WO2019161591A1 - 间充质干细胞的分离培养方法及冻存、复苏方法 - Google Patents

间充质干细胞的分离培养方法及冻存、复苏方法 Download PDF

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WO2019161591A1
WO2019161591A1 PCT/CN2018/080589 CN2018080589W WO2019161591A1 WO 2019161591 A1 WO2019161591 A1 WO 2019161591A1 CN 2018080589 W CN2018080589 W CN 2018080589W WO 2019161591 A1 WO2019161591 A1 WO 2019161591A1
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mesenchymal stem
cells
stem cells
cell
culture
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PCT/CN2018/080589
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French (fr)
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杨涛
隋昳
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深圳至博生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
    • 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
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
    • 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
    • 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
    • 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
    • C12N5/0668—Mesenchymal stem cells from other natural sources
    • 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
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes

Definitions

  • the present application relates to the field of biotechnology, and in particular, to a method for separating and culturing mesenchymal stem cells, and a method for cryopreservation and resuscitation.
  • Stem cells are a class of cells that have different differentiation potential and self-renew in an undifferentiated state.
  • Stem cell therapy refers to the process of treating a disease after treatment (or implantation) of a stem cell derived from a human or an allogeneic source.
  • Such in vitro procedures include isolation, purification, amplification, cryopreservation, and resuscitation of cryopreserved stem cells.
  • the main diseases involved include bone and joint diseases, liver cirrhosis, graft host rejection (GVHD), spinal cord injury and degenerative nervous system diseases and diabetes.
  • GVHD graft host rejection
  • MSCs mesenchymal stem cells
  • MSCs mesenchymal stem cells
  • stem cell preparations follow a common development process, like other cell preparations, from cell preparation, in vitro experiments, in vivo animal experiments, to clinical studies implanted in humans, ie, the process of clinical treatment.
  • this particularity is that stem cells are the most primitive cells, and any subtle changes in the microenvironment will affect their function.
  • the process of stem cell preparation after the stem cells are separated from the body, the nature of the stem cells begins to change. Therefore, how to operate to ensure that the stem cells have good cell quality, safety and biological effects is an urgent problem to be solved.
  • the purpose of the present application is to provide a method for isolating and culturing mesenchymal stem cells, and a method for cryopreservation and resuscitation, which can obtain mesenchymal stem cells with good cell quality, safety and biological effects, and can be well preserved and resuscitated.
  • the mesenchymal stem cells are highly repetitive cells that are produced by the production of mesenchymal stem cells.
  • a method for isolating and cultivating mesenchymal stem cells comprising:
  • mesenchymal stem cells primary cells After 5-10 days of primary cell culture of mesenchymal stem cells, replace them with serum-free DMEM nutrient solution, and add 65 to 75% of the cells to the cells, remove the culture supernatant, and then add the first A digestive juice, digested for 0.5-2 min, the cells are shrunk, added to the removed culture supernatant and neutralized, centrifuged, re-suspended in complete medium, inoculated according to 1 to 1.5, and cultured for 4 to 5 days, When the cells are 65 to 75% confluent, they are subcultured at a ratio of 1:6 to 8;
  • Isolation of Mesenchymal Stem Cells Collecting cells in the P2 or P3 logarithmic growth phase and not more than 80% of the cell fusion in the culture and expansion of mesenchymal stem cells, digesting, neutralizing, and washing according to the requirements of passage. The desired mesenchymal stem cells can then be collected.
  • the first digestive juice is 0.25% trypsin-EDTA.
  • the centrifugation is performed by centrifugation at 800 to 1200 rpm for 6 to 10 minutes.
  • the mesenchymal stem cell primary cell is a bone marrow mesenchymal stem cell primary cell or an autologous adipose-derived mesenchymal stem cell primary cell or a umbilical cord mesenchymal stem cell primary cell.
  • the mesenchymal stem cell primary cell is a bone marrow mesenchymal stem cell primary cell
  • the obtained mesenchymal stem cell primary cell comprises:
  • the bone marrow solution was taken and diluted: the bone marrow solution was extracted with a syringe containing 500 U/ml heparin in PBS, mixed uniformly, and diluted with PBS containing 20 U/ml heparin to obtain a bone marrow buffer solution;
  • the bone marrow buffer solution was added to a lymphocyte separation liquid having a specific gravity of 1.073 g/ml, and the mononuclear cell population was purified by centrifugation, and then centrifuged to wash the DMEM medium to obtain a precipitate;
  • Cell expansion culture The precipitate obtained by centrifugation is suspended by serum-free culture medium, inoculated at a density of 5 ⁇ 10 6 /mL, and cultured under the conditions of 35-40 ° C, 5% CO 2 and saturated humidity; After 3 days of culture, the medium was changed for the first time, and the unattached cells were discarded. The culture medium was replaced once every 3 days, and the primary cells of the bone marrow mesenchymal stem cells were obtained after 6 days of co-culture.
  • the mesenchymal stem cell primary cell is an autologous adipose-derived mesenchymal stem cell primary cell, and the obtained mesenchymal stem cell primary cell comprises:
  • Autologous fat particles were extracted and cut: autologous fat particles were taken, visible microvessels and muscle tissue were removed, washed with PBS, and then the adipose tissue was cut into a paste to make it less than 1 mm 3 ;
  • Isolation and purification of cells adding a second digestive juice to the paste-like adipose tissue for digestion, digesting, collecting and collecting the filtrate by filtration, and then neutralizing with a low-sugar DMEM medium containing 10% FBS, and then centrifuging to obtain a precipitate;
  • Cell expansion culture serum-free medium is added to the precipitate obtained after separation and purification of the cells, inoculated at a density of 1 ⁇ 10 6 /mL, and at 35 to 40 ° C, 5% CO 2 and saturated humidity. The culture was carried out under the condition; after the culture for 3 days, the liquid was changed for the first time, the unattached cells were discarded, and the culture solution was changed once every 3 days, and the primary cells of the autologous adipose-derived mesenchymal stem cells were obtained after 6 days of co-culture.
  • the second digestive juice is serum-free DMEM containing 0.1% collagenase type I and 0.05% trypsin.
  • the mesenchymal stem cell primary cell is a umbilical cord mesenchymal stem cell primary cell
  • the obtained mesenchymal stem cell primary cell comprises:
  • Treatment of umbilical cord Take the umbilical cord and cut it.
  • the total length of the umbilical cord after cutting is more than 15cm.
  • Cell expansion culture The shredded Walton gum is applied to the culture dish, added to serum-free complete medium, cultured at 35-40 ° C, 5% CO 2 and saturated humidity, and cultured for 7 to 10 days.
  • the primary cells of umbilical cord mesenchymal stem cells can be obtained.
  • a method for cryopreservation and resuscitation of mesenchymal stem cells wherein the mesenchymal stem cells obtained by the above-described isolation and culture method of mesenchymal stem cells are cryopreserved and resuscitated, including:
  • Cryopreservation of mesenchymal stem cells resuspend the mesenchymal stem cells with a cell cryopreservation solution, and then placed in a container filled with isopropyl alcohol, and then transferred to a liquid nitrogen tank at -90 to -70 ° C overnight;
  • Resuscitation of mesenchymal stem cells The frozen mesenchymal stem cells are immediately thawed in a 40-45 ° C water bath, then added to the complete medium and mixed. After centrifugation, the supernatant is discarded and resuspended in complete medium. The cells are cultured at 35 to 40 ° C, 5% CO 2 and saturated humidity, and 65 to 75% of the cells are allowed to adhere.
  • the cell cryopreservation solution in the cryopreservation of the mesenchymal stem cells consists of 90% fetal bovine serum and 10% DMSO.
  • the present application provides a method for isolating and culturing mesenchymal stem cells, which can obtain mesenchymal stem cells with good cell quality, safety and biological effects. Further, the mesenchymal stem cells obtained by the method for isolating and culturing the mesenchymal stem cells further provide a method for cryopreservation and resuscitation of mesenchymal stem cells, which can well preserve and resuscitate the mesenchymal stem cells.
  • FIG. 1 is a schematic flow chart of a method for separating and culturing mesenchymal stem cells according to an embodiment of the present application.
  • FIG. 2 is a schematic flow chart of obtaining mesenchymal stem cell primary cells in the first embodiment of the first embodiment of the present application.
  • FIG. 3 is a schematic flow chart of obtaining mesenchymal stem cell primary cells in a second embodiment of the first embodiment of the present application.
  • FIG. 4 is a schematic flow chart of obtaining mesenchymal stem cell primary cells in a third embodiment of the first embodiment of the present application.
  • FIG. 5 is a schematic flow chart of a method for cryopreservation and resuscitation of mesenchymal stem cells according to the second embodiment of the present application.
  • a method for separating and culturing mesenchymal stem cells comprises:
  • Step S1 obtaining primary cells of mesenchymal stem cells
  • Step S2 Culture and expansion of primary cells of mesenchymal stem cells: After primary culture of mesenchymal stem cells for 5-10 days, the cells are replaced with serum-free DMEM nutrient solution, and 65 to 75% of the cells are fused, and the culture supernatant is removed. Then, the first digestive juice is added, and the cells are digested for 0.5-2 min. After the cells are shrunk, the removed culture supernatant is added to neutralize, and then centrifuged, re-suspended in complete medium, inoculated according to 1 to 1.5, and cultured 4 to After 5 days, the cells were subcultured at a ratio of 1:6-8 when 65 to 75% of the cells were fused;
  • Step S3 Isolation of Mesenchymal Stem Cells: Collecting cells in the P2 or P3 logarithmic growth phase and not more than 80% of the cell fusion in the culture and expansion of mesenchymal stem cells, and digesting according to the requirements of the passage. After washing, the desired mesenchymal stem cells can be collected.
  • the method for isolating and culturing a mesenchymal stem cell obtaineds good cell quality and safety by determining the culture and expansion of mesenchymal stem cell primary cells and the specific operation in the separation of mesenchymal stem cells. Sexual and biological effects of mesenchymal stem cells.
  • the mesenchymal stem cell primary cell may be a bone marrow mesenchymal stem cell primary cell or an autologous adipose-derived mesenchymal stem cell primary cell or a umbilical cord mesenchymal stem cell primary cell. Therefore, there are three embodiments for obtaining mesenchymal stem cell primary cells. All three implementation methods need to evaluate and screen the donor's health, strictly exclude HBV, syphilis-positive, high-risk groups susceptible to HIV (such as drug abusers, homosexuals, multiple sexual partners), various Patients with tuberculosis (such as tuberculosis, kidney tuberculosis, lymphatic tuberculosis, and bone tuberculosis).
  • the obtaining the mesenchymal stem cell primary cell comprises:
  • Step S11 extracting the bone marrow solution and diluting: the bone marrow solution is extracted with a syringe containing 500 U/ml heparin in PBS, mixed uniformly, and diluted with PBS containing 20 U/ml heparin to obtain a bone marrow buffer solution;
  • Step S12 isolation and purification of cells: the bone marrow buffer solution is added to a lymphocyte separation liquid having a specific gravity of 1.073 g/ml, and the mononuclear cell population is purified by centrifugation, and then washed by DMEM culture solution to obtain a precipitate;
  • Step S13 Cell expansion culture: The precipitate obtained by centrifugation is suspended by a serum-free culture solution, inoculated at a density of 5 ⁇ 10 6 /mL, and at 35 to 40 ° C, 5% CO 2 and saturated humidity conditions. The culture was carried out for the first time after the culture for 3 days, and the unadhered cells were discarded, and the culture medium was replaced once every 3 days, and the primary cells of the bone marrow mesenchymal stem cells were obtained after 6 days of co-culture.
  • the bone marrow fluid is extracted specifically: the prone position is taken, the upper spine is taken as the puncture point, the iodophor is routinely disinfected, and the 2% lidocaine is locally anesthetized. Puncture the upper spine with a bone through the needle, and pull out the needle core after a breakthrough.
  • the bone marrow solution was extracted with a 20 ml sterile syringe containing 2 ml of PBS containing 500 U/ml heparin, shaken, and poured into a 50 ml sterile centrifuge tube to extract about 50 ml. Immediately after the bone marrow fluid is taken, it can be sent to the laboratory for dilution.
  • the dilution of the bone marrow fluid is preferably: diluting the bone marrow solution with an equal amount of PBS containing 20 U/ml heparin to obtain a bone marrow buffer.
  • step S12 it is preferred that the bone marrow buffer is slowly added to the lymphocyte separation solution along the wall of the tube. More preferably, the bone marrow buffer and the lymphocyte separation solution are in equal volume.
  • Purification of the mononuclear cell population by centrifugation is preferably carried out by centrifugation at 2000 rpm for 30 minutes to purify the mononuclear cell population.
  • the precipitate was preferably washed by centrifugation with DMEM medium, and washed twice with DMEM (L) medium for 1000 rpm, centrifuged for 5 minutes, and the supernatant was discarded to obtain a precipitate.
  • step S13 it is preferred to cultivate at 37 ° C, 5% CO 2 and saturated humidity after inoculation.
  • the specific embodiment may be: taking a prone position, taking the upper spine as a puncture point, iodophor conventional disinfection, and 2% lidocaine local anesthesia. Puncture the upper spine with a bone through the needle, and pull out the needle core after a breakthrough.
  • the bone marrow solution was extracted with a 20 ml sterile syringe containing 2 ml of PBS containing 500 U/ml heparin, shaken, and poured into a 50 ml sterile centrifuge tube to extract about 50 ml.
  • the collected bone marrow fluid is stored in a refrigerator at 4 ° C for a short period of time (no more than 24 hours) or immediately sent to the laboratory.
  • the bone marrow was diluted with an equal volume of 20 U/ml heparin in PBS, and a lymphocyte separation solution having an equal volume specific gravity of 1.073 g/ml was slowly added dropwise along the wall of the tube.
  • the mononuclear cell population was purified by centrifugation for 30 minutes at 2000 rpm.
  • the cells were washed twice with DMEM (L), centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded to take a precipitate.
  • the serum-free medium was suspended, inoculated into a culture flask at 5 ⁇ 10 6 /mL, and cultured at 37 ° C under 5% CO 2 saturated humidity. After 3 days, the medium was changed for the first time, and the unattached cells were discarded, and the culture solution was changed once every 3 days. A total of 6d, for transplantation, passaging or cryopreservation.
  • the obtaining mesenchymal stem cell primary cell includes:
  • Step S11' extracting autologous fat particles and cutting: extracting autologous fat particles, removing visible microvessels and muscle tissue, washing with PBS, and then cutting the adipose tissue into a paste to make it less than 1 mm 3 ;
  • Step S12' separation and purification of cells: adding a second digestive juice to the paste-like adipose tissue for digestion, digesting, collecting and collecting the filtrate by filtration, and then neutralizing with a low-sugar DMEM medium containing 10% FBS, and then centrifuging to obtain a precipitate. ;and
  • Step S13' Cell expansion culture: a serum-free medium is added to the precipitate obtained after separation and purification of the cells, and inoculated at a density of 1 ⁇ 10 6 /mL, and at 35 to 40 ° C, 5% CO 2 The culture was carried out under the condition of saturated humidity; after the culture for 3 days, the cells were first changed, the unattached cells were discarded, and the culture solution was replaced once every 3 days, and the primary cells of autologous adipose-derived mesenchymal stem cells were obtained after 6 days of co-culture.
  • step S11' it is preferred to wash three times with PBS.
  • the second digestive juice is preferably serum-free DMEM containing 0.1% collagenase type I and 0.05% trypsin. More preferably, the digestion is carried out in a water bath at 37 ° C and stirred for a period of 40 to 45 minutes. The post-digestion filtration is preferably carried out by filtration using a 200 mesh filter. Preferably, the low sugar DMEM medium is used in an amount equal to the filtrate; the centrifugation after neutralization using the low sugar DMEM medium is preferably carried out by centrifugation at 1500 rpm for 10 minutes.
  • step S13' it is preferred to cultivate at 37 ° C, 5% CO 2 and saturated humidity after inoculation.
  • a specific embodiment may be: 15 to 20 mL of autologous fat particles are taken from the lower abdomen of the patient, visible microvessels and muscle tissue are removed, PBS is washed 3 times, and then the adipose tissue is cut into a paste with scissors, and the requirement is ⁇ 1mm 3 . Transfer the paste adipose tissue to the Erlenmeyer flask, add the digestive solution (serum-free DMEM containing 0.1% collagenase type I and 0.05% trypsin), stir at 40 ° C for 40 to 45 minutes in a 37 ° C water bath until the tissue block is digested. .
  • the filtrate was collected by filtration through a 200-mesh sieve, and then neutralized in an equal amount using a low-sugar DMEM medium containing 10% FBS, and centrifuged at 1500 rpm for 10 min, and the supernatant was discarded.
  • Fresh serum-free medium was added to the precipitate, and the cells were cultured at a cell density of 1 ⁇ 10 6 /mL at 37 ° C under 5% CO 2 . After 3 days, the medium was changed for the first time, the unattached cells were discarded, and the culture solution was changed once every 3 days. A total of 6d, for transplantation, passaging or cryopreservation.
  • the obtaining mesenchymal stem cell primary cell includes:
  • Step S11′′ Processing the umbilical cord: taking the umbilical cord and cutting it.
  • the total length of the umbilical cord after cutting is more than 15 cm.
  • the blood stain is removed by using 2 times double-anti-normal saline, then the blood vessel and the outer membrane are removed, and the Walton is removed. Glue and cut into 0.5 ⁇ 1mm 3 ;
  • Step S12′′ cell expansion culture: the shredded Walton gum is applied to the culture dish, added to the serum-free complete medium, cultured at 35-40 ° C, 5% CO 2 and saturated humidity, and cultured.
  • Primary cells of umbilical cord mesenchymal stem cells can be obtained in 7 to 10 days.
  • step S11 after washing with physiological saline, it can be immediately sent to the laboratory for dilution, and if it needs to be temporarily stored, it can be temporarily stored in a refrigerator at 4 ° C.
  • step S12 serum-free complete medium is added. Thereafter, it is preferably cultured under the conditions of 37 ° C, 5% CO 2 and saturated humidity.
  • the specific embodiment may be: within 1 minute after the placenta is delivered, the umbilical cord is cut after ligation near the placenta, and the total length of the collected umbilical cord is not less than 15 cm.
  • the umbilical cord was transferred to a sterile Petri dish by sterile forceps, and the blood stain was substantially removed with 2 times of double-resistant physiological saline. Remove the blood vessels and the outer membrane, peel off the Walton gum, and cut into 0.5 to 1 mm 3 .
  • the above-mentioned shredded Walton gum was evenly spread on a 10 cm culture dish, attached at room temperature for 5 to 10 minutes, and then added with 5 ml of complete medium (without serum).
  • the CO 2 saturated humidity incubator at 37 ° C and a volume fraction of 5% was cultured for 7 to 10 days.
  • the first digestive juice is preferably 0.25% trypsin-EDTA.
  • the centrifugation is performed by centrifugation at 800 to 1200 rpm for 6 to 10 minutes.
  • the primary cells are cultured for 7 days, then changed to medium (serum-free DMEM (L)), 70% of the cells are fused, and the culture supernatant is removed to a 50 ml centrifuge tube, and 0.25% trypsin is added to the culture flask.
  • a method for cryopreservation and resuscitation of mesenchymal stem cells wherein the mesenchymal stem cells obtained by the method for isolating and culturing the mesenchymal stem cells of the first embodiment are cryopreserved and resuscitated, comprising:
  • Step T1 cryopreservation of mesenchymal stem cells: resuspend the mesenchymal stem cells with a cell cryopreservation solution, and then placed in a container filled with isopropyl alcohol, and then transferred to liquid nitrogen at -90 to -70 ° C overnight. Cans; and
  • Step T2 Resuscitation of Mesenchymal Stem Cells: The frozen mesenchymal stem cells are immediately placed in a 40-45 ° C water bath, then added to the complete medium and mixed. After centrifugation, the supernatant is discarded to complete the medium. After resuspending, the cells are cultured under the conditions of 35-40 ° C, 5% CO 2 and saturated humidity, and 65 to 75% of the cells are attached to the cells.
  • step T1 preferably, the cell cryopreservation consists of 90% fetal bovine serum and 10% DMSO.
  • step T1 is to resuspend mesenchymal stem cells (MSCs) with cell cryopreservation solution (90% fetal bovine serum, 10% DMSO), and mix well, 1 ml per tube (1 15 cm culture dish) ) Packed in a cryotube seal. Mark the cryotubes, place the cryotubes in a freezer box containing isopropyl alcohol, overnight at -80 °C, and transfer to a liquid nitrogen tank the next day.
  • MSCs mesenchymal stem cells
  • cell cryopreservation solution 90% fetal bovine serum, 10% DMSO
  • step T2 the centrifugation treatment is preferably performed by centrifugation at 1000 rpm for 8 minutes. After resuspending in complete medium, it is preferably cultured under conditions of 37 ° C, 5% CO 2 and saturated humidity. In some embodiments, step T2 is such that the MSCs are removed from the liquid nitrogen and immediately thawed in a 42 ° C water bath. Add an equal volume of complete medium and mix. After centrifugation at 1000 rpm for 8 minutes, discard the supernatant, resuspend in complete medium, and incubate in a CO 2 saturated humidity incubator at 37 ° C in a volume fraction of 5%. 70% of the cells can be attached.

Abstract

提供一种间充质干细胞的分离培养方法，该分离培养方法包括：获取间充质干细胞原代细胞；间充质干细胞原代细胞的培养扩增；间充质干细胞的分离。还提供一种间充质干细胞的冻存、复苏方法，能保存并复苏该间质干细胞。

Description

间充质干细胞的分离培养方法及冻存、复苏方法
本申请是以申请号为201810155579.7，申请日为2018年2月23日的中国专利申请为基础，并主张其优先权，该申请的全部内容在此作为整体引入本申请中。
技术领域
本申请涉及生物技术领域，尤其涉及一种间充质干细胞的分离培养方法及冻存、复苏方法。
背景技术
干细胞是一类具有不同分化潜能，并在非分化状态下自我更新的细胞。干细胞治疗是指应用人自体或异体来源的干细胞经体外操作后(或植入)人体，用于疾病治疗的过程。这种体外操作包括干细胞的分离、纯化、扩增、冻存和冻存后的复苏等过程。目前国内外已经开展了多种干细胞类型的临床应用研究，涉及的主要疾病类型包括骨关节疾病、肝硬化、移植物宿主排斥反应(GVHD)、脊髓损伤及退行性神经系统疾病和糖尿病等。其中由于间充质干细胞(MSCs)具有一定的多向分化潜能及抗炎和免疫调控能力等，广泛应用于临床各种疾病的治疗。
开展临床试验治疗，需要制备大量的细胞，而用于干细胞治疗的细胞制备技术具有多样性、复杂性和特殊性。作为一种新型的生物治疗产品，干细胞制剂除了像其他细胞制品一样遵循一个共同的研发过程，即从细胞制剂的制备、体外实验、体内动物实验，到植入人体的临床研究即临床治疗的过程外，还具备其特殊性。这种特殊性就是干细胞是最原始的细胞，任何细微的微环境改变都会影响其功能的发挥。而在干细胞制备过程中，从体内干细胞分离出来后，干细胞的性质就开始发生变化，因此如何操作以确保干细胞具有良好的细胞质量、安全性以及生物学效应等，是一个亟需解决的问题。
申请内容
本申请的目的在于提供一种间充质干细胞的分离培养方法及冻存、复苏方法，能得到具有良好的细胞质量、安全性以及生物学效应的间质干细胞，并且能很好的保存并复苏该间质干细胞。
为解决上述技术问题，申请采用如下所述的技术方案。一种间充质干细胞的分离培养方法，包括：
获取间充质干细胞原代细胞；
间充质干细胞原代细胞的培养扩增：间充质干细胞原代细胞培养5-10天后换成无血清DMEM营养液，至细胞65～75％融合，移去培养上清液，然后添加第一消化液，消化0.5-2min，细胞收缩后加入所移去的培养上清液中和后进行离心分离，重新加入完全培养基重悬，按照1传1～1.5接种，培养4～5天后，细胞65～75％融合时以1∶6～8的比例进行传代培养；及
间充质干细胞的分离：收集间充质干细胞原代细胞的培养扩增中的处于P2或P3对数生长期且细胞融合程度不超过80％的细胞，按传代的要求消化、中和、洗涤后即可收集得到所需的间充质干细胞。
优选地，所述第一消化液为0.25％胰蛋白酶-EDTA。
优选地，所述的进行离心分离为在800～1200转/分下离心6～10min。
优选地，所述间充质干细胞原代细胞为骨髓间充质干细胞原代细胞或自体脂肪间充质干细胞原代细胞或脐带间充质干细胞原代细胞。
优选地，所述间充质干细胞原代细胞为骨髓间充质干细胞原代细胞，所述获取间充质干细胞原代细胞包括：
抽取骨髓液并稀释：用含500U/ml肝素的PBS液的注射器抽取骨髓液，混合均匀后用含20U/ml肝素的PBS液稀释得到骨髓缓冲液；
细胞的分离与纯化：将骨髓缓冲液中加入比重为1.073g/ml的淋巴细胞分离液上，通过离心分离纯化单个核细胞群，然后用DMEM培养液离心洗涤得到沉淀；及
细胞扩增培养：将离心分离后得到的沉淀利用无血清培养液混悬，按5× 10 6/mL的密度进行接种并于35～40℃、5％CO 2及饱和湿度的条件下培养；培养3天后首次换液，弃去未贴壁细胞，以后每3d更换培养液1次，共培养6天即可得到骨髓间充质干细胞原代细胞。
优选地，所述间充质干细胞原代细胞为自体脂肪间充质干细胞原代细胞，所述获取间充质干细胞原代细胞包括：
抽取自体脂肪颗粒并剪碎：抽取自体脂肪颗粒，剔除可见微血管及肌肉组织，用PBS进行洗涤，然后将脂肪组织剪碎至糊状使其小于1mm 3；
细胞的分离与纯化：往糊状的脂肪组织中添加第二消化液进行消化，消化后过滤收集滤液，然后使用含有10％FBS的低糖DMEM培养基进行中和，再离心得到沉淀；及
细胞扩增培养：往经过细胞的分离与纯化后得到的沉淀中加入无血清培养基，以1×10 6/mL的密度进行接种，并于35～40℃、5％CO 2及饱和湿度的条件下培养；培养3天后首次换液，弃去未贴壁细胞，以后每3d更换培养液1次，共培养6天即可得到自体脂肪间充质干细胞原代细胞。
优选地，所述第二消化液为含有0.1％胶原酶I型和0.05％胰蛋白酶的无血清DMEM。
优选地，所述间充质干细胞原代细胞为脐带间充质干细胞原代细胞，所述获取间充质干细胞原代细胞包括：
处理脐带：取脐带并剪断，剪断后的脐带总长度大于15cm，用生理盐水清洗后，再用含2倍双抗生理盐水去除血渍，接着去除血管与外膜，剥离华尔通胶并剪成0.5～1mm 3；及
细胞扩增培养：将剪碎的华尔通胶涂布于培养皿上，加入无血清完全培养基，于35～40℃、5％CO 2及饱和湿度的条件下培养，培养7～10天即可得到脐带间充质干细胞原代细胞。
一种间充质干细胞的冻存、复苏方法，将上述间充质干细胞的分离培养方法所得到的间充质干细胞进行冻存及复苏，包括：
间充质干细胞的冻存：用细胞冻存液重悬所述间充质干细胞，然后置于装 有异丙醇的容器中，于-90～-70℃下过夜后移入液氮罐；及
间充质干细胞的复苏：取冻存的间充质干细胞立即投入40～45℃水浴中融化，然后加入完全培养基并混匀，离心处理后弃去上清液，以完全培养基重悬后于35～40℃、5％CO 2及饱和湿度的条件下培养，待65～75％的细胞贴壁即可。
优选地，所述间充质干细胞的冻存中的细胞冻存液由90％胎牛血清及10％DMSO组成。
本申请的有益效果在于：
本申请提供一种间充质干细胞的分离培养方法，能得到具有良好的细胞质量、安全性以及生物学效应的间质干细胞。并且针对上述间充质干细胞的分离培养方法所得到的间质干细胞，还提供一种间充质干细胞的冻存、复苏方法，能很好的保存并复苏该间质干细胞。
附图说明
图1是本申请实施例一中间充质干细胞的分离培养方法的流程示意图。
图2是本申请实施例一的第一种实施方式中获取间充质干细胞原代细胞的流程示意图。
图3是本申请实施例一的第二种实施方式中获取间充质干细胞原代细胞的流程示意图。
图4是本申请实施例一的第三种实施方式中获取间充质干细胞原代细胞的流程示意图。
图5是本申请实施例二中间充质干细胞的冻存、复苏方法的流程示意图。
具体实施方式
为使本领域的普通技术人员更加清楚地理解申请的目的、技术方案和优点，以下结合附图和实施例对申请做进一步的阐述。
实施例一
如图1所示，一种间充质干细胞的分离培养方法，包括：
步骤S1：获取间充质干细胞原代细胞；
步骤S2：间充质干细胞原代细胞的培养扩增：间充质干细胞原代细胞培养5-10天后换成无血清DMEM营养液，至细胞65～75％融合，移去培养上清液，然后添加第一消化液，消化0.5-2min，细胞收缩后加入所移去的培养上清液中和后进行离心分离，重新加入完全培养基重悬，按照1传1～1.5接种，培养4～5天后，细胞65～75％融合时以1∶6～8的比例进行传代培养；及
步骤S3：间充质干细胞的分离：收集间充质干细胞原代细胞的培养扩增中的处于P2或P3对数生长期且细胞融合程度不超过80％的细胞，按传代的要求消化、中和、洗涤后即可收集得到所需的间充质干细胞。
本申请所提供的一种间充质干细胞的分离培养方法，通过确定间充质干细胞原代细胞的培养扩增以及间充质干细胞的分离中的具体操作，从而得到具有良好的细胞质量、安全性以及生物学效应的间质干细胞。
在步骤S1中，所述间充质干细胞原代细胞可以是骨髓间充质干细胞原代细胞或自体脂肪间充质干细胞原代细胞或脐带间充质干细胞原代细胞。因此获取间充质干细胞原代细胞有三种实施方式。该三种实施方式均需要对供者的健康情况进行评估和筛查，严格排除HBV、梅毒阳性者、易感染HIV的高危人群(如吸毒史者、同性恋者、多个性伴侣者)、各种结核病(如肺结核、肾结核、淋巴结核及骨结核等)患者。
第一种实施方式
请一并参阅图2，当所述间充质干细胞原代细胞为骨髓间充质干细胞原代细胞时，所述获取间充质干细胞原代细胞包括：
步骤S11：抽取骨髓液并稀释：用含500U/ml肝素的PBS液的注射器抽取骨髓液，混合均匀后用含20U/ml肝素的PBS液稀释得到骨髓缓冲液；
步骤S12：细胞的分离与纯化：将骨髓缓冲液中加入比重为1.073g/ml的淋巴细胞分离液上，通过离心分离纯化单个核细胞群，然后用DMEM培养液离心洗涤得到沉淀；及
步骤S13：细胞扩增培养：将离心分离后得到的沉淀利用无血清培养液混悬， 按5×10 6/mL的密度进行接种并于35～40℃、5％CO 2及饱和湿度的条件下培养；培养3天后首次换液，弃去未贴壁细胞，以后每3d更换培养液1次，共培养6天即可得到骨髓间充质干细胞原代细胞。
其中，步骤S11中，抽取骨髓液具体为：取俯卧位，取髂后上棘为穿刺点，碘伏常规消毒，2％利多卡因局部麻醉。用骨穿针在髂后上棘穿刺，有突破感后拔出针芯。用装有2ml含500U/ml肝素的PBS液的20ml无菌注射器抽取骨髓液，摇匀后注入50ml的无菌离心管中，共抽取50ml左右。在抽取骨髓液之后可以立即送至实验室进行稀释，而如需短暂保存，可以是短暂存放于4℃冰箱内。骨髓液的稀释优选为：用等量含20U/ml肝素的PBS液稀释骨髓液得到骨髓缓冲液。
步骤S12中，优选的是，将骨髓缓冲液沿管壁徐徐滴流叠加入淋巴细胞分离液上，更好的是，所述骨髓缓冲液与淋巴细胞分离液等体积。通过离心分离纯化单个核细胞群优选为，以2000转/分的转速离心30分钟，纯化单个核细胞群。用DMEM培养液离心洗涤得到沉淀优选为，用DMEM(L)培养液离心洗涤2次，每次1000转/分，离心5分钟，弃去上清液，取沉淀。
步骤S13中，优选的是接种后于37℃、5％CO 2及饱和湿度的条件下培养。
在该实施方式中，具体的实施例可以是：取俯卧位，取髂后上棘为穿刺点，碘伏常规消毒，2％利多卡因局部麻醉。用骨穿针在髂后上棘穿刺，有突破感后拔出针芯。用装有2ml含500U/ml肝素的PBS液的20ml无菌注射器抽取骨髓液，摇匀后注入50ml的无菌离心管中，共抽取50ml左右。采集的骨髓液，短暂存放于4℃冰箱内(不超过24小时)或立即送至实验室。用等量含20U/ml肝素的PBS液稀释骨髓后，沿管壁徐徐滴流叠加入等体积比重为1.073g/ml的淋巴细胞分离液上。2000转/分，离心30分钟，纯化单个核细胞群。用DMEM(L)培养液离心洗涤2次，每次1000转/分，离心5分钟，弃去上清液，取沉淀。无血清培养液混悬，按5×10 6/mL接种于培养瓶中，置37℃5％CO 2饱和湿度培养。3天后首次换液，弃去未贴壁细胞，以后每3d更换培养液1次。共6d，以备移植、传代或冻存。
第二种实施方式
请一并参阅图3，当所述间充质干细胞原代细胞为自体脂肪间充质干细胞原代细胞时，所述获取间充质干细胞原代细胞包括：
步骤S11′：抽取自体脂肪颗粒并剪碎：抽取自体脂肪颗粒，剔除可见微血管及肌肉组织，用PBS进行洗涤，然后将脂肪组织剪碎至糊状使其小于1mm 3；
步骤S12′：细胞的分离与纯化：往糊状的脂肪组织中添加第二消化液进行消化，消化后过滤收集滤液，然后使用含有10％FBS的低糖DMEM培养基进行中和，再离心得到沉淀；及
步骤S13′：细胞扩增培养：往经过细胞的分离与纯化后得到的沉淀中加入无血清培养基，以1×10 6/mL的密度进行接种，并于35～40℃、5％CO 2及饱和湿度的条件下培养；培养3天后首次换液，弃去未贴壁细胞，以后每3d更换培养液1次，共培养6天即可得到自体脂肪间充质干细胞原代细胞。
其中，步骤S11′中，优选为用PBS洗涤3遍。
在步骤S12′中，所述第二消化液优选为含有0.1％胶原酶I型和0.05％胰蛋白酶的无血清DMEM。进一步优选的是，在37℃的水浴中进行消化，且进行搅拌，消化时长为40～45分钟。消化后过滤优选为选用200目过滤网进行过滤。优选地，所述低糖DMEM培养基的用量与滤液等体积；利用低糖DMEM培养基中和后所进行的离心优选为，以1500转/分的转速离心10分钟。
在步骤S13′中，优选的是接种后于37℃、5％CO 2及饱和湿度的条件下培养。
在该实施方式中，具体的实施例可以是：患者下腹部抽取15～20mL自体脂肪颗粒，剔除可见微血管及肌肉组织，PBS洗涤3遍，然后用剪刀将脂肪组织剪碎至糊状，要求＜1mm 3。将糊状脂肪组织转移至锥形瓶中，添加消化液(含有0.1％胶原酶I型和0.05％胰蛋白酶的无血清DMEM)，37℃水浴中匀速搅拌40～45分钟，至组织块消化干净。200目筛网过滤收集滤液，然后使用含有10％FBS的低糖DMEM培养基等量中和，1500转/分离心10min，弃上清。向沉淀中加入新鲜的无血清培养基，以1×10 6/mL的细胞密度，置37℃5％CO 2 饱和湿度培养。3天后首次换液，弃去未贴壁细胞，以后每3天更换培养液1次。共6d，以备移植、传代或冻存。
第三种实施方式
请一并参阅图4，当所述间充质干细胞原代细胞为脐带间充质干细胞原代细胞时，所述获取间充质干细胞原代细胞包括：
步骤S11″：处理脐带：取脐带并剪断，剪断后的脐带总长度大于15cm，用生理盐水清洗后，再用含2倍双抗生理盐水去除血渍，接着去除血管与外膜，剥离华尔通胶并剪成0.5～1mm 3；及
步骤S12″：细胞扩增培养：将剪碎的华尔通胶涂布于培养皿上，加入无血清完全培养基，于35～40℃、5％CO 2及饱和湿度的条件下培养，培养7～10天即可得到脐带间充质干细胞原代细胞。
其中，在步骤S11″中，用生理盐水清洗后可以立即送至实验室进行稀释，而如需短暂保存，可以是短暂存放于4℃冰箱内。在步骤S12″中，加入无血清完全培养基后，优选为于37℃、5％CO 2及饱和湿度的条件下培养。
在该实施方式中，具体的实施例可以是：待胎盘娩出1分钟内，在靠近胎盘处结扎后剪断脐带，保证采集的脐带总长度不少于15cm。用生理盐水简单清洗后置一次性无菌采集容器内，拧紧瓶盖，短暂存放于4℃冰箱内(不超过24小时)或立即送至实验室。无菌镊子转移脐带至无菌培养皿中，用含2倍双抗生理盐水基本去除血渍。去除血管与外膜，剥离华尔通胶，剪成0.5～1mm 3。按照2～3cm长的脐带一皿的量，将上述剪碎的华尔通胶均匀涂布于10cm的培养皿上，室温5～10分钟贴附，后加入5ml完全培养基(无血清)，37℃、体积分数为5％的CO 2饱和湿度培养箱7～10天培养。
在步骤S2中，所述第一消化液优选为0.25％胰蛋白酶-EDTA。优选地，所述的进行离心分离为在800～1200转/分下离心6～10min。在一些具体实施例中为，原代细胞培养7天后换液(无血清DMEM(L))，至细胞70％融合，移去培养上清液至50ml离心管，培养瓶中加0.25％胰蛋白酶-EDTA3ml，消化1min，细胞收缩脱离瓶壁，加入先前移去的培养上清液稀释，使用移液管轻轻吹打， 将脐带间充质干细胞悬液移入50ml离心管。1000转/分离心8分钟，弃上清。重新加入完全培养基重悬，按照1传1～1.5接种10cm培养皿。培养4～5天后，细胞融合70％左右时传代培养，以1∶(6～8)传代至15cm培养皿中。
实施例二
一种间充质干细胞的冻存、复苏方法，将实施例一中所述间充质干细胞的分离培养方法所得到的间充质干细胞进行冻存及复苏，包括：
步骤T1：间充质干细胞的冻存：用细胞冻存液重悬所述间充质干细胞，然后置于装有异丙醇的容器中，于-90～-70℃下过夜后移入液氮罐；及
步骤T2：间充质干细胞的复苏：取冻存的间充质干细胞立即投入40～45℃水浴中融化，然后加入完全培养基并混匀，离心处理后弃去上清液，以完全培养基重悬后于35～40℃、5％CO 2及饱和湿度的条件下培养，待65～75％的细胞贴壁即可。
在步骤T1中，优选地，所述细胞冻存液由90％胎牛血清及10％DMSO组成。在一些具体实施例中，步骤T1为，用细胞冻存液(90％胎牛血清、10％DMSO)重悬间充质干细胞(MSCs)，充分混匀，每管1ml(1个15cm培养皿)分装于冻存管密封。在冻存管上做好标记，将冻存管置于装有异丙醇的冻存盒中，于-80℃冰箱过夜，次日移入液氮罐。
在步骤T2中，离心处理优选为在1000转/分下离心8分钟。以完全培养基重悬后优选为于37℃、5％CO 2及饱和湿度的条件下培养。在一些具体实施例中，步骤T2为，从液氮中取出MSCs，立即投入42℃水浴中融化。加入等体积完全培养基并混匀，1000转离心8分钟后，弃去上清液，以完全培养基重悬后于37℃、体积分数为5％的CO 2饱和湿度培养箱中培养，待70％细胞贴壁即可。

Claims (10)

  1. 一种间充质干细胞的分离培养方法，其特征在于：包括：
    获取间充质干细胞原代细胞；
    间充质干细胞原代细胞的培养扩增：间充质干细胞原代细胞培养5-10天后换成无血清DMEM营养液，至细胞65～75％融合，移去培养上清液，然后添加第一消化液，消化0.5-2min，细胞收缩后加入所移去的培养上清液中和后进行离心分离，重新加入完全培养基重悬，按照1传1～1.5接种，培养4～5天后，细胞65～75％融合时以1∶6～8的比例进行传代培养；及
    间充质干细胞的分离：收集间充质干细胞原代细胞的培养扩增中的处于P2或P3对数生长期且细胞融合程度不超过80％的细胞，按传代的要求消化、中和、洗涤后即可收集得到所需的间充质干细胞。
  2. 如权利要求1所述的间充质干细胞的分离培养方法，其特征在于：所述第一消化液为0.25％胰蛋白酶-EDTA。
  3. 如权利要求1所述的间充质干细胞的分离培养方法，其特征在于：所述的进行离心分离为在800～1200转/分下离心6～10min。
  4. 如权利要求1所述的间充质干细胞的分离培养方法，其特征在于：所述间充质干细胞原代细胞为骨髓间充质干细胞原代细胞或自体脂肪间充质干细胞原代细胞或脐带间充质干细胞原代细胞。
  5. 如权利要求4所述的间充质干细胞的分离培养方法，其特征在于：所述间充质干细胞原代细胞为骨髓间充质干细胞原代细胞，所述获取间充质干细胞原代细胞包括：
    抽取骨髓液并稀释：用含500U/ml肝素的PBS液的注射器抽取骨髓液，混合均匀后用含20U/ml肝素的PBS液稀释得到骨髓缓冲液；
    细胞的分离与纯化：将骨髓缓冲液中加入比重为1.073g/ml的淋巴细胞分离液上，通过离心分离纯化单个核细胞群，然后用DMEM培养液离心洗涤得到沉淀；及
    细胞扩增培养：将离心分离后得到的沉淀利用无血清培养液混悬，按5× 10 6/mL的密度进行接种并于35～40℃、5％CO 2及饱和湿度的条件下培养；培养3天后首次换液，弃去未贴壁细胞，以后每3d更换培养液1次，共培养6天即可得到骨髓间充质干细胞原代细胞。
  6. 如权利要求4所述的间充质干细胞的分离培养方法，其特征在于：所述间充质干细胞原代细胞为自体脂肪间充质干细胞原代细胞，所述获取间充质干细胞原代细胞包括：
    抽取自体脂肪颗粒并剪碎：抽取自体脂肪颗粒，剔除可见微血管及肌肉组织，用PBS进行洗涤，然后将脂肪组织剪碎至糊状使其小于1mm 3；
    细胞的分离与纯化：往糊状的脂肪组织中添加第二消化液进行消化，消化后过滤收集滤液，然后使用含有10％FBS的低糖DMEM培养基进行中和，再离心得到沉淀；及
    细胞扩增培养：往经过细胞的分离与纯化后得到的沉淀中加入无血清培养基，以1×10 6/mL的密度进行接种，并于35～40℃、5％CO 2及饱和湿度的条件下培养；培养3天后首次换液，弃去未贴壁细胞，以后每3d更换培养液1次，共培养6天即可得到自体脂肪间充质干细胞原代细胞。
  7. 如权利要求6所述的间充质干细胞的分离培养方法，其特征在于：所述第二消化液为含有0.1％胶原酶I型和0.05％胰蛋白酶的无血清DMEM。
  8. 如权利要求4所述的间充质干细胞的分离培养方法，其特征在于：所述间充质干细胞原代细胞为脐带间充质干细胞原代细胞，所述获取间充质干细胞原代细胞包括：
    处理脐带：取脐带并剪断，剪断后的脐带总长度大于15cm，用生理盐水清洗后，再用含2倍双抗生理盐水去除血渍，接着去除血管与外膜，剥离华尔通胶并剪成0.5～1mm 3；及
    细胞扩增培养：将剪碎的华尔通胶涂布于培养皿上，加入无血清完全培养基，于35～40℃、5％CO 2及饱和湿度的条件下培养，培养7～10天即可得到脐带间充质干细胞原代细胞。
  9. 一种间充质干细胞的冻存、复苏方法，其特征在于：将权利要求1所述 间充质干细胞的分离培养方法所得到的间充质干细胞进行冻存及复苏，包括：
    间充质干细胞的冻存：用细胞冻存液重悬所述间充质干细胞，然后置于装有异丙醇的容器中，于-90～-70℃下过夜后移入液氮罐；及
    间充质干细胞的复苏：取冻存的间充质干细胞立即投入40～45℃水浴中融化，然后加入完全培养基并混匀，离心处理后弃去上清液，以完全培养基重悬后于35～40℃、5％CO 2及饱和湿度的条件下培养，待65～75％的细胞贴壁即可。
  10. 如权利要求9所述的间充质干细胞的冻存、复苏方法，其特征在于：所述间充质干细胞的冻存中的细胞冻存液由90％胎牛血清及10％DMSO组成。
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