CN108410803B - A kind of induced lipolysis stem cell at cartilage differentiation cultural method and culture solution - Google Patents
A kind of induced lipolysis stem cell at cartilage differentiation cultural method and culture solution Download PDFInfo
- Publication number
- CN108410803B CN108410803B CN201810265912.XA CN201810265912A CN108410803B CN 108410803 B CN108410803 B CN 108410803B CN 201810265912 A CN201810265912 A CN 201810265912A CN 108410803 B CN108410803 B CN 108410803B
- Authority
- CN
- China
- Prior art keywords
- cell
- culture
- stem cell
- cartilage
- complete medium
- 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.)
- Expired - Fee Related
Links
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
- C12N5/0655—Chondrocytes; Cartilage
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/999—Small molecules not provided for elsewhere
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
- C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
- C12N2506/1384—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from adipose-derived stem cells [ADSC], from 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
- 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)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Rheumatology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Developmental Biology & Embryology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of induced lipolysis stem cells into the cultural method and culture solution of cartilage differentiation, include the following steps: that (1) takes subcutaneus adipose tissue, it rinses, removing adipose tissue film and blood vessel are cut into fragment, enzymic digestion, complete medium is added, centrifugation removal fat cell and fat drips, it is resuspended in complete medium, it is inoculated in culture bottle to cultivate and changes the non-attached cell of culture solution removing afterwards for 24 hours, change liquid within every 3 days, with 37 DEG C of 3~5min of digestion of 0.25% pancreatin and 1mmol/L EDTA after cell fusion, complete medium, which terminates, to be digested, the passage of 1:2 ratio；(2) passage cell replaces medium to incomplete culture medium after being inoculated in the culture bottle culture 48h containing complete medium, and Sox9 gene activation is added and continues to cultivate, every 3d changes a subculture.Method provided by the invention can effectively induced lipolysis stem cell at cartilage differentiation, a large amount of cartilage cells can be prepared using fat stem cell as seed cell with meet it is clinical to cartilage cell the needs of.
Description
Technical field
The invention belongs to biological field, it is related to the cultural method and culture of a kind of induced lipolysis stem cell at cartilage differentiation
Liquid.
Background technique
Currently, in the field of research mescenchymal stem cell, main object or mesenchymal stem cell, and just
The method for foring separation and culture is walked, but mass propgation mesenchymal stem cell receives certain limitation.Especially
In cartilage tissue engineered research, because of the separation and culture difficulty of mesenchymal stem cell, it is limited as good group
Weaver's journey seed cell source.So nearest people have found the cell in the research of the adipose-derived stem cell of research steering
With very strong proliferation and differentiation capability, it is relatively easy to separation and culture.In clinical studies, fat stem cell is in cell transplantation
And organizational project etc. also has a wide range of applications.
Summary of the invention
It is an object of that present invention to provide a kind of induced lipolysis stem cells into the cultural method and culture solution of cartilage differentiation.
Above-mentioned purpose is achieved through the following technical solutions:
A kind of induced lipolysis stem cell includes the following steps: at the cultural method of cartilage differentiation
(1) fat stem cell is separately cultured: taking patient or healthy volunteer's subcutaneus adipose tissue, PBS buffer solution rinses 2
~3 times, adipose tissue film and blood vessel are removed with eye scissors and ophthalmic tweezers and is cut into 1mm3The fragment of size, 0.1% Type I collagen enzyme
With 0.25% pancreatin, 37 DEG C of digestion 40min, during which stir 4~5 times, be added complete medium after digestion, 1500r/min from
Heart 10min, abandons fat cell and fat drips that supernatant removal suspends, and cell precipitation is resuspended in complete medium, is inoculated in culture bottle
In, in 37 DEG C, 5%CO2It is cultivated in incubator, changes culture solution afterwards for 24 hours and remove non-attached cell, change liquid within every 3 days later, cell melts
With 0.25% pancreatin and 1mmol/LEDTA37 DEG C of 3~5min of digestion after conjunction, complete medium, which terminates, to be digested, the passage of 1:2 ratio；
(2) fat stem cell is induced at cartilage differentiation: taking passage cell with 1 × 106The density of/mL is inoculated in containing completely
In the culture bottle of culture medium, 37 DEG C, 5%CO2After cultivating 48h, incomplete culture medium is replaced medium to, while Sox9 base is added
Because activator continues culture 2~4 weeks, during which every 3d changes a subculture (i.e. drug containing DMEM).
Preferably, complete medium is the DMEM containing 10% fetal calf serum.
Preferably, incomplete culture medium is the DMEM without fetal calf serum.
Preferably, Sox9 gene activation is red sesame aldehyde B.
Preferably, take the 3rd~5 generation passage cell for induction.
A kind of induced lipolysis stem cell at cartilage differentiation culture medium, for the DMEM culture for adding Sox9 gene activation
Base.
Preferably, Sox9 gene activation is red sesame aldehyde B.
Method provided by the invention can induced lipolysis stem cell can be effectively kind with fat stem cell at cartilage differentiation
A large amount of cartilage cells are prepared to meet the needs of clinic is to cartilage cell in daughter cell.
Detailed description of the invention
Fig. 1 is the band comparison diagram that Western blot detects II Collagen Type VI of group of cells and proteoglycans expression.
Specific embodiment
Technical solution of the present invention is introduced combined with specific embodiments below.Non- special emphasis tests material in following embodiments
Material is routine experiment material, is had no special requirements, and is all the conventional material that those skilled in the art are easy to get.
One, experimental material
Red sesame aldehyde A, the self-control of red sesame aldehyde B standard product, for purity 98% or more, chemical information is as follows.
DMEM culture medium and fetal calf serum are purchased from Gibco company.
Two, experimental method
1, fat stem cell is separately cultured
The subcutaneus adipose tissue of healthy volunteer is taken, PBS buffer solution is rinsed 3 times, removes fat with eye scissors and ophthalmic tweezers
Tissue film and blood vessel are simultaneously cut into 1mm3The fragment of size, 37 DEG C of digestion 40min of 0.1% Type I collagen enzyme and 0.25% pancreatin, during which
Complete medium (DMEM containing 10% fetal calf serum, similarly hereinafter) is added after digestion in stirring 5 times, 1500r/min centrifugation
10min abandons fat cell and fat drips that supernatant removal suspends, and cell precipitation is resuspended in complete medium, is inoculated in culture bottle,
In 37 DEG C, 5%CO2It is cultivated in incubator, changes culture solution afterwards for 24 hours and remove non-attached cell, change liquid, cell fusion within every 3 days later
4min is digested with 0.25% pancreatin and 1mmol/L EDTA37 DEG C afterwards, complete medium terminates digestion, the passage of 1:2 ratio.
2, the induction differentiation of fat stem cell
Take the 4th generation passage cell with 1 × 106The density of/mL is inoculated in the culture bottle containing complete medium, and 37 DEG C,
5%CO2It after cultivating 48h, replaces medium to incomplete culture medium (DMEM without fetal calf serum), grouping culture:
Red sesame aldehyde A group: final concentration of 25 μM of red sesame aldehyde A is added in incomplete culture medium；
Red sesame aldehyde B group: final concentration of 25 μM of red sesame aldehyde B is added in incomplete culture medium；
Control group: being only incomplete culture medium.
Continue culture 3 weeks, during which every 3d changes a subculture.
3, it is detected at cartilage differentiation
After cultivating 3 weeks, western blot detects the expression quantity of II Collagen Type VI and proteoglycans (aggrecan).Step is such as
Under: after the cell of each group is cracked by RIPA, BCA is quantitative, after SDS-PAGE is separated, using half dry type electrotransfer method by albumen
It is transferred on PVDF from gel.Film being placed in 5% defatted milk (being dissolved in PBS, pH7.4), overnight, TBST washes film for 4 DEG C of closings,
Each 5min, is repeated 3 times.Be added the diluted primary antibody collagen-II (1:2000) of TBST, aggrecan (1:1000) and
GAPDH (1:2000), 4 DEG C of overnight incubations.TBST washes film, and each 5min is repeated 3 times, horseradish peroxidase (HRP) label
The sheep anti-Mouse secondary antibody (1:2000) of goat-anti rabbit secondary antibody (1:2000) and HRP label is incubated at room temperature 2h.TBST washes film, every time
10min is repeated 3 times, and reacts 2min, gel imaging system development imaging in chemiluminescence detection reagent.Calculate II Collagen Type VI/
GAPDH, proteoglycans/GAPDH relative expression quantity.
4, RT-qPCR method detects Sox9mRNA relative expression quantity
Each group is added Trizol and extracts cell total rna, (anti-with PrimeScrip RT Master Mix after measuring concentration
Transcript reagent box), according to the system reverse transcription of 500ng/10 μ l at the cDNA of 50 μ l, with SYBRPremix Ex Taq, (fluorescence is fixed
Measure PCR kit) Sox9mRNA relative expression quantity is detected on ABI7500 real-time fluorescence quantitative PCR instrument with 20 μ l systems.With
For GAPDH gene expression dose as internal reference, the ratio for calculating Sox9mRNA/GAPDH gene represents the relative expression of Sox9mRNA
Level, gene expression relative quantification, which is calculated, carries software using instrument.
Primer sequence is as follows:
5, statistical analysis
Data processing carries out one-way analysis of variance using 19.0 software of SPSS, is as a result indicated with mean ± standard deviation, group
Between compare P < 0.05 and think there is statistical difference.
Three, experimental result
1, II Collagen Type VI of group of cells and proteoglycans expression quantity
Western blot testing result is as shown in Figure 1.II Collagen Type VI of control group and red sesame aldehyde A group, proteoglycans expression
It is extremely weak, visible obvious II Collagen Type VI of red sesame aldehyde B group, proteoglycans expression.
The above results show red sesame aldehyde B group fat stem cell obviously to Chondrocyte Differentiation.
2, each group Sox9mRNA relative expression quantity
Compared to control group (1.00 ± 0.08, normalized), red sesame aldehyde B group Sox9mRNA relative expression quantity (3.41
± 0.13) significant to increase, and red sesame aldehyde A group is without significantly raised (1.09 ± 0.07).
The above results show that red sesame aldehyde B significantly has activated the expression of Sox9 gene in fat stem cell.
To sum up, red sesame aldehyde B can by activate Sox9 gene expressing promoting into fat stem cell at cartilage differentiation, and
Red sesame aldehyde A does not have this effect.Therefore, red sesame aldehyde B may be used as fat stem cell into chondrocyte induction differentiation agent, can add
Fat stem cell is prepared into DMEM culture medium into cartilage differentiation induced medium.
The above-mentioned technical solution that the specific embodiments are only for explaining the present invention, it will be appreciated by those skilled in the art that, this hair
Bright protection scope is not limited to above-mentioned specific embodiment.
Claims (1)
1. a kind of induced lipolysis stem cell is at the culture medium of cartilage differentiation, for the DMEM culture medium for adding Sox9 gene activation,
It is characterized by: the Sox9 gene activation is red sesame aldehyde B.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810265912.XA CN108410803B (en) | 2018-03-28 | 2018-03-28 | A kind of induced lipolysis stem cell at cartilage differentiation cultural method and culture solution |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810265912.XA CN108410803B (en) | 2018-03-28 | 2018-03-28 | A kind of induced lipolysis stem cell at cartilage differentiation cultural method and culture solution |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108410803A CN108410803A (en) | 2018-08-17 |
| CN108410803B true CN108410803B (en) | 2019-02-05 |
Family
ID=63133651
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810265912.XA Expired - Fee Related CN108410803B (en) | 2018-03-28 | 2018-03-28 | A kind of induced lipolysis stem cell at cartilage differentiation cultural method and culture solution |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108410803B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111518751A (en) * | 2019-11-20 | 2020-08-11 | 广东先康达生物科技有限公司 | Method for in vitro activating adipose-derived stem cells to convert adipose-derived stem cells into protochondrocytic cells |
| CN112430570B (en) * | 2020-11-25 | 2022-07-26 | 西部医美生物科技成都有限公司双流医疗分公司 | Use of adipose-derived stem cells edited by gene editing technology for improving differentiation efficiency |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SG11201500188YA (en) * | 2012-08-08 | 2015-02-27 | Univ Nanyang Tech | Methods of manufacturing hydrogel microparticles having living cells, and compositions for manufacturing a scaffold for tissue engineering |
| KR20160002681A (en) * | 2013-01-16 | 2016-01-08 | 인썸(인스티튜트 내셔날 드 라 싼테 에 드 라 리셰르셰메디칼르) | A soluble fibroblast growth factor receptor 3 (fgr3) polypeptide for use in the prevention or treatment of skeletal growth retardation disorders |
| CN108602866A (en) * | 2015-09-18 | 2018-09-28 | 生命制药有限责任公司 | Expression and folding in CD-RAP preparation process is improved by using CD-RAP precursor proteins |
-
2018
- 2018-03-28 CN CN201810265912.XA patent/CN108410803B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN108410803A (en) | 2018-08-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Yu et al. | Yield and characterization of subcutaneous human adipose-derived stem cells by flow cytometric and adipogenic mRNA analyzes | |
| Luo et al. | Neural differentiation of bone marrow mesenchymal stem cells with human brain‐derived neurotrophic factor gene‐modified in functionalized self‐assembling peptide hydrogel in vitro | |
| CN109749986A (en) | A method of broken up by human pluripotent stem cells and obtains diabetes and beta Cell of islet | |
| Nippert et al. | Isolation and cultivation of adult rat cardiomyocytes | |
| Siebzehnrubl et al. | Isolation and characterization of adult neural stem cells | |
| AU2018215170B2 (en) | Differentiation control method for pluripotent stem cells | |
| Watson et al. | Comparison of markers and functional attributes of human adipose-derived stem cells and dedifferentiated adipocyte cells from subcutaneous fat of an obese diabetic donor | |
| Chen et al. | Single‐cell and spatial transcriptomics decodes Wharton's jelly‐derived mesenchymal stem cells heterogeneity and a subpopulation with wound repair signatures | |
| CN102168065A (en) | Method for inducing human umbilical cord mesenchymal stem cells in vitro into liver cells and application thereof | |
| CN106661552B (en) | Cardiac cell culture material | |
| CN108410803B (en) | A kind of induced lipolysis stem cell at cartilage differentiation cultural method and culture solution | |
| CN111088222B (en) | Preparation method of single-cell suspension of adipose tissue | |
| Pizzute et al. | Ascorbate-dependent impact on cell-derived matrix in modulation of stiffness and rejuvenation of infrapatellar fat derived stem cells toward chondrogenesis | |
| Zhong et al. | Stagewise keratinocyte differentiation from human embryonic stem cells by defined signal transduction modulators | |
| Jossen et al. | An approach towards a gmp compliant in-vitro expansion of human adipose stem cells for autologous therapies | |
| Li et al. | Skin biopsy and patient-specific stem cell lines | |
| Kim et al. | Ameliorating fibrotic phenotypes of keloid dermal fibroblasts through an epidermal growth factor-mediated extracellular matrix remodeling | |
| Panella et al. | Chemically defined xeno-and serum-free cell culture medium to grow human adipose stem cells | |
| JP2010158206A (en) | Method for sorting human myocardial precursor cell | |
| CN1638640A (en) | Human fetal bladder-derived epithelial cells | |
| CN108486039A (en) | The method that small molecule induction human adipose-derived stem cell is divided into interstitial glands | |
| Jiang et al. | Differentiation of E14 mouse embryonic stem cells into thyrocytes in vitro | |
| Qian et al. | One-step simple isolation method to obtain both epidermal and dermal stem cells from human skin specimen | |
| Yang et al. | An improved method for the isolation and culture of rat epidermal stem cells | |
| Damjanovski et al. | Role of ECM remodeling in thyroid hormone‐dependent apoptosis during anuran metamorphosis |
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 | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20181212 Address after: 410100 Dormitory Building 205, 98 Lixiang Road, Quantang Street, Changsha Economic and Technological Development Zone, Changsha City, Hunan Province Applicant after: HUNAN YUANPIN CELL BIOTECHNOLOGY Co.,Ltd. Address before: 211505 D1 building, 9 Chuang Chuang Road, Zhongshan science and Technology Park, Liuhe District, Nanjing, Jiangsu. Applicant before: NANJING QIANNIANJIAN STEM CELL GENE ENGINEERING Co.,Ltd. |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20190205 |