CN111518760A - Culture medium for promoting in-vitro proliferation and differentiation of umbilical cord mesenchymal stem cells - Google Patents
Culture medium for promoting in-vitro proliferation and differentiation of umbilical cord mesenchymal stem cells Download PDFInfo
- Publication number
- CN111518760A CN111518760A CN202010445542.5A CN202010445542A CN111518760A CN 111518760 A CN111518760 A CN 111518760A CN 202010445542 A CN202010445542 A CN 202010445542A CN 111518760 A CN111518760 A CN 111518760A
- Authority
- CN
- China
- Prior art keywords
- mscs
- huc
- promoting
- differentiation
- stem cells
- 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.)
- Withdrawn
Links
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
- C12N5/0665—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
- C12N5/0654—Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
- C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
- C12N2506/1369—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from blood-borne mesenchymal stem cells, e.g. MSC from umbilical blood
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Developmental Biology & Embryology (AREA)
- Rheumatology (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Hematology (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a culture medium for promoting in-vitro proliferation and differentiation of umbilical cord mesenchymal stem cells. The invention discovers that the L-histidine derivative 5 has the function of promoting the proliferation of the hUC-MSCs in vitro and can induce the osteogenic differentiation of the hUC-MSCs. Therefore, the L-histidine derivative 5 can be used for preparing hUC-MSCs seed cells in vitro and promoting osteogenic differentiation of the hUC-MSCs seed cells, and has the prospect of preparing a culture medium for promoting the hUC-MSCs to proliferate in vitro and inducing the osteogenic differentiation of the hUC-MSCs.
Description
Technical Field
The invention belongs to the field of stem cells, relates to in-vitro proliferation and induced differentiation of umbilical cord mesenchymal stem cells, and particularly relates to a culture medium for promoting in-vitro proliferation and differentiation of umbilical cord mesenchymal stem cells.
Background
Stem cell engineering refers to the manipulation of stem cells in vitro, including in vitro proliferation, directed induction, transverse differentiation, gene modification, tissue formation, and the like. The scientific value lies in the attractive application prospect, and the treatment of diseases by using the cell technology is the development direction of future medicine. At present, the main contents of stem cell engineering research are ES cell isolation culture, directed induced differentiation, gene manipulation, embryo engineering, nuclear transplantation, etc., and the aim is to deeply research the differentiation mechanism of pluripotent stem cells, establish in vitro three-dimensional culture conditions, construct different tissues and organs, and develop novel biomaterials which can be applied in vivo.
Human umbilical cord mesenchymal stem cells (hUC-MSCs) are a class of mesenchymal stem cells with multipotential differentiation capacity and self-renewal capacity. The umbilical cord has the advantages of easily-obtained materials, difficulty in pollution, no relation to moral, ethical and legal problems and the like, and is widely used for extracting the hUC-MSCs. Moreover, researches find that the in vitro amplification and multidirectional differentiation capacity of the hUC-MSCs is stronger than that of mesenchymal stem cells from other tissues, and the hUC-MSCs are very suitable for being used as seed cells in stem cell engineering.
The problem to be solved in the first place for stem cell research and application is how to rapidly obtain a large number of stem cell seed cells in vitro; the second problem to be solved is if the differentiation of the seed cells into the target cells is induced.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a culture medium for promoting the in-vitro proliferation and differentiation of umbilical cord mesenchymal stem cells.
The technical scheme of the invention is as follows:
the application of the L-histidine derivative with the following chemical structure in promoting the in vitro proliferation of the umbilical cord mesenchymal stem cells and inducing the osteogenic differentiation of the umbilical cord mesenchymal stem cells.
A culture medium for promoting the in vitro proliferation and inducing the osteogenic differentiation of umbilical cord mesenchymal stem cells, which contains an L-histidine derivative with the following chemical structure.
The beneficial technical effects are as follows:
the invention discovers that the L-histidine derivative 5 has the function of promoting the proliferation of the hUC-MSCs in vitro and can induce the osteogenic differentiation of the hUC-MSCs. Therefore, the L-histidine derivative 5 can be used for preparing hUC-MSCs seed cells in vitro and promoting osteogenic differentiation of the hUC-MSCs seed cells, and has the prospect of preparing a culture medium for promoting the hUC-MSCs to proliferate in vitro and inducing the osteogenic differentiation of the hUC-MSCs.
Drawings
FIG. 1 is a chemical structural formula of L-histidine derivative 5;
FIG. 2 is a diagram of phenotypic flow assays for hUC-MSCs;
FIG. 3 shows the proliferation promoting rate of hUC-MSCs by different concentrations of L-histidine derivative 5;
FIG. 4 shows alizarin red staining results.
Detailed Description
The following examples are intended to illustrate the essence of the present invention, but should not be construed as limiting the scope of the present invention.
First, test materials
DMEM/F12 medium and fetal bovine serum were purchased from Gibco.
Glutamine and diabody were purchased from Nanjing Senega Biotech, Inc.
The PBS buffer solution is prepared according to the formula, stored at 4 ℃ and used up within 24 h.
Collagenase type iv and pancreatin were purchased from shanghai diligent kang biotechnology limited, and used as described herein.
The chemical structural formula of the L-histidine derivative 5 is shown in figure 1, and the purity is not lower than 98%.
Second, test method
1. Extraction culture and identification of hUC-MSCs
The hUC-MSCs used are the same as those in patents 2020104333269 and 2020104333714, and the preparation and identification methods are as follows:
collecting umbilical cord of newborn produced by normal full-term caesarean section at about 12cm (storing umbilical cord in PBS buffer solution containing 1% double antibody at 4 deg.C, extracting stem cells within 6h, culturing), washing with PBS buffer solution containing 1% double antibody to remove umbilical artery and vein and umbilical cord adventitia, and shearing to about 1mm3Placing the tissue blocks in a constant temperature shaking instrument at 37 deg.C, adding collagenase type IV and pancreatin, digesting for 60min and 30min respectively to extract cells, resuspending the cells in DMEM/F12 medium containing 20% FBS, 25mmol/L glutamine and 1% double antibody, and adding 1.0 × 106and/mL, inoculating the cells into a cell culture bottle, changing the culture medium after culturing for 4 days, changing the culture medium for 1 time after 2-3 days, and carrying out passage when 80% of the cells are fused. The 5 th generation cells were used for the experiment.
Taking 5 th generation hUC-MSCs, carrying out pancreatin digestion, fully and uniformly blowing to prepare single cell suspension, and adding CD34-PE, CD45-PE, CD73-PE, CD90-PE and CD 105-FITC. Incubating for 30min at room temperature in a dark place, fixing paraformaldehyde, and detecting by a flow cytometer.
2. In vitro proliferation of hUC-MSCs
2.1 grouping
Low concentration drug group: culture with DMEM/F12 medium containing 10. mu. M L-histidine derivative 5(DMSO Co-solvent), 20% FBS, 25mmol/L glutamine and 1% double antibody;
high concentration drug group: culturing with DMEM/F12 medium containing 20 μ M L-histidine derivative 5(DMSO Co-dissolved), 20% FBS, 25mmol/L glutamine and 1% double antibody;
control group: DMEM/F12 medium containing 20% FBS, 25mmol/L glutamine and 1% double antibody was used for culture, and DMSO solvent with the same volume as that of the low-concentration and high-concentration drug groups was added to the medium.
2.2 measurement of cell proliferation Activity by MTT method
Taking 5 th generation hUC-MSCs, digesting with pancreatin, fully and uniformly blowing, and preparing into single cell suspension with each well being 4 × 104The cells were plated in 96-well plates at 5% CO2Culturing in an incubator at 37 ℃; after 24h, replacing the corresponding culture medium according to the grouping, continuously culturing for 48h, adding 20 mu L of MTT solution (5mg/mL) into each hole, continuously culturing for 4h, discarding the supernatant, adding 150 mu L of LDMSO into each hole, oscillating the shaker at low speed for 10min to fully dissolve the crystal, measuring the absorbance value (OD) of each hole at 490nm wavelength by using an enzyme-linked immunosorbent detector, and calculating the proliferation promoting rate of the L-histidine derivative 5 to the hUC-MSCs according to a formula:
proliferation promoting rate (%) - (drug OD 490-control OD490 value) ÷ control OD490 value × 100%.
Each set of 5 replicates and the test was run in 3 replicates.
3. Alizarin red staining determination of hUC-MSCs osteogenic differentiation
Taking 5 th generation hUC-MSCs, digesting with pancreatin, fully and uniformly blowing, and preparing into single cell suspension with each well being 5 × 104Inoculum at 24-well plates at concentration/mL in 5% CO2Culturing in an incubator at 37 ℃; after 24h, the control group and the high concentration drug group (abbreviated as drug) in the above groups are selectedGroup) medium was changed and the culture was continued, and the medium was changed 1 time every 3 days. After 18d, alizarin red staining is adopted to observe the formation of mineralized nodules in the culture plate.
4. Data analysis
In SPSS 17.0, data are presented as mean. + -. SD and tested for t, with P < 0.05 indicating that the difference is statistically significant.
Third, test results
1. Extraction culture and identification results of hUC-MSCs
Phenotypic identification of hUC-MSCs flow results As shown in Table 1 and FIG. 2, CD34-PE and CD45-PE negative expression, CD73-PE, CD90-PE and CD105-FITC positive expression, consistent with the phenotypic characteristics of hUC-MSCs.
TABLE 1 phenotypic identification results of hUC-MSCs
Expression rate | |
CD34-PE | 0.35% |
CD45-PE | 0.47% |
CD73-PE | 95.2% |
CD90-PE | 96.8% |
CD105-FITC | 98.5% |
2. Proliferation promoting rate of hUC-MSCs by L-histidine derivative 5
The proliferation promoting rate of the L-histidine derivative 5 on the hUC-MSCs is shown in Table 2 and FIG. 3, and it can be seen that the L-histidine derivative 5 has the function of promoting the in vitro proliferation of the hUC-MSCs and has a dose effect.
TABLE 2 proliferation-promoting Rate of L-histidine derivative 5 on hUC-MSCs
Proliferation promoting rate | |
L-histidine derivative 5 (10. mu.M) | (118.2±6.9)% |
L-histidine derivative 5 (20. mu.M) | (205.5±8.4)% |
3. Osteogenesis inducing effect of L-histidine derivative 5 on hUC-MSCs
The alizarin red staining result is shown in fig. 4, and no obvious bone mineralization nodules are seen in the control group and obvious bone mineralization nodules are seen in the drug group. The alizarin red staining method is a method for reflecting the osteogenic differentiation degree by measuring the bone mineralization nodule degree, and the higher the number of the bone mineralization nodules is, the higher the osteogenic differentiation degree is.
The above test results show that the L-histidine derivative 5 has the effect of promoting the proliferation of hUC-MSCs in vitro and can induce the osteogenic differentiation of the hUC-MSCs. Therefore, the L-histidine derivative 5 can be used for preparing hUC-MSCs seed cells in vitro and promoting osteogenic differentiation of the hUC-MSCs seed cells, and has the prospect of preparing a culture medium for promoting the hUC-MSCs to proliferate in vitro and inducing the osteogenic differentiation of the hUC-MSCs.
Claims (2)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010445542.5A CN111518760A (en) | 2020-05-24 | 2020-05-24 | Culture medium for promoting in-vitro proliferation and differentiation of umbilical cord mesenchymal stem cells |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010445542.5A CN111518760A (en) | 2020-05-24 | 2020-05-24 | Culture medium for promoting in-vitro proliferation and differentiation of umbilical cord mesenchymal stem cells |
Publications (1)
Publication Number | Publication Date |
---|---|
CN111518760A true CN111518760A (en) | 2020-08-11 |
Family
ID=71908185
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202010445542.5A Withdrawn CN111518760A (en) | 2020-05-24 | 2020-05-24 | Culture medium for promoting in-vitro proliferation and differentiation of umbilical cord mesenchymal stem cells |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN111518760A (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112391341A (en) * | 2020-11-30 | 2021-02-23 | 张川 | Application of SDF-1 protein activator in promoting human umbilical cord mesenchymal stem cell proliferation and differentiation in vitro |
-
2020
- 2020-05-24 CN CN202010445542.5A patent/CN111518760A/en not_active Withdrawn
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112391341A (en) * | 2020-11-30 | 2021-02-23 | 张川 | Application of SDF-1 protein activator in promoting human umbilical cord mesenchymal stem cell proliferation and differentiation in vitro |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN110592022B (en) | Special culture medium for lung tumor organoid and 3D culture method without stent | |
CN112195152B (en) | Culture method and application of human colorectal cancer tissue organoid | |
CN111534483B (en) | Application of insulin-like growth factor binding protein 7 activator in chondrogenic differentiation of human umbilical cord mesenchymal stem cells | |
CN114292816A (en) | Lung cancer organoid culture solution, and culture reagent combination and culture method thereof | |
CN109182257A (en) | A kind of mechanical environment cultural method for improving chondrocyte proliferation activity, maintaining cartilage phenotype | |
CN111334469A (en) | PBMC (peripheral blood mononuclear cell) in-vitro 3D (three-dimensional) methylcellulose agarose hydrogel culture medium and preparation method thereof | |
CN109706115B (en) | Construction method of mouse bone marrow mesenchymal stem cell line | |
CN111518760A (en) | Culture medium for promoting in-vitro proliferation and differentiation of umbilical cord mesenchymal stem cells | |
CN109749993B (en) | Culture method of umbilical cord mesenchymal stem cells | |
CN112852709B (en) | Method for culturing mouse lung organoid | |
CN109234230A (en) | A kind of primary separation method of skin mesenchymal stem cells | |
CN111534482B (en) | Culture medium for chondrogenic differentiation and chondrogenic differentiation of umbilical cord mesenchymal stem cells | |
CN105441386A (en) | Culture and identification method for very small porcine embryonic-like stem cells | |
CN111534485A (en) | Culture medium for promoting in-vitro proliferation of umbilical cord mesenchymal stem cells and maintaining characteristics of stem cells | |
RU2384618C2 (en) | Method for making fibroblast-like cells of umbilical cord of newborn | |
CN114703132A (en) | Application of extracellular vesicles in preparation of culture medium for relieving iPSC-MSCs (induced pluripotent stem cells-mesenchymal stem cells) aging | |
Aydoğdu et al. | Isolation, culture, cryopreservation, and preparation of skin-derived fibroblasts as a final cellular product under good manufacturing practice–compliant conditions | |
CN101186899A (en) | Method applied for tumour cell and stem cell co-culture | |
CN104060329A (en) | Immortalized quality-control cell bank for chromosome karyotype analysis and construction method thereof | |
CN106754672A (en) | A kind of cultural method of attached cell | |
CN110484496A (en) | A kind of purposes of natural small molecule compounds inducing umbilical cord mesenchymal stem Osteoblast Differentiation | |
CN112941017A (en) | Culture medium for inducing human mesenchymal stem cells to form fat and differentiate and preparation method thereof | |
CN106350484B (en) | A method of mesenchymal stem cell is improved into rouge differentiation efficiency | |
CN105368772A (en) | Culture medium, application thereof and method for cultivating dental pulp stem cells | |
CN104120106A (en) | Method of forming skeletal muscle cell by induction differentiation by utilization of pig dedifferentiated adipocyte |
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 | ||
WW01 | Invention patent application withdrawn after publication |
Application publication date: 20200811 |
|
WW01 | Invention patent application withdrawn after publication |