图2E为高浓度bFGF培养基培养细胞在传代后细胞形态。
图3是使用试剂盒中的TME培养基培养hUC-MSC的图，其中图3A为接种2小时后的细胞形态，图3B为接种24小时后的细胞形态，图3C为接种48小时后的细胞形态。
图4是使用试剂盒中的TMD培养基培养hUC-MSC的图，其中图4A为接种2小时后的细胞形态，图4B为接种24小时后的细胞形态，图4C为接种48小时后的细胞形态。
图5是利用本发明的试剂盒分步培养hUC-MSC的图，其中图5A为使用试剂盒中的TME培养基接种2小时后的细胞形态，图5B为使用试剂 盒中的TME培养基接种4小时后更换为TMD培养基继续培养至24小时的细胞形态，图5C为使用试剂盒中的TME培养基接种4小时后更换为TMD培养基继续培养至48小时的细胞形态。
图6(6A至6I)为采用本发明的试剂盒中的进行无血清分步培养获得的hUC-MSC经流式细胞仪分析细胞表面分子的结果，显示所述hUC-MSC表达CD29、CD44、CD73、CD90、CD105、HLA-ABC的阳性比例大于99％；表达CD45、CD34、HLA-DR的阳性比例小于1％。
图7为Vi-Cell细胞活力分析仪对获得的hUC-MSC的细胞活力、生长特性分析结果，其中图7A为hUC-MSC的直径分布图，图7B为hUC-MSC的生长曲线，图7C为hUC-MSC的实时活力分析，结果表明所述hUC-MSC的活性在99.7％以上，细胞直径分布在13μm左右，并且具有潜伏期、对数增长期、平台期的增殖特性。
图8为获得的hUC-MSC向成骨细胞和成骨细胞的定向诱导分化结果，其中图8A示出了茜素红与成骨过程的钙结节发生显色反应产生的深红色化合物，图8B示出了油红O对成脂细胞的脂肪泡特异性着色。
图9为免疫荧光染色分析获得的hUC-MSC多能性特异蛋白，从左到右，从上到下依次为SSEA-4、SOX-2、OCT-4、NANOG。
实施发明的最佳方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。
未注明具体条件的，按照本发明所属领域的常规条件或仪器试剂供应商的建议条件进行；为注明商购来源的，为可以市售购得的常规产品。
实施例1 常规有血清培养基培养hUC-MSC
受试培养基：89体积份的α-MEM，10％胎牛血清(FBS)、100U/ml青霉素、100U/ml链霉素，0.1体积份的β-巯基乙醇，10ng/ml的b-FGF，1体积份的非必需氨基酸水溶液(11140，Gibco公司)。
在生物安全柜内，取分离于自然分娩新生儿脐带华通式胶组织的第3 代的hUC-MSC，以2×104个细胞/cm2密度接种于T75细胞培养瓶，加15mL受试培养基，移入CO2浓度为5％的37℃恒温培养箱中。接种2小时后观察细胞贴壁情况，hUC-MSC大量细胞贴壁，有触角伸出；在48小时后观察，hUC-MSC达90％汇合；细胞触角舒展，明亮。
细胞形态参见图1。但是，在细胞培养过程中，引入血清会造成传播异种病原体风险，并且因血清批次差异也会导致细胞生长过程不稳定。
实施例2 常规市售无血清培养基培养hUC-MSC
参考实施例1方法，以相同细胞源、相同密度接种，加15mL市售无血清培养基(赛业公司产品，货号HUXUC-90061)培养细胞。接种2小时后观察细胞已贴壁，细胞明亮，多呈圆形，触角为伸展；在接种24小时后观察细胞，hUC-MSC在显微镜下明亮，触角延伸，扩增不明显；在接种48小时后，细胞汇合率达50％左右；接种72小时后观察细胞，hUC-MSC细胞明亮，达90％以上汇合，胰酶消化收集细胞冻存。
选择地，细胞达100％汇合后，继续培养后，细胞从培养瓶边缘处开始卷曲脱落。由此可知，当缺乏血清成分时，细胞易脱落，难以维持良好贴壁状态。
实施例3 培养基组成的筛选
(一)TME培养基组成的筛选
受试培养基：0.01、0.02、0.05、0.1、0.15、0.2、0.3或0.5体积份的β-巯基乙醇，1体积份的非必需氨基酸水溶液(11140，Gibco公司)，99体积份的a-MEM。
参考实施例1方法，以相同细胞源、相同密度接种，加15mL受试培养基培养细胞。观察细胞贴壁情况。
结果：在培养基中分别含0.01和0.02体积份β-巯基乙醇的两个浓度组中，细胞贴壁速度较慢，在接种4小时后，仍有部分细胞未贴壁，约8小时后，细胞基本全部贴壁；在培养基中分别含0.05、0.1、0.15和0.2体积份β-巯基乙醇的四个浓度组中，在接种4小时后细胞已完全贴壁，细胞 明亮，伸出触角；在培养基中分别含0.3和0.5体积份β-巯基乙醇的两个浓度组中，在接种4小时后，细胞也已经贴壁，但部分细胞状态变差，出现分化早期症状(见图2A)。
(二)TMD培养基组成的筛选
TME培养基：0.1体积份的β-巯基乙醇，1体积份的非必需氨基酸水溶液(11140，Gibco公司)，99体积份的a-MEM。
受试培养基：0.1体积份的β-巯基乙醇，10ng/ml的重组人碱性成纤维生长因子(b-FGF，Peprotech公司)，1体积份的非必需氨基酸水溶液(11140，Gibco公司)，1、2、5、8、10、12、15或20体积份的Knockout FBS血清替代物(10828-028，Gibco公司)，89体积份的a-MEM。
参考实施例1方法，以相同细胞源、相同密度接种，加15mL TME培养基培养细胞。接种2小时后观察细胞已贴壁，继续培养，接种约4小时后细胞即完全贴壁，更换15mL受试培养基。观察细胞生长情况。
结果：在培养基中分别含1、2、5体积份血清替代物的三个浓度组中，细胞增殖缓慢，在接种24小时后观察细胞，hUC-MSC部分细胞聚集，细胞扁平，折光率差，汇合度达20％左右，接种48小时后观察细胞，hUC-MSC细胞明亮，达60％左右汇合后，基本停止增殖(见图2B)；在培养基中分别含8、10、12体积份血清替代物的三个浓度组中，细胞生长状态良好，在接种24小时后观察细胞，hUC-MSC呈梭形旋涡状聚集，伸展度高，细胞明亮，汇合度达40-60％，接种48小时后观察细胞，hUC-MSC细胞明亮，达90％以上汇合；在培养基中分别含15、20体积份血清替代物的两个浓度组中，出现和低浓度组相同的状况，细胞生长缓慢，细胞扁平化，轮廓清晰(见图2C)。
(三)TMD培养基组成的筛选
TME培养基：0.1体积份的β-巯基乙醇，1体积份的非必需氨基酸水溶液(11140，Gibco公司)，99体积份的a-MEM。
受试培养基：0.1体积份的β-巯基乙醇，1、2、5、8、10、12、15、18或20ng/ml的重组人碱性成纤维生长因子(b-FGF，Peprotech公司)，1体积份的非必需氨基酸水溶液(11140，Gibco公司)，10体积份的Knockout  FBS血清替代物(10828-028，Gibco公司)，89体积份的a-MEM。
参考实施例1方法，以相同细胞源、相同密度接种，加15mL TME培养基培养细胞。接种2小时后观察细胞已贴壁，继续培养，接种约4小时后细胞即完全贴壁，更换15mL受试培养基。观察细胞生长情况。
结果：在培养基中分别含1、2ng/ml bFGF的两个浓度组中，细胞增殖缓慢，细胞状态差，呈现营养不足状态(参见图2D)；在培养基中分别含5、8、10、12、15ng/ml bFGF的浓度组中，细胞正常生长，亮度高，生长好；在培养基中分别含18、20ng/ml bFGF的浓度组中，细胞增殖良好，明亮，但经过多次传代过程中，细胞易分化，细胞会成团状汇集，或触角变长(参见图2E)。
实施例4 利用试剂盒中的TME培养基培养hUC-MSC
TME培养基：0.1体积份的β-巯基乙醇，1体积份的非必需氨基酸水溶液(11140，Gibco公司)，99体积份的a-MEM。
在生物安全柜内，取分离于自然分娩新生儿脐带华通式胶组织的第3代的hUC-MSC，以2×104个细胞/cm2密度接种于T75细胞培养瓶，加15mL TME培养基，移入CO2浓度为5％的37℃恒温培养箱中。接种2小时后观察细胞已贴壁伸出触角，在24小时和48小时移出培养箱观察，细胞贴壁状况良好，但出现大量漂浮死细胞。增殖不明显；接种后第3天，更换新鲜ME培养基继续培养，细胞逐渐从瓶底脱落死亡，增殖不明显。
细胞形态参见图3。
实施例5 利用试剂盒中的TMD培养基培养hUC-MSC
TMD培养基：0.1体积份的β-巯基乙醇，10ng/ml的重组人碱性成纤维生长因子(b-FGF，Peprotech公司)，1体积份的非必需氨基酸水溶液(11140，Gibco公司)，10体积份的Knockout FBS血清替代物(10828-028，Gibco公司)，89体积份的a-MEM
参考实施例4方法，以相同细胞源、相同密度接种，加15mL TMD培养基培养细胞。同样在接种2小时后观察细胞贴壁情况，hUC-MSC仍有 大量细胞漂浮未贴壁；在24小时后观察细胞，细胞贴壁不均匀，局部聚集；在48小时后观察，hUC-MSC细胞贴壁更大面积聚集；接种后第3天，更换新鲜TMD培养基继续培养，部分细胞在换液过程中脱落，少部分细胞呈局部老化死亡，培养至96小时后，细胞汇合度约90％。
细胞形态参见图4。
实施例6 利用本发明的试剂盒进行hUC-MSC的无血清分步培养
TME培养基：0.1体积份的β-巯基乙醇，1体积份的非必需氨基酸水溶液(11140，Gibco公司)，99体积份的a-MEM；
TMD培养基：0.1体积份的β-巯基乙醇，10ng/ml的重组人碱性成纤维生长因子(b-FGF，Peprotech公司)，1体积份的非必需氨基酸水溶液(11140，Gibco公司)，10体积份的Knockout FBS血清替代物(10828-028，Gibco公司)，89体积份的a-MEM。
参考实施例4方法，以相同细胞源、相同密度接种，加15mL TME培养基培养细胞。接种2小时后观察细胞已贴壁，继续培养，接种约4小时后细胞即完全贴壁，更换新鲜TMD培养基；在接种24小时后观察细胞，hUC-MSC呈梭形旋涡状聚集，伸展度高，细胞明亮，汇合度达40-60％；接种48小时后观察细胞，hUC-MSC细胞明亮，达90％以上汇合，胰酶消化收集细胞冻存。
选择地，细胞达100％汇合后，继续培养后，细胞未卷起脱落，维持长时间良好贴壁。
细胞形态参见图5。
将实施例6与实施例1相比较可知，本发明采取TME培养基和TMD培养基进行无血清分步培养，实现了与常规有血清培养的相同结果，但同时又避免了在培养细胞由于引入血清造成传播异种病原体风险，也避免了培养过程中因血清批次差异导致细胞生长过程不稳定的情况。
实施例7 流式细胞仪分析hUC-MSC的表面标志
取实施例6培养的第3代细胞，待细胞生长至90％汇合后，2mL 0.125％ 胰酶消化，然后在4℃下1200rpm离心6分钟，弃上清收集细胞，PBS清洗两次，将细胞每管1×105个转移至流式管，分别加5μL CD34-PE、CD45-FITC、CD29-FITC、CD44-PE、CD73-PE、CD105-PE、CD90-FITC、HLA-ABC-FITC、HLA-DR-PE、IgG1-PE(同型对照)和IgG1-FITC(同型对照)抗体，混匀4℃下避光孵育30分钟，PBS清洗一次，离心去上清，加500μL PBS缓冲液重悬混匀，上机检测(流式细胞仪XL，Beckman公司)，每个样本收集1×104个细胞。
结果参见图6。
实施例8 细胞活力仪分析hUC-MSC的细胞活力、生长特性
取实施例6培养的第3代细胞接种到T25培养瓶中，待细胞达到95％-100％汇合后，0.125％胰酶消化，收集细胞以1×105个/孔密度接种于两个6孔板。待细胞全部贴壁且部分生长10小时后，收集两孔细胞加500μL PBS制成细胞悬液，上机分析(细胞活力分析仪Vi-Cell XR，Beckman公司)。此后每12小时取样分析，绘制生长曲线。
结果参见图7，表明hUC-MSC活性在99.7％以上，细胞直径分布在在9-15μm，并且具有潜伏期、对数增长期、平台期的增殖特性。
实施例9 hUC-MSC多向分化潜能的鉴定
1)成骨诱导分化
取实施例6培养的第3代hUC-MSC以3×104个细胞/cm2接种至6孔细胞培养板，24小时后，每孔添加新鲜配制的人UC MSC成骨诱导分化培养基(HUXUC-90021，赛业产品)2mL，此后每3天更换新鲜的成骨分化诱导培养基，2周后多聚甲醛固定，茜素红染色3-5min。
结果参见图8A，表明以本发明方法得到的hUC-MSC在成骨诱导两周后，茜素红与成骨过程的钙结节发生深红色显色反应。
2)成脂诱导分化
取实施例6培养的第3代hUC-MSC以2×104个细胞/cm2接种至6孔细胞培养板，待细胞达到100％汇合后，每孔添加成脂诱导分化培养基A 液(HUXUC-90031，赛业产品)开始诱导，3天后换成成脂诱导分化培养基B液进行维持24小时，如此循环。当脂滴出现较多但较小时，用成脂诱导液B维持7天，诱导结束后4％多聚甲醛固定，油红O染色。
结果参见图8B，表明以本发明方法得到的hUC-MSC在成脂诱导两周后，油红O对成脂细胞着色明显。
实施例10 免疫荧光染色分析hUC-MSC特异性蛋白
取实施例6培养的第5代hUC-MSC以5×103个细胞每孔的密度接种于24孔细胞培养板，待细胞生长至30％～50％汇合，以4％多聚甲醛固定15分钟后用0.25％TritonX-100打孔处理20分钟，山羊血清封闭后加稀释好的鼠抗人一抗(抗-SOX2抗体、抗-OCT4抗体、抗-NANOG抗体和抗-NANOG抗体)，在4℃下过夜避光孵育；之后加FITC标记的山羊抗鼠二抗，在室温下避光孵育2小时；然后以DAPI/PI染核，在室温避光孵育20min，荧光显微镜下观察。
结果参见图9，表明以本发明方法分步培养的hUC-MSC表达SOX-2、OCT-4、NANOG以及SSEA-4特异性蛋白。
以上对本发明具体实施方式的描述并不限制本发明，本领域技术人员可以根据本发明作出各种改变或变形，只要不脱离本发明的精神，均应属于本发明所附权利要求的范围。

Claims (9)

  1. 一种用于分步培养hUC-MSC的试剂盒，其特征在于，所述试剂盒包括分开放置的TME培养基和TMD培养基，其中所述TME培养基含有a-MEM、β-巯基乙醇和非必需氨基酸，所述TMD培养基含有a-MEM/DMEM-F12、β-巯基乙醇、非必需氨基酸、重组人碱性成纤维生长因子(b-FGF)和血清替代物。
  2. 根据权利要求1所述的试剂盒，其特征在于，所述hUC-MSC为从自然分娩或剖宫产的健康新生儿脐带组织分离出的人脐带间充质干细胞。
  3. 根据权利要求1或2所述的试剂盒，其特征在于，所述TME培养基含有0.05-0.2体积份的β-巯基乙醇、0.5-2体积份的非必需氨基酸水溶液和95-100体积份的a-MEM，其中，所述非必需氨基酸水溶液包含浓度各为8-12mM的甘氨酸、丙氨酸、L-天门酰胺、L-天冬氨酸、谷氨酸、脯氨酸和丝氨酸；
    优选地，所述TME培养基含有0.1体积份的β-巯基乙醇、1体积份的非必需氨基酸水溶液和99体积份的a-MEM；
    优选地，所述TME培养基由所述a-MEM、β-巯基乙醇和非必需氨基酸水溶液组成。
  4. 根据权利要求1至3中任一项所述的试剂盒，其特征在于，所述TMD培养基包含0.05-0.2体积份的β-巯基乙醇、0.5-2体积份的非必需氨基酸水溶液、8-12体积份的血清替代物、85-95体积份的a-MEM/DMEM-F12和终浓度为5-15ng/ml的重组人碱性成纤维生长因子，其中，所述非必需氨基酸水溶液包含浓度各为8-12mM的甘氨酸、丙氨酸、L-天门酰胺、L-天冬氨酸、谷氨酸、脯氨酸和丝氨酸；
    优选地，所述TMD培养基包含0.1体积份的β-巯基乙醇、1体积份的非必需氨基酸水溶液、10体积份的血清替代物、89体积份的a-MEM/DMEM-F12和终浓度为10ng/ml的重组人碱性成纤维生长因子；
    优选地，所述TMD培养基由所述a-MEM/DMEM-F12、β-巯基乙醇、非必需氨基酸水溶液、重组人碱性成纤维生长因子和血清替代物组成。
  5. 使用权利要求1至4中任一项所述的试剂盒分步培养hUC-MSC的方法，其特征在于，所述方法包括：先使用所述试剂盒中的TME培养基培养hUC-MSC，然后使用所述试剂盒中的TMD培养基进行培养。
  6. 根据权利要求5所述的方法，其特征在于，所述方法包括以下步骤：
    (1)将hUC-MSC以0.5-4×104个细胞/cm2的密度接种于所述试剂盒中的TME培养基中培养3-6小时；细胞在此步骤中进行显著的贴壁；
    (2)弃去TME培养基，PBS清洗，更换为所述试剂盒中的TMD培养基，每3-5天更换新鲜的TMD培养基；细胞在此步骤中进行显著的扩增；
    (3)待细胞达70-90％汇合后，收集细胞备用、冻存或重复步骤(1)和(2)传代培养；
    可选择地，取步骤(3)收集的hUC-MSC细胞，检测以下项目中的一项或多项：分化能力、细胞活性、细胞纯度、细胞污染和增殖特性。
  7. 根据权利要求5或6所述的方法，其特征在于，所述方法包括以下步骤：
    (1)将hUC-MSC以2×104个细胞/cm2的密度接种于所述试剂盒中的TME培养基中培养3-4小时；
    (2)弃去TME培养基，PBS清洗1次，更换为预先37℃孵育的所述试剂盒中的TMD培养基，每3天更换新鲜的TMD培养基；
    (3)待细胞达90％汇合后，收集细胞备用、冻存或重复步骤(1)和(2)传代培养；
    可选择地，取步骤(3)收集的hUC-MSC细胞，检测以下项目中的全部：分化能力、细胞活性、细胞纯度、细胞污染和增殖特性。
  8. 通过权利要求5至7中任一项所述的方法获得的hUC-MSC。
  9. 根据权利要求8所述的hUC-MSC，其特征在于，所述hUC-MSC具有以下特征：
    (1)粘附于塑料培养器皿成梭形旋涡状生长；
    (2)表达CD29、CD44、CD73、CD90、CD105和HLA-ABC的阳性比例大于99.0％；表达CD45、CD34和HLA-DR的阳性比例小于1.0％；
    (3)体外可诱导分化为成骨细胞和成脂细胞；
    (4)活细胞检测比率达99％以上；
    (5)呈典型的“S型”生长曲线特性；
    (6)多能性基因表达，所述多能性基因为选自SSEA-4、OCT-4、NANOG和SOX-2中的一种或多种。
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