CN113106058A - Screening and identifying method of human umbilical cord-derived Muse cells - Google Patents
Screening and identifying method of human umbilical cord-derived Muse cells Download PDFInfo
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- CN113106058A CN113106058A CN202110206416.9A CN202110206416A CN113106058A CN 113106058 A CN113106058 A CN 113106058A CN 202110206416 A CN202110206416 A CN 202110206416A CN 113106058 A CN113106058 A CN 113106058A
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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
- C12N5/0668—Mesenchymal stem cells from other natural sources
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- 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
- C12N5/0653—Adipocytes; Adipose tissue
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0654—Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
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- 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
Abstract
The invention discloses a screening and identifying method of human umbilical cord-derived Muse cells, which comprises the steps of determining participators, determining experimental instruments and reagents, sorting Muse cells, amplifying the Muse cells in vitro and identifying the Muse cells. The invention has the beneficial effects that: sorting primary cultured dermal fibroblast SSEA-3 positive cells by a flow cytometer, culturing Muse cell clusters under the conditions of MC culture medium and poly-HEMA coating plate, and observing the self-renewal capacity of suspension-adherence-suspension, proliferation and amplification. The cells can be differentiated into fat cells and bone cells by the induction of the 3 rd generation Muse cells through bone and fat formation. The project is proved to successfully establish a human umbilical cord source Muse cell culture scheme.
Description
Technical Field
The invention relates to a screening and identifying method of human umbilical cord-derived Muse cells, in particular to a screening and identifying method of human umbilical cord-derived Muse cells, belonging to the technical field of cell research.
Background
With the development of stem cell technology in the treatment of disease, the promotion of stem cell technology to human health is increasingly recognized. In recent years, Muse cells derived from mesenchymal stem cells have the characteristics of strong anti-stress capability, good homing effect, low tumorigenicity and the like in the aspect of stem cell transplantation, and open a new direction for further improving the stem cell treatment technology. The umbilical cord mesenchymal stem cells have the advantages of rich sources, convenient material acquisition, no wound to a donor, easy separation culture and in-vitro amplification, less ethical limitation, more original cells and the like, so that the extraction, purification and amplification of Muse cells are more effective. At present, related reports on culture of Muse cells in umbilical cord source mesenchymal stem cells are rare, and researches on screening, identification and the like of the cells are also rarely reported. The prospect of separating and culturing Muse cells from umbilical cord tissues is wide, so a complete and stable operation process is set up through experiments in the project to obtain the Muse cells with higher purity. Provides a certain reference for clinical application of Muse cells in mesenchymal stem cell treatment tissue regeneration, immunoregulation and the like in future.
Disclosure of Invention
The invention aims to provide a method for screening and identifying human umbilical cord-derived Muse cells in order to solve the problems.
The invention realizes the purpose through the following technical scheme: a method for screening and identifying human umbilical cord-derived Muse cells comprises the following steps:
step one, determining participators.
And step two, determining experimental instruments and reagents.
And step three, sorting Muse cells, namely sorting primary cultured dermal fibroblast SSEA-3 positive cells by a flow cytometer, culturing Muse cell clusters under the conditions of MC culture medium and poly-HEMA coated plate, and observing the self-renewal capacity of suspension-adherence-suspension, proliferation and amplification.
And step four, in-vitro amplification of the Muse cells, wherein the Muse cells obtained by separation are amplified by adopting a suspension culture-adherent culture-suspension culture mode.
Step five, identifying Muse cells, namely sorting anti-SSEA-3 antibody positive cells (Muse cells) from cultured umbilical cord-derived mesenchymal cells by using FACS (FACS); the alkaline phosphatase staining was performed, and the pluripotency was examined by immunofluorescence staining, reverse transcription-polymerase chain reaction (RT-PCR) and Western-blot.
As a still further scheme of the invention: in the second step, the experimental apparatus and the reagent comprise the following components:
flow cytometry, a CO2 incubator, a desktop centrifuge, a biological safety cabinet, a microscope, a fluorescence microscope and a photographic system, a microplate reader mesenchymal stem cell serum-free conditioned medium (Hangzhou baitong), 0.25% pancreatin (Ginogo), Anti-CD29, Anti-CD45, Anti-CD90, Anti-CD105 (BD Biosciences), Anti-CD34 (Santa Cruz Biotechnology), Anti-CD44 (Abcam), a cell cycle instant detection kit (Kaiki Biosciences), osteogenic and adipogenic induced differentiation culture solution, alizarin red, oil red O Cyagen, Anti-SSEA3, high-glucose DMEM and fetal bovine serum.
As a still further scheme of the invention: in the fourth step, the in vitro amplification of Muse cells comprises the following steps:
(1) the obtained MSC cells were passed through a 200 mesh cell filter. Incubation with SSEA 3-FITC-conjugated antibody on a shaker for 20 min at room temperature;
(2) human Ig G-FITC was used as an isotype control, PI staining excluded dead cells in FACS analysis, and the individual cells obtained were sorted;
(3) culturing the cells in a Poly-HEMA coated 6-well plate in a suspension manner by using an MC culture medium, adding 500ul of the MC culture medium every two days, and centrifuging at a low speed of 1000 rpm for 3 minutes, 800 rpm for 5 minutes and 600 rpm for 5 minutes after four days to remove dead single cells;
(4) and then continuing suspension culture, after 7 days, centrifuging and re-planting the Muse cell clusters which do not grow up to a 6-well plate coated with matrigel, observing the growth condition after the Muse cell clusters are attached to the wall, digesting the cells with pancreatin after the cells reach 90 percent fusion, and then re-inoculating the cells to the 6-well plate coated with Poly-HEMA for suspension culture by using an MC culture medium.
As a still further scheme of the invention: in the fifth step, the identification of Muse cells comprises the following steps:
(1) and taking 3 rd generation cells with good growth state to respectively perform the following identification:
(2) detecting the activity of the cells by a CCK-8 method;
(3) detecting the cell cycle by a flow cytometer;
(4) flow cytometry detection of cell surface CD29, CD34, CD44, CD45, CD90, CD105 and SSEA3 antigens;
(5) and adding adipogenic and osteogenic induced differentiation liquid to detect adipogenic and osteogenic induced differentiation capacity.
The invention has the beneficial effects that: the screening and identifying method of the human umbilical cord-derived Muse cells is reasonable in design, primary cultured dermal fibroblast SSEA-3 positive cells are sorted by a flow cytometer, Muse cell clusters are cultured under the conditions of MC culture medium and poly-HEMA coating plate, and the self-renewal capacity of suspension-adherence-suspension, proliferation and amplification is observed. The cells can be differentiated into fat cells and bone cells by the induction of the 3 rd generation Muse cells through bone and fat formation. The project is proved to successfully establish a human umbilical cord source Muse cell culture scheme.
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FIG. 1 is a schematic flow chart of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to fig. 1, a method for screening and identifying human umbilical cord-derived Muse cells includes the following steps:
step one, determining participators.
And step two, determining experimental instruments and reagents.
And step three, sorting Muse cells, namely sorting primary cultured dermal fibroblast SSEA-3 positive cells by a flow cytometer, culturing Muse cell clusters under the conditions of MC culture medium and poly-HEMA coated plate, and observing the self-renewal capacity of suspension-adherence-suspension, proliferation and amplification.
And step four, in-vitro amplification of the Muse cells, wherein the Muse cells obtained by separation are amplified by adopting a suspension culture-adherent culture-suspension culture mode.
Step five, identifying Muse cells, namely sorting anti-SSEA-3 antibody positive cells (Muse cells) from cultured umbilical cord-derived mesenchymal cells by using FACS (FACS); the alkaline phosphatase staining was performed, and the pluripotency was examined by immunofluorescence staining, reverse transcription-polymerase chain reaction (RT-PCR) and Western-blot.
Further, in the embodiment of the present invention, in the second step, the laboratory instruments and the reagents include the following:
flow cytometry, a CO2 incubator, a desktop centrifuge, a biological safety cabinet, a microscope, a fluorescence microscope and a photographic system, a microplate reader mesenchymal stem cell serum-free conditioned medium (Hangzhou baitong), 0.25% pancreatin (Ginogo), Anti-CD29, Anti-CD45, Anti-CD90, Anti-CD105 (BD Biosciences), Anti-CD34 (Santa Cruz Biotechnology), Anti-CD44 (Abcam), a cell cycle instant detection kit (Kaiki Biosciences), osteogenic and adipogenic induced differentiation culture solution, alizarin red, oil red O Cyagen, Anti-SSEA3, high-glucose DMEM and fetal bovine serum.
Further, in the present embodiment, in the fourth step, the in vitro amplification of Muse cells comprises the following steps:
(1) the obtained MSC cells were passed through a 200 mesh cell filter. Incubation with SSEA 3-FITC-conjugated antibody on a shaker for 20 min at room temperature;
(2) human Ig G-FITC was used as an isotype control, PI staining excluded dead cells in FACS analysis, and the individual cells obtained were sorted;
(3) culturing the cells in a Poly-HEMA coated 6-well plate in a suspension manner by using an MC culture medium, adding 500ul of the MC culture medium every two days, and centrifuging at a low speed of 1000 rpm for 3 minutes, 800 rpm for 5 minutes and 600 rpm for 5 minutes after four days to remove dead single cells;
(4) and then continuing suspension culture, after 7 days, centrifuging and re-planting the Muse cell clusters which do not grow up to a 6-well plate coated with matrigel, observing the growth condition after the Muse cell clusters are attached to the wall, digesting the cells with pancreatin after the cells reach 90 percent fusion, and then re-inoculating the cells to the 6-well plate coated with Poly-HEMA for suspension culture by using an MC culture medium.
Further, in the present embodiment, in the fifth step, the identification of Muse cells includes the following steps:
(1) and taking 3 rd generation cells with good growth state to respectively perform the following identification:
(2) detecting the activity of the cells by a CCK-8 method;
(3) detecting the cell cycle by a flow cytometer;
(4) flow cytometry detection of cell surface CD29, CD34, CD44, CD45, CD90, CD105 and SSEA3 antigens;
(5) and adding adipogenic and osteogenic induced differentiation liquid to detect adipogenic and osteogenic induced differentiation capacity.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (4)
1. A method for screening and identifying human umbilical cord-derived Muse cells is characterized by comprising the following steps: the method comprises the following steps:
step one, determining participants;
step two, determining experimental instruments and reagents;
step three, sorting Muse cells, namely sorting primary cultured dermal fibroblast SSEA-3 positive cells by a flow cytometer, culturing Muse cell clusters under the conditions of MC culture medium and poly-HEMA coated plate, and observing the self-renewal capacity of suspension-adherence-suspension, proliferation and amplification;
step four, in-vitro amplification of Muse cells, wherein the Muse cells obtained by separation are amplified by adopting a suspension culture-adherent culture-suspension culture mode;
step five, identifying Muse cells, namely sorting anti-SSEA-3 antibody positive cells from cultured umbilical cord-derived mesenchymal cells by FACS; and (3) carrying out alkaline phosphatase staining, and detecting the pluripotency of the DNA by immunofluorescence staining, reverse transcription polymerase chain reaction and Western-blot.
2. The method for screening and identifying human umbilical cord-derived Muse cells according to claim 1, wherein the method comprises the following steps: in the second step, the experimental apparatus and the reagent comprise the following components:
the kit comprises a flow cytometer, a CO2 incubator, a desktop centrifuge, a biological safety cabinet, a microscope, a fluorescence microscope, a photographic system, a serum-free conditioned medium of mesenchymal stem cells of an enzyme labeling instrument, 0.25% pancreatin, anti-CD29, anti-CD45, anti-CD90, anti-CD105, anti-CD34, anti-CD44, a cell cycle instant detection kit, an osteogenic and adipogenic induced differentiation culture solution, alizarin red, oil red O, Anti-SSEA3, high-sugar DMEM and fetal calf serum.
3. The method for screening and identifying human umbilical cord-derived Muse cells according to claim 1, wherein the method comprises the following steps: in the fourth step, the in vitro amplification of Muse cells comprises the following steps:
(1) the obtained MSC cells were passed through a 200 mesh cell filter. Incubation with SSEA 3-FITC-conjugated antibody on a shaker for 20 min at room temperature;
(2) human Ig G-FITC was used as an isotype control, PI staining excluded dead cells in FACS analysis, and the individual cells obtained were sorted;
(3) culturing the cells in a Poly-HEMA coated 6-well plate in a suspension manner by using an MC culture medium, adding 500ul of the MC culture medium every two days, and centrifuging at a low speed of 1000 rpm for 3 minutes, 800 rpm for 5 minutes and 600 rpm for 5 minutes after four days to remove dead single cells;
(4) and then continuing suspension culture, after 7 days, centrifuging and re-planting the Muse cell clusters which do not grow up to a 6-well plate coated with matrigel, observing the growth condition after the Muse cell clusters are attached to the wall, digesting the cells with pancreatin after the cells reach 90 percent fusion, and then re-inoculating the cells to the 6-well plate coated with Poly-HEMA for suspension culture by using an MC culture medium.
4. The method for screening and identifying human umbilical cord-derived Muse cells according to claim 1, wherein the method comprises the following steps: in the fifth step, the identification of Muse cells comprises the following steps:
(1) and taking 3 rd generation cells with good growth state to respectively perform the following identification:
(2) detecting the activity of the cells by a CCK-8 method;
(3) detecting the cell cycle by a flow cytometer;
(4) flow cytometry detection of cell surface CD29, CD34, CD44, CD45, CD90, CD105 and SSEA3 antigens;
(5) and adding adipogenic and osteogenic induced differentiation liquid to detect adipogenic and osteogenic induced differentiation capacity.
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| CN202110206416.9A CN113106058A (en) | 2021-02-24 | 2021-02-24 | Screening and identifying method of human umbilical cord-derived Muse cells |
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| CN202110206416.9A CN113106058A (en) | 2021-02-24 | 2021-02-24 | Screening and identifying method of human umbilical cord-derived Muse cells |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117187174A (en) * | 2023-11-08 | 2023-12-08 | 广州正源生物技术有限公司 | Muse cell culture medium and extraction method of fat Muse cells |
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- 2021-02-24 CN CN202110206416.9A patent/CN113106058A/en not_active Withdrawn
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117187174A (en) * | 2023-11-08 | 2023-12-08 | 广州正源生物技术有限公司 | Muse cell culture medium and extraction method of fat Muse cells |
| CN117187174B (en) * | 2023-11-08 | 2024-02-06 | 广州正源生物技术有限公司 | Muse cell culture medium and extraction method of fat Muse cells |
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