CN112029713A - Chorionic mesenchymal stem cell isolation culture amplification method - Google Patents
Chorionic mesenchymal stem cell isolation culture amplification method Download PDFInfo
- Publication number
- CN112029713A CN112029713A CN202010864039.3A CN202010864039A CN112029713A CN 112029713 A CN112029713 A CN 112029713A CN 202010864039 A CN202010864039 A CN 202010864039A CN 112029713 A CN112029713 A CN 112029713A
- Authority
- CN
- China
- Prior art keywords
- mesenchymal stem
- stem cells
- culture
- chorionic
- tissue
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 210000002901 mesenchymal stem cell Anatomy 0.000 title claims abstract description 95
- 238000000034 method Methods 0.000 title claims abstract description 39
- 230000003321 amplification Effects 0.000 title claims abstract description 17
- 238000003199 nucleic acid amplification method Methods 0.000 title claims abstract description 17
- 238000002955 isolation Methods 0.000 title description 6
- 238000004140 cleaning Methods 0.000 claims abstract description 26
- 238000000926 separation method Methods 0.000 claims abstract description 13
- 238000012258 culturing Methods 0.000 claims abstract description 12
- 210000004027 cell Anatomy 0.000 claims description 62
- 210000001136 chorion Anatomy 0.000 claims description 49
- 210000001519 tissue Anatomy 0.000 claims description 48
- 239000000243 solution Substances 0.000 claims description 46
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 34
- 239000012894 fetal calf serum Substances 0.000 claims description 34
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 claims description 26
- 210000003954 umbilical cord Anatomy 0.000 claims description 21
- 210000001691 amnion Anatomy 0.000 claims description 17
- 238000004113 cell culture Methods 0.000 claims description 17
- 210000005059 placental tissue Anatomy 0.000 claims description 16
- 239000008055 phosphate buffer solution Substances 0.000 claims description 14
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 13
- 229930182555 Penicillin Natural products 0.000 claims description 13
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims description 13
- 229960001484 edetic acid Drugs 0.000 claims description 13
- 238000011081 inoculation Methods 0.000 claims description 13
- 229940049954 penicillin Drugs 0.000 claims description 13
- 229960005322 streptomycin Drugs 0.000 claims description 13
- 238000005406 washing Methods 0.000 claims description 13
- 230000004927 fusion Effects 0.000 claims description 12
- 238000001514 detection method Methods 0.000 claims description 11
- 230000000694 effects Effects 0.000 claims description 9
- 238000013441 quality evaluation Methods 0.000 claims description 9
- 230000004069 differentiation Effects 0.000 claims description 8
- 238000011156 evaluation Methods 0.000 claims description 8
- 238000010008 shearing Methods 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 208000007536 Thrombosis Diseases 0.000 claims description 7
- 210000004369 blood Anatomy 0.000 claims description 7
- 239000008280 blood Substances 0.000 claims description 7
- 239000012535 impurity Substances 0.000 claims description 7
- 108010019160 Pancreatin Proteins 0.000 claims description 6
- 239000002777 nucleoside Substances 0.000 claims description 6
- 150000003833 nucleoside derivatives Chemical class 0.000 claims description 6
- 229940055695 pancreatin Drugs 0.000 claims description 6
- 241000204031 Mycoplasma Species 0.000 claims description 5
- 230000001580 bacterial effect Effects 0.000 claims description 5
- 210000003754 fetus Anatomy 0.000 claims description 5
- 238000003908 quality control method Methods 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 230000007480 spreading Effects 0.000 claims description 2
- 238000003892 spreading Methods 0.000 claims description 2
- 230000029087 digestion Effects 0.000 description 17
- 210000000130 stem cell Anatomy 0.000 description 12
- 230000008569 process Effects 0.000 description 9
- 210000002826 placenta Anatomy 0.000 description 8
- 230000006870 function Effects 0.000 description 6
- 230000035755 proliferation Effects 0.000 description 6
- 238000005520 cutting process Methods 0.000 description 5
- 230000009286 beneficial effect Effects 0.000 description 4
- 230000008827 biological function Effects 0.000 description 4
- 230000007774 longterm Effects 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 102100022464 5'-nucleotidase Human genes 0.000 description 3
- 102100037241 Endoglin Human genes 0.000 description 3
- 101000678236 Homo sapiens 5'-nucleotidase Proteins 0.000 description 3
- 101000881679 Homo sapiens Endoglin Proteins 0.000 description 3
- 230000007365 immunoregulation Effects 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 description 2
- 102000006354 HLA-DR Antigens Human genes 0.000 description 2
- 108010058597 HLA-DR Antigens Proteins 0.000 description 2
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 2
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 2
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 2
- 101001046686 Homo sapiens Integrin alpha-M Proteins 0.000 description 2
- 101000800116 Homo sapiens Thy-1 membrane glycoprotein Proteins 0.000 description 2
- 102100022338 Integrin alpha-M Human genes 0.000 description 2
- 102100033523 Thy-1 membrane glycoprotein Human genes 0.000 description 2
- 230000009815 adipogenic differentiation Effects 0.000 description 2
- 210000001185 bone marrow Anatomy 0.000 description 2
- 230000009816 chondrogenic differentiation Effects 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 210000003716 mesoderm Anatomy 0.000 description 2
- 230000009818 osteogenic differentiation Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 1
- 102100037850 Interferon gamma Human genes 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- RGCKGOZRHPZPFP-UHFFFAOYSA-N alizarin Chemical group C1=CC=C2C(=O)C3=C(O)C(O)=CC=C3C(=O)C2=C1 RGCKGOZRHPZPFP-UHFFFAOYSA-N 0.000 description 1
- 230000000735 allogeneic effect Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 230000009194 climbing Effects 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 102000038379 digestive enzymes Human genes 0.000 description 1
- 108091007734 digestive enzymes Proteins 0.000 description 1
- 210000003981 ectoderm Anatomy 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 210000001654 germ layer Anatomy 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 208000024908 graft versus host disease Diseases 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 210000000822 natural killer cell Anatomy 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 210000001428 peripheral nervous system Anatomy 0.000 description 1
- 230000003169 placental effect Effects 0.000 description 1
- 238000005498 polishing Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 210000001525 retina Anatomy 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 238000013517 stratification Methods 0.000 description 1
- 230000010415 tropism Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
Images
Classifications
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Developmental Biology & Embryology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Rheumatology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses a chorionic mesenchymal stem cell separation culture and amplification method, which comprises the steps of 1) cleaning, 2) treating, 3) culturing and 4) inoculating and subculturing.
Description
Technical Field
The invention relates to the technical field of stem cell isolated culture, in particular to a chorionic mesenchymal stem cell isolated culture amplification method.
Background
Mesenchymal Stem Cells (MSCs) are a population of cells with the potential for self-renewal, high proliferation and multipotentiality, and are the cells of origin for animal organisms and various tissue organs. Recent studies have shown that mesenchymal stem cells are present in numerous adult tissues (e.g., bone marrow, muscle, gastrointestinal tract, skin, retina, peripheral and central nervous system, etc.). The MSCs have wide sources, are easy to separate and amplify in vitro, and have multidirectional differentiation potential to three germ layer cells; has low immunogenicity, no ability to stimulate allogeneic immune response, no killing by cytotoxic T cells and NK cells, and has repairing, tumor tropism and immunoregulation effects.
Mesenchymal stem cells are derived from the mesoderm and ectoderm in the early stages of development, while the main part of the placenta is the amnion and chorion mesoderm, which theoretically confirms the possibility of containing mesenchymal stem cells in the placenta. Therefore, the placental chorionic mesenchymal stem cells (CV-MSCs) can be obtained by amplification from placenta, express the characteristics of the mesenchymal stem cells from biological characteristics, proliferation capacity and surface markers, have the potential of differentiation into cell lines such as bones and fat, have the immunoregulation function similar to the bone marrow mesenchymal stem cells (BM-MSCs) and can effectively inhibit peripheral blood mononuclear cells from secreting IFN-gamma. Compared with umbilical cord mesenchymal stem cells, the human umbilical cord mesenchymal stem cells have stronger immunoregulation function, so the human umbilical cord mesenchymal stem cells have better prospect in treating various GVHD and autoimmune diseases. At present, methods commonly adopted for separating chorionic mesenchymal stem cells are a tissue mass adherence method and an enzyme digestion method.
The economic cost of enzyme digestion separation is high, the digestion time is long, generally 4-6 hours are needed, the longest digestion time is 16 hours, the size of a tissue block can influence the digestion effect of cells, if the digestion is insufficient, a sufficient number of cells cannot be obtained, and the digestion time is too long, the damage of digestive enzymes to the cells is too large, and a large number of cells die. The tissue block culture method primary culture scheme is simple and easy to operate, and has obvious advantages in the aspects of shortening the operation time, reducing the cost, and being high in cell yield.
Disclosure of Invention
The invention provides a separation, culture and amplification method of chorionic mesenchymal stem cells.
The scheme of the invention is as follows:
a chorionic mesenchymal stem cell isolation culture amplification method comprises the following steps:
1) cleaning up
After the fetus is delivered, obtaining placenta tissues under an aseptic condition, washing the surface of the placenta by using a cleaning solution, and cleaning and removing residual blood and other impurities;
2) treatment of
And (3) cutting a cross incision of the cleaned placenta tissue by taking the root of the umbilical cord as a cross center, slightly stripping the amniotic membrane layer, separating the umbilical cord from the amniotic membrane, and carefully separating the chorion.
3) Repeatedly washing the stripped chorion in PBS (phosphate buffer solution) containing 100U/ml penicillin and 100ug/ml streptomycin to remove blood clots, and shearing chorion tissue into pieces with the size of 1-3 cm by using an aseptic tissue shear3. The chorion is a thin layer membrane-like tissue block, the operation method is simple, and the time is saved;
4) culturing
Flatly paving the chorion tissue block in a cell culture dish, and adding the chorion tissue block into a culture solution containing 10-20% fetal calf serum for culture after water is volatilized; the operation is characterized in that the tissue block is small, the tissue adherence is firm, the mesenchymal stem cells can climb out of the tissue block, and a large amount of primary cells can be obtained;
5) inoculating and subculturing, wherein after the growth of the primary separated mesenchymal stem cells of the chorionic tissue block reaches 80-90% fusion, subculturing and expanding culture can be carried out to obtain chorionic mesenchymal stem cells, digesting the cells with 0.25% pancreatin/EDTA (ethylene diamine tetraacetic acid) according to the ratio of 5000-7000 cells/cm2And (3) inoculating, wherein 95% of fusion can be achieved 4-5 days after the chorion mesenchymal stem cells are inoculated, and more than 95% of the chorion mesenchymal stem cells can be expanded to more than 20 generations. The digestion time is short, the damage to cells in the digestion process is small, the inoculation density can well control the growth cycle of the cells, and the chorionic mesenchymal stem cells can be subcultured according to the digestion and the inoculation densityTo amplify for more than 20 generations.
As a preferable technical scheme, the cleaning solution in the step 1) contains phosphate buffer solution of penicillin and streptomycin.
As a preferable technical scheme, the culture solution of 10-20% fetal calf serum in the step 3) contains the alpha-MEM basic culture solution containing nucleoside and the fetal calf serum, which is more beneficial to the separation and proliferation of stem cells, and can better maintain the dryness and biological functions of the stem cells in the long-term amplification culture process.
As an optimal technical scheme, in the step 3), the culture solution of 10-20% fetal calf serum is changed once every 3 days after the cells are cultured in the cell culture dish, then the culture solution of 10-20% fetal calf serum is changed every 2-3 days after the cells are attached to the wall, the chorionic mesenchymal stem cells can be seen to climb out of the tissue block after 4-5 days, and the chorionic mesenchymal stem cells can be fused by 80-90% after 7-10 days.
As a preferable technical scheme, the using amount of the culture solution containing 10-20% of fetal calf serum in the step 3) is 20-50 ml/dish.
As an improvement of the technical scheme, the method also comprises the step of evaluating the quality of the mesenchymal stem cells obtained in the step 5), wherein the quality evaluation comprises cell quantity, cell activity, cell surface markers, differentiation function, bacterial detection, mycoplasma detection and safety evaluation
The method is adopted to establish a production standard, a quality control system and an evaluation system of chorionic mesenchymal stem cells.
Due to the adoption of the technical scheme, the chorionic mesenchymal stem cell separation culture amplification method comprises the steps of 1) cleaning, obtaining placenta tissues under an aseptic condition, washing the surface of the placenta with cleaning fluid, and cleaning to remove residual blood and other impurities; 2) and (3) processing, namely cutting a cross incision on the cleaned placenta tissue by taking the root of the umbilical cord as a cross center, slightly stripping the amniotic membrane layer, separating the umbilical cord from the amniotic membrane, and carefully separating the chorion. Repeatedly washing the stripped chorion in PBS containing double antibody (100U/ml penicillin, 100ug/ml streptomycin) to remove blood clot, and shearing chorion tissue into 1cm with aseptic tissue scissors3Left and right size(ii) a 3) Culturing, namely flatly paving the chorion tissue blocks in a cell culture dish, and adding the chorion tissue blocks into a culture solution containing 10-20% fetal calf serum for culturing after water is volatilized; 4) inoculating and subculturing, wherein after 80-90% of mesenchymal stem cells in the primary separation culture of the chorionic tissue mass are fused, subculturing and amplification culture can be carried out to obtain chorionic mesenchymal stem cells, digesting the cells with 0.25% of pancreatin/EDTA (ethylene diamine tetraacetic acid) according to the ratio of 5000-7000 cells/cm2Inoculating, wherein 95% of fusion can be achieved 4-5 days after the chorionic mesenchymal stem cells are inoculated, and more than 95% of the chorionic mesenchymal stem cells can be expanded to more than 20 generations; the method for obtaining the chorionic mesenchymal stem cells by the tissue mass separation method is simple, low in cost, good in cell activity and high in purity, the concentration of the finally obtained cells is high, the obtained mesenchymal stem cells are simple, the culture time is shortened, the cells can be seen to climb out of the tissue mass after being cultured for 4-5 days, the concentration of the finally obtained cells is high, the whole stem cell separation, preparation and culture process can quickly and efficiently obtain the high-purity mesenchymal stem cells from the chorionic tissue, the cell quality completely meets the national promulgated stem cell preparation quality control and preclinical research guide principle (trial), and the guarantee is provided for clinical application; the method comprises the steps of obtaining placenta tissues, cleaning, gently polishing a glass amniotic membrane layer, separating an umbilical cord and an amniotic membrane, separating the chorion, and controlling the size of a tissue block to be 1-3 cm3The cell culture dish is adopted for spreading, and the solution is replenished after the water is completely volatilized, so that the yield of the primary cells is obviously improved.
Drawings
FIG. 1 is the chorion tissue stratification of example 3;
FIG. 2 is the peeled chorion of example 3;
FIG. 3 is a climbing out of the tissue mass of umbilical cord mesenchymal stem cells of example 3;
FIG. 4 is a drawing of the umbilical cord mesenchymal stem cell P10 flow-generation detection of Negative, CD90, CD105 and CD73 in example 3;
FIG. 5 is a graph of identification of the multipotentiality of umbilical cord mesenchymal stem cells of example 4;
Detailed Description
In order to make up for the above deficiencies, the present invention provides a chorionic mesenchymal stem cell isolation, culture and amplification method to solve the above problems in the background art.
A chorionic mesenchymal stem cell isolation culture amplification method comprises the following steps:
1) cleaning, namely obtaining placenta tissues under an aseptic condition after delivery of the fetus, washing the surface of the placenta by using a cleaning solution, and cleaning and removing residual blood and other impurities;
2) and (3) processing, namely cutting a cross incision on the cleaned placenta tissue by taking the root of the umbilical cord as a cross center, slightly stripping the amniotic membrane layer, separating the umbilical cord from the amniotic membrane, and carefully separating the chorion. Repeatedly washing the stripped chorion in PBS containing double antibody (100U/ml penicillin, 100ug/ml streptomycin) to remove blood clot, and shearing chorion tissue into 1cm with aseptic tissue scissors3Left and right size. The chorion is a thin layer membrane-like tissue block, the operation method is simple, and the time is saved;
3) culturing, namely flatly paving the chorion tissue blocks in a cell culture dish, and adding the chorion tissue blocks into a culture solution containing 10-20% fetal calf serum for culturing after water is volatilized; the operation is characterized in that the tissue block is small, the tissue adherence is firm, the mesenchymal stem cells can climb out of the tissue block, and a large amount of primary cells can be obtained;
4) inoculating and subculturing, wherein after the growth of the primary separated mesenchymal stem cells of the chorionic tissue block reaches 80-90% fusion, subculturing and expanding culture can be carried out to obtain chorionic mesenchymal stem cells, digesting the cells with 0.25% pancreatin/EDTA (ethylene diamine tetraacetic acid) according to the ratio of 5000-7000 cells/cm2And (3) inoculating, wherein 95% of fusion can be achieved 4-5 days after the chorion mesenchymal stem cells are inoculated, and more than 95% of the chorion mesenchymal stem cells can be expanded to more than 20 generations. The digestion time is short, the damage to cells in the digestion process is small, the inoculation density can well control the growth cycle of the cells, and the chorionic mesenchymal stem cells can be amplified for more than 20 generations after the chorionic mesenchymal stem cells are subjected to inoculation density passage according to the digestion.
The cleaning solution in the step 1) contains phosphate buffer solution of penicillin and streptomycin.
The culture solution of 10-20% fetal calf serum in the step 3) contains alpha-MEM basic culture solution containing nucleoside and fetal calf serum, which is more beneficial to separation and proliferation of stem cells, and can better maintain the dryness and biological functions of the stem cells in the long-term amplification culture process.
Further comprising the step of performing quality evaluation on the mesenchymal stem cells obtained in the step 4), wherein the quality evaluation comprises cell number, cell activity, cell surface markers, differentiation function, bacterial detection, mycoplasma detection and safety evaluation.
And 3) replacing the culture solution of 10-20% fetal calf serum once every 3 days of cell culture in the cell culture dish in the step 3), replacing the culture solution of 10-20% fetal calf serum every 2-3 days, allowing the chorionic mesenchymal stem cells to climb out of the tissue block after adhering to the wall for 4-5 days, and allowing the chorionic mesenchymal stem cells to reach 80-90% fusion after 7-10 days.
The amount of the culture solution containing 10-20% fetal calf serum in the step 3) is 20-50 ml/dish.
The method is adopted to establish a production standard, a quality control system and an evaluation system of chorionic mesenchymal stem cells.
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.
Example 1:
1) cleaning, namely obtaining placenta tissues under an aseptic condition after delivery of the fetus, washing the surface of the placenta by using a cleaning solution, and cleaning and removing residual blood and other impurities;
2) and (3) processing, namely cutting a cross incision on the cleaned placenta tissue by taking the root of the umbilical cord as a cross center, slightly stripping the amniotic membrane layer, separating the umbilical cord from the amniotic membrane, and carefully separating the chorion. Repeatedly washing the stripped chorion in PBS containing 100U/ml penicillin and 100ug/ml streptomycin to remove blood clot, and shearing chorion tissue into pieces of 2cm in size by aseptic tissue scissors3. The chorion is a thin layer membrane-like tissue block, the operation method is simple, and the time is saved;
3) culturing, namely flatly paving the chorion tissue blocks in a cell culture dish, and adding the chorion tissue blocks into a culture solution containing 10-20% fetal calf serum for culturing after water is volatilized; the operation is characterized in that the tissue block is small, the tissue adherence is firm, the mesenchymal stem cells can climb out of the tissue block, and a large amount of primary cells can be obtained;
4) inoculating and subculturing, wherein after the growth of the primary separated mesenchymal stem cells of the chorionic tissue block reaches 80-90% and the cells are fused, subculturing can be carried out to obtain chorionic mesenchymal stem cells, the cells are digested by 0.25% pancreatin/EDTA (ethylene diamine tetraacetic acid) according to the ratio of 5000 cells/cm2And (3) inoculating, wherein 95% of fusion can be achieved 4-5 days after the chorion mesenchymal stem cells are inoculated, and more than 95% of the chorion mesenchymal stem cells can be expanded to more than 20 generations. The digestion time is short, the damage to cells in the digestion process is small, the inoculation density can well control the growth cycle of the cells, and the chorionic mesenchymal stem cells can be amplified for more than 20 generations after the chorionic mesenchymal stem cells are subjected to inoculation density passage according to the digestion.
The cleaning solution in the step 1) contains phosphate buffer solution of penicillin and streptomycin.
The culture solution of 10-20% fetal calf serum in the step 3) contains alpha-MEM basic culture solution containing nucleoside and fetal calf serum, which is more beneficial to separation and proliferation of stem cells, and can better maintain the dryness and biological functions of the stem cells in the long-term amplification culture process.
Further comprising the step of performing quality evaluation on the mesenchymal stem cells obtained in the step 4), wherein the quality evaluation comprises cell number, cell activity, cell surface markers, differentiation function, bacterial detection, mycoplasma detection and safety evaluation.
And 3) replacing the culture solution of 10% fetal calf serum once every 3 days after cell culture in the cell culture dish in the step 3), replacing the culture solution of 10% fetal calf serum every 2-3 days after wall adhesion, and allowing the chorionic mesenchymal stem cells to climb out of the tissue block after 4-5 days of wall adhesion, wherein the chorionic mesenchymal stem cells can reach 80-90% fusion after 7-10 days.
In the step 3), 20ml of culture solution containing 10% fetal calf serum is added in each dish.
Example 2:
a chorionic mesenchymal stem cell isolation culture amplification method comprises the following steps:
1) cleaning, namely obtaining placenta tissues under an aseptic condition after delivery of the fetus, washing the surface of the placenta by using a cleaning solution, and cleaning and removing residual blood and other impurities;
2) and (3) processing, namely cutting a cross incision on the cleaned placenta tissue by taking the root of the umbilical cord as a cross center, slightly stripping the amniotic membrane layer, separating the umbilical cord from the amniotic membrane, and carefully separating the chorion. Repeatedly washing the stripped chorion in PBS containing 100U/ml penicillin and 100ug/ml streptomycin to remove blood clot, and shearing chorion tissue into pieces with size of 3cm by aseptic tissue scissors3. The chorion is a thin layer membrane-like tissue block, the operation method is simple, and the time is saved;
3) culturing, namely flatly paving the chorion tissue blocks in a cell culture dish, and adding the chorion tissue blocks into a culture solution containing 10-20% fetal calf serum for culturing after water is volatilized; the operation is characterized in that the tissue block is small, the tissue adherence is firm, the mesenchymal stem cells can climb out of the tissue block, and a large amount of primary cells can be obtained;
4) inoculating and subculturing, wherein after the growth of the primary separated mesenchymal stem cells of the chorionic tissue block reaches 80-90% and the cells are fused, subculturing can be carried out to obtain chorionic mesenchymal stem cells, the cells are digested by 0.25% pancreatin/EDTA (ethylene diamine tetraacetic acid) according to the ratio of 5000-7000 cells/cm2And (3) inoculating, wherein 95% of fusion can be achieved 4-5 days after the chorion mesenchymal stem cells are inoculated, and more than 95% of the chorion mesenchymal stem cells can be expanded to more than 20 generations. The digestion time is short, the damage to cells in the digestion process is small, the inoculation density can well control the growth cycle of the cells, and the chorionic mesenchymal stem cells can be amplified for more than 20 generations after the chorionic mesenchymal stem cells are subjected to inoculation density passage according to the digestion.
The cleaning solution in the step 1) contains phosphate buffer solution of penicillin and streptomycin.
The culture solution of 10-20% fetal calf serum in the step 3) contains alpha-MEM basic culture solution containing nucleoside and fetal calf serum, which is more beneficial to separation and proliferation of stem cells, and can better maintain the dryness and biological functions of the stem cells in the long-term amplification culture process.
Further comprising the step 5) of performing quality evaluation on the mesenchymal stem cells obtained in the step 4), wherein the quality evaluation comprises cell number, cell activity, cell surface markers, differentiation function, bacterial detection, mycoplasma detection and safety evaluation.
And 3) replacing the culture solution of 10% fetal calf serum once every 3 days after cell culture in the cell culture dish in the step 3), replacing the culture solution of 10% fetal calf serum every 2-3 days after wall adhesion, and allowing the chorionic mesenchymal stem cells to climb out of the tissue block after 4-5 days of wall adhesion, wherein the chorionic mesenchymal stem cells can reach 80-90% fusion after 7-10 days.
In the step 3), a culture solution containing 10% fetal calf serum is added in a volume of 50 ml/dish.
Example 3:
after screening infectious diseases and the like, signing an informed consent and a donation protocol, aseptically collecting placenta tissues after delivery, flushing the surface with Phosphate Buffer Solution (PBS) containing penicillin and streptomycin under an aseptic condition, and cleaning to remove blood and other impurities remained in blood vessels.
A cross incision was cut with the root of the umbilical cord as the center of the cross, the amniotic membrane layer was gently peeled off, and after separating the umbilical cord from the amniotic membrane, the chorion was carefully separated (see fig. 1). Repeatedly washing the stripped chorion in PBS containing double antibody (100U/ml penicillin, 100ug/ml streptomycin) to remove blood clot, and shearing chorion tissue into 1cm with aseptic tissue scissors3Tissue mass of size (see fig. 2).
The tissue is spread in a cell culture dish and after the water is evaporated, complete culture solution containing 10-20% fetal calf serum is added.
Changing the liquid every 2-3 days after culturing the cells, wherein the cells can be seen to climb out of the tissue block after adhering the walls for 4-5 days (see figure 3), and the chorionic mesenchymal stem cells can be fused by 80-90% after 7-10 days;
digesting the cells with 0.25% trypsin/EDTA at 5000-2Inoculation, and the cell can reach 95% fusion passage 4-5 days after inoculation.
The culture solution of 20% fetal calf serum contains alpha-MEM basic culture solution containing nucleoside and fetal calf serum.
The mesenchymal stem cell dryness maintenance is carried out on the chorion simple mesenchymal stem cells obtained in the example 3, and the stem cell surface antigens after the three culture solutions are cultured are analyzed in a surface marker flow mode, so that the results show that the homogeneity of the obtained MSCs is good. All express CD73, CD105, CD 90; does not express CD45, CD34, CD11b, CD19 and HLA-DR. The result is that the chorionic mesenchymal stem cells P10 are positive in expression of CD73, CD90 and CD105 and Negative in expression of Negative- -CD45, CD34, CD11b, CD19 and HLA-DR by flow assay, and is shown in figure 4.
Example 4:
performing multi-directional differentiation potential identification on the chorionic mesenchymal stem cells in example 3, wherein fig. 5A is osteogenic differentiation control, and fig. 5B is alizarin red staining after osteogenic differentiation; FIG. 5C is a adipogenic differentiation control, and FIG. 5D is oil red "O" staining after adipogenic differentiation; fig. 5E is a chondrogenic differentiation control and fig. 5F is an allothrin blue staining after chondrogenic differentiation.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (7)
1. A separation, culture and amplification method of mesenchymal stem cells of villus is characterized by comprising the following steps:
1) cleaning up
After the fetus is delivered, obtaining a placenta tissue under an aseptic condition, washing the surface of the placenta tissue by using a cleaning solution, and cleaning to remove residual blood and other impurities on the surface of the placenta tissue;
2) treatment of
Shearing a cross incision on the cleaned placenta tissue by taking the root of the umbilical cord as a cross center, slightly stripping the amniotic membrane layer, separating the umbilical cord from the amniotic membrane, and carefully separating the chorion;
3) repeatedly washing the stripped chorion in PBS containing 100U/ml penicillin and 100ug/ml streptomycin to remove blood clots;
4) culturing
Spreading chorion in a plate, and shearing chorion tissue into pieces with size of 1-3 cm by using sterile tissue scissors3Flatly paving the tissue block in a cell culture dish, and adding the tissue block into a culture solution containing 10-20% fetal calf serum for culture after water is volatilized;
5) passage of inoculation
After 80-90% of mesenchymal stem cells in the primary separation culture of chorionic tissues are fused, subculture can be performed to obtain chorionic mesenchymal stem cells, the cells are digested by 0.25% pancreatin/EDTA (ethylene diamine tetraacetic acid) according to the ratio of 5000-7000 cells/cm2And (3) inoculation, wherein after 4-5 days of inoculation, 95% of the mesenchymal stem cells can be fused, and more than 95% of the mesenchymal stem cells can be expanded to more than 20 generations.
2. The isolated culture and expansion method of chorionic mesenchymal stem cells according to claim 1, wherein: the cleaning solution in the step 1) contains phosphate buffer solution of penicillin and streptomycin.
3. The isolated culture and expansion method of chorionic mesenchymal stem cells according to claim 1, wherein: the culture solution of 10-20% fetal calf serum in the step 3) contains alpha-MEM basic culture solution containing nucleoside and fetal calf serum.
4. The isolated culture and expansion method of chorionic mesenchymal stem cells according to claim 1, wherein: and 3) replacing the culture solution of 10-20% fetal calf serum once after 3 days of cell culture in the cell culture dish in the step 3), replacing the culture solution of 10-20% fetal calf serum every 2-3 days later, allowing the chorionic mesenchymal stem cells to climb out of the tissue block after 4-5 days of wall attachment, and allowing the chorionic mesenchymal stem cells to reach 80-90% fusion after 7-10 days.
5. The isolated culture and expansion method of chorionic mesenchymal stem cells according to claim 1, wherein: the amount of the culture solution containing 10-20% fetal calf serum in the step 3) is 20-50 ml/dish.
6. The isolated culture and expansion method of chorionic mesenchymal stem cells according to claim 1, wherein: further comprising the step of performing quality evaluation on the mesenchymal stem cells obtained in the step 5), wherein the quality evaluation comprises cell number, cell activity, cell surface markers, differentiation function, bacterial detection, mycoplasma detection and safety evaluation.
7. The isolated culture and expansion method of chorionic mesenchymal stem cells according to any one of claims 1 to 6, characterized by: the method is adopted to establish a production standard, a quality control system and an evaluation system of chorionic mesenchymal stem cells.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010864039.3A CN112029713A (en) | 2020-08-25 | 2020-08-25 | Chorionic mesenchymal stem cell isolation culture amplification method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010864039.3A CN112029713A (en) | 2020-08-25 | 2020-08-25 | Chorionic mesenchymal stem cell isolation culture amplification method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112029713A true CN112029713A (en) | 2020-12-04 |
Family
ID=73581240
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010864039.3A Pending CN112029713A (en) | 2020-08-25 | 2020-08-25 | Chorionic mesenchymal stem cell isolation culture amplification method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112029713A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114149966A (en) * | 2021-12-08 | 2022-03-08 | 华夏源细胞工程集团股份有限公司 | Method for obtaining placenta mesenchymal stem cells |
| CN114752559A (en) * | 2022-06-14 | 2022-07-15 | 北京国卫生物科技有限公司 | Isolation culture amplification method of human placental chorionic mesenchymal stem cells |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110079498A (en) * | 2019-05-05 | 2019-08-02 | 山西省干细胞基因工程有限公司 | A kind of Human plactnta mescenchymal stem cell and its preparation method and application |
| CN110540959A (en) * | 2019-10-08 | 2019-12-06 | 孟明耀 | Umbilical cord mesenchymal stem cell isolation culture amplification method |
-
2020
- 2020-08-25 CN CN202010864039.3A patent/CN112029713A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110079498A (en) * | 2019-05-05 | 2019-08-02 | 山西省干细胞基因工程有限公司 | A kind of Human plactnta mescenchymal stem cell and its preparation method and application |
| CN110540959A (en) * | 2019-10-08 | 2019-12-06 | 孟明耀 | Umbilical cord mesenchymal stem cell isolation culture amplification method |
Non-Patent Citations (1)
| Title |
|---|
| 马榕 等: "胎盘绒毛间充质干细胞在大鼠急性心肌梗死修复中的作用", 南通大学学报（医学版) * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114149966A (en) * | 2021-12-08 | 2022-03-08 | 华夏源细胞工程集团股份有限公司 | Method for obtaining placenta mesenchymal stem cells |
| CN114752559A (en) * | 2022-06-14 | 2022-07-15 | 北京国卫生物科技有限公司 | Isolation culture amplification method of human placental chorionic mesenchymal stem cells |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110540959A (en) | Umbilical cord mesenchymal stem cell isolation culture amplification method | |
| CN105238751B (en) | Isolated culture method of umbilical cord tissue mesenchymal stem cells | |
| CN106754674B (en) | The method and its application of amnion mesenchymal stem cell are prepared from Human plactnta amnion | |
| US7915039B2 (en) | Method for obtaining mesenchymal stem cells | |
| CN105765060B (en) | Method for producing amniotic mesenchymal cell composition, cryopreservation method, and therapeutic agent | |
| US9139813B2 (en) | Renovation and repopulation of decellularized tissues and cadaveric organs by stem cells | |
| CN109234229B (en) | Method for separating mesenchymal stem cells from placental blood vessels and digestive enzyme composition used in same | |
| CN101629165B (en) | Preparation method of original mesenchymal stem cell | |
| CN108220229B (en) | Preparation method for improving output of umbilical cord source mesenchymal stem cell primary cells | |
| CN113249317A (en) | Isolated culture and amplification method and system for human umbilical cord mesenchymal stem cells | |
| CN108685948B (en) | Preparation method of medical cell repairing agent | |
| CN104762257B (en) | A kind of method preparing mescenchymal stem cell from umbilical cord | |
| CN112029713A (en) | Chorionic mesenchymal stem cell isolation culture amplification method | |
| CN108865988A (en) | A kind of separation of human amnion mesenchymal stem cell, culture and purification process | |
| CN111346051A (en) | Preparation method of umbilical cord mesenchymal stem cell injection for treating cerebral infarction | |
| Widowati et al. | Comparative analysis of Wharton’s Jelly mesenchymal stem cell (WJ-MSCs) isolated using explant and enzymatic methods | |
| CN115851587A (en) | Optimized culture medium, kit and culture method of human placenta-derived mesenchymal stem cells | |
| CN107354130B (en) | Human placenta chorion mesenchymal stem cell separation method | |
| CN109628388B (en) | Isolation of mesenchymal stem cells from placental blood vessels with digestive enzyme composition | |
| CN104726403A (en) | Method for separating and culturing human placental-derived mesenchymal stem cell | |
| CN114752559B (en) | Isolation culture amplification method of human placental chorionic mesenchymal stem cells | |
| CN111979188A (en) | Placenta mesenchymal stem cell isolation culture amplification method | |
| RU2645255C1 (en) | Method for obtaining of biosafe culture of mesenchimal stem cells from human chorionic villae | |
| WO2022056991A1 (en) | Mesenchymal stem cells derived from umbilical cord, and preparation method therefor and use thereof | |
| CN109771697B (en) | Dermal fibroblast skin sheet and construction method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20240606 Address after: Room 50350, Building D, Phase II, New Drug Creation Base, No.1 Yaocheng Avenue, Medical High tech Industrial Development Zone, Taizhou City, Jiangsu Province, 225316 Applicant after: Jiangsu Saiyi Biotechnology Co.,Ltd. Country or region after: China Address before: Building C2-1, Building 16, Shuwu, No. 73 Tanmi Road, Jiangbei New District, Nanjing City, Jiangsu Province, 211500 Applicant before: Jiangsu Saiyi Cell Technology Research Institute Co.,Ltd. Country or region before: China |
|
| TA01 | Transfer of patent application right |