CN113025566A - Endothelial cell osteogenesis induced differentiation culture medium and preparation method thereof - Google Patents
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Abstract
The invention discloses an endothelial cell osteogenesis induced differentiation culture medium and a preparation method thereof, belonging to the technical field of culture media. The chemolysis culture medium is prepared from the following components: alpha-MEM culture medium, fetal bovine serum 5-50% by volume, Insulin-Transferrin-Selenium (ITS-G) 0.1-5% by volume, bFGF 1-10 ng/ml, EGF 1-10 ng/ml, VEGF 5-50 ng/ml, TGF-beta 5-50 ng/ml, MSCGS 0.1-5% by volume, sodium pyruvate 0.1-10mM, mercaptoethanol 0.01-1mM, Dexamethane 10-200nM concentration, Vc 10-150 ug/ml concentration, and beta-sodium glycerophosphate 1-100mM concentration. The preparation method of the culture medium is simple and convenient, the induction speed is high, and the process is stable.
Description
Technical Field
The invention relates to the technical field of culture media, in particular to an endothelial cell osteogenesis induced differentiation culture medium and a preparation method thereof.
Background
At present, the existing osteogenesis induction culture medium mainly aims at various tissue mesenchymal stem cells including bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells and adipose mesenchymal stem cells, but at present, no culture medium directly aiming at differentiation from endothelial cells to osteoblasts exists. The present technology is characterized by that firstly, the endothelial cells are induced and differentiated into stem cells by means of different reagents, so that the endothelial cells can be differentiated into osteoblasts, then the osteoblasts are differentiated into osteoblasts by means of osteogenic induction culture medium.
Disclosure of Invention
The invention aims to overcome the technical defects of the existing culture medium and provide a convenient and efficient culture medium for inducing differentiation from endothelial cells to osteoblasts and a preparation method thereof.
The purpose of the invention is realized as follows:
the ectopic ossification lineage tracing indicates that the cell type causing the ectopic ossification is derived from endothelial cells, but the endothelial cells do not have stem cell characteristics, and the current research shows that the vascular endothelial cells can be converted into pluripotent stem cell-like cells through activating a kinase-2 (ALK2) receptor-dependent mechanism. (1) Chondrocytes and osteoblasts express endothelial markers in lesions of Fibrodysplasia Ossificans Progressiva (FOP) or in lesions of transgenic mice expressing constitutively active ALK 2. (2) Mouse ectopic ossification lineage tracings constructed using Tie2-Cre also indicate endothelial origin of these cell types. (3) Expression of constitutively active ALK2 in endothelial cells results in a transition from endothelial cells to mesenchymal cells and acquires a stem cell-like phenotype. (4) Similar results were obtained with untransfected endothelial cells treated with transforming growth factor b2 (TGF-b2) or a ligand for bone morphogenetic protein 4 (BMP4) in an alk2 dependent manner. These stem cell-like cells can be triggered to differentiate into osteoblasts, chondrocytes or adipocytes. The invention simulates the differentiation of in vivo vascular endothelial cells to stem cells and further to osteoblasts in vitro by a special induction culture medium.
The invention provides an endothelial cell osteogenesis induced differentiation culture medium which is prepared from the following components,
alpha-MEM culture medium, fetal bovine serum 5-50% by volume percentage, Insulin-Transferrin-Selenium (ITS-G) 0.1-5% by volume percentage, bFGF 1-10 ng/ml, EGF 1-10 ng/ml, VEGF 5-50 ng/ml, TGF-beta 5-50 ng/ml concentration, MSCGS 0.1-5% by volume percentage, sodium pyruvate 0.1-10mM, mercaptoethanol 0.01-1mM, Dexamethane 10-200nM concentration, Vc 10-150 ug/ml concentration, beta-sodium glycerophosphate 1-100mM concentration, L-glutamine 10-50mM concentration, and streptomycin 0.1-5% by volume percentage.
Preferably, the osteogenic differentiation induction medium for endothelial cells of the present invention is prepared from,
alpha-MEM medium, fetal bovine serum 20% volume percent, Insulin-Transferrin-Selenium (ITS-G) 1% volume percent, bFGF 5 ng/ml, EGF 1 ng/ml, VEGF 50 ng/ml, TGF-beta 25 ng/ml concentration, MSCGS 1% volume percent, sodium pyruvate 0.5mM, mercaptoethanol 0.05mM, Dexamethane 80nM concentration, Vc 70 ug/ml concentration, beta-sodium glycerophosphate 10mM concentration, L-glutamine 20mM concentration, streptomycin 1% volume percent.
Wherein the TGF-beta is TGF-beta 2.
The invention also provides a preparation method of the endothelial cell osteogenesis induced differentiation culture medium, which comprises the following steps:
step one, component configuration. Preparing VEGF mother liquor, TGF-beta mother liquor, sodium pyruvate mother liquor, mercaptoethanol mother liquor, Dexamethasone mother liquor, Vc mother liquor, beta-sodium glycerophosphate mother liquor, L-glutamine mother liquor and streptomycin mother liquor according to reagent requirements, and filtering and sterilizing the mother liquor by using a 0.22 mu m filter membrane;
step two, preparing a working concentration culture solution of the endothelial cells of the skin microvascular in a super clean bench according to the formula of the invention, wherein the working concentration culture solution comprises an alpha-MEM culture medium, 20% volume percentage of fetal bovine serum, 5 ng/ml of bFGF, 1 ng/ml of EGF, 50 ng/ml of VEGF, 1% volume ratio of Insulin-Transferrin-Selenium (ITS-G), 25 ng/ml of TGF-beta, 1% volume ratio of MSCGS, 0.5mM of sodium pyruvate, 0.05mM of mercaptoethanol, 80nM concentration of Dexamethasone, 70 ug/ml of Vc, 10mM of beta-sodium glycerophosphate, 20mM of L-glutamine and 1% volume percentage of streptomycin;
and step three, pretreatment. Reviving the endothelial cells of the skin microvasculature from a liquid nitrogen tank, adjusting the cell state to reach the logarithmic phase of growth, digesting the cells by using 0.25 percent of pancreatin, and resuspending the cells by using an endothelial cell osteogenesis induced differentiation culture solution to ensure that the concentration of the cells is 1X10^ 6/mL;
step four, performing grouped culture, namely adding 100ul of cell suspension in the step three, namely adding 1x10^5 cells into a 12-hole plate, supplementing 900ul of endothelial cell osteogenesis induced differentiation culture medium into the 12-hole plate, placing the cells in an incubator at 37 ℃ under the condition of 5% CO2 for culture;
replacing the culture solution, removing the culture solution in the 12-hole plate every 3 days, adding a new endothelial cell osteogenesis induction culture solution, replacing every 3 days, and ending 21 days to form osteoblasts;
and step six, detection.
Further, the skin microvascular endothelial cells are selected from the group consisting of epidermal microvascular endothelium and dermal microvascular endothelium.
Further, in the second step, the TGF-beta is TGF-beta 2.
Further, the composition also comprises 1% of nonessential amino acid by volume percentage or 1% of antibiotic by volume percentage.
The invention discloses a medium formula and an induction method for osteogenic induced differentiation of vascular endothelial cells.
Advantageous effects
The culture medium realizes the breakthrough of induced differentiation from the vascular endothelial cells to the osteoblasts, can further research cell sources of different diseases by screening components for induced differentiation from the vascular endothelial cells to the osteoblasts through a large number of experiments, and provides an effective tool for basic research of medicine and cognition of disease mechanisms. The preparation method of the culture medium for inducing differentiation of the vascular endothelial cells into osteogenesis is simple and convenient, simple and efficient in operation, rapid in induction speed and stable in process, and can realize the induction differentiation of the vascular endothelial cells into osteogenesis; the invention utilizes the a-MEM culture medium containing 10% FBS to culture the vascular endothelial cells, and the provided preparation method of the culture medium for the osteogenic induced differentiation of the vascular endothelial cells can adapt to various culture raw materials, has high induced differentiation degree and high speed, provides a novel stem cell induction mode, and provides a new way for the research of bioscience.
Drawings
FIG. 1 shows the effect of the medium for osteogenic differentiation induction of vascular endothelial cells on cell morphology for 48 hours;
FIG. 2 shows the expression of cell dryness-related proteins vWF, VE-cadherin, FSP-1 and alpha-SMA in a skin microvascular endothelial cell osteogenesis-induced differentiation medium according to the formulation of the present invention after 48 hours of induction;
FIG. 3 shows the ALP expression of osteoblasts after 7 days of induction of vascular endothelial cells induced by the medium of the present invention;
FIG. 4 shows the formation of mineralized osteoblast nodules in the vascular endothelial cells induced by the culture medium of the present invention for 21 days.
Detailed Description
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.
The components involved in the invention are conventional products sold in the market. The specific implementation case information is as follows:
the endothelial cell osteogenesis inducing differentiation culture medium is prepared from an alpha-MEM culture medium, 5-50% by volume of fetal bovine serum, 0.1-5% by volume of Insulin-Transferrin-Selenium (ITS-G), 1-10 ng/ml of bFGF, 1-10 ng/ml of EGF, 5-50 ng/ml of VEGF, 5-50 ng/ml of TGF-beta, 0.1-5% by volume of MSCGS, 0.1-10mM of sodium pyruvate, 0.01-1mM of mercaptoethanol, 10-200nM of Dexamethasone, 10-150 ug/ml of Vc, 1-100mM of beta-sodium glycerophosphate, 10-50mM of L-glutamine and 0.1-5% by volume of streptomycin.
Preferably, the medium for inducing differentiation of vascular endothelial cell osteogenesis is prepared from alpha-MEM medium, 20% volume percent of fetal bovine serum, 1% volume ratio of Insulin-Transferrin-Selenium (ITS-G), 5 ng/ml bFGF, 1 ng/ml EGF, 50 ng/ml VEGF, 25 ng/ml TGF-beta, 1% volume percent MSCGS, 0.5mM sodium pyruvate, 0.05mM mercaptoethanol, 80nM Dexamethane, 70 ug/ml Vc, 10mM sodium beta-glycerophosphate, 20mM L-glutamine, and 1% volume percent streptomycin. Typically, the TGF-. beta.is TGF-. beta.2.
Wherein, a-MEM is 12571063 for the product number of hyclone company, 10099-141 for the product number of GIBCO for fetal bovine serum, 100-18B for the product number of peprotech for bFGF, 315-09 for EGF, 500-M88 for VEGF, 41400045 for Insulin-Transferrin-Selenium (ITS-G); the product number of the MSCGS is 7552 for science ll, the product number of the Dexamethasone is D4902 for sigma, the product number of the Vc is A4403 for sigma, the product number of the L-glutamine is V900419 for sigma, the product number of the beta-sodium glycerophosphate is 50020 for sigma, the product number of the sodium pyruvate is 50020 for sigma, and the product number of the mercaptoethanol is M3148 for sigma.
The invention relates to a preparation method of a culture medium for osteogenesis induced differentiation of skin microvascular endothelial cells, which comprises the following steps:
step one, component configuration. Preparing VEGF mother liquor, TGF-beta mother liquor, sodium pyruvate mother liquor, mercaptoethanol mother liquor, Dexamethasone mother liquor, Vc mother liquor, beta-sodium glycerophosphate mother liquor, L-glutamine mother liquor and streptomycin mother liquor according to reagent requirements, and filtering and sterilizing the mother liquor by using a 0.22 mu m filter membrane;
step two, preparing a working concentration culture solution of the endothelial cells of the skin microvascular in a super clean bench according to the formula of the invention, wherein the working concentration culture solution comprises an alpha-MEM culture medium, 20% volume percentage of fetal bovine serum, 5 ng/ml of bFGF, 1 ng/ml of EGF, 50 ng/ml of VEGF, 1% volume ratio of Insulin-Transferrin-Selenium (ITS-G), 25 ng/ml of TGF-beta, 1% volume ratio of MSCGS, 0.5mM of sodium pyruvate, 0.05mM of mercaptoethanol, 80nM concentration of Dexamethasone, 70 ug/ml of Vc, 10mM of beta-sodium glycerophosphate, 20mM of L-glutamine and 1% volume percentage of streptomycin;
and step three, pretreatment. Reviving the endothelial cells of the skin microvasculature from a liquid nitrogen tank, adjusting the cell state to reach the logarithmic phase of growth, digesting the cells by using 0.25 percent of pancreatin, and resuspending the cells by using an endothelial cell osteogenesis induced differentiation culture solution to ensure that the concentration of the cells is 1X10^ 6/mL;
step four, performing grouped culture, namely adding 100ul of cell suspension in the step three, namely adding 1x10^5 cells into a 12-hole plate, supplementing 900ul of endothelial cell osteogenesis induced differentiation culture medium into the 12-hole plate, placing the cells in an incubator at 37 ℃ under the condition of 5% CO2 for culture; (ii) a
And step five, changing the culture solution, removing the culture solution in the 12-hole plate every 3 days, adding a new endothelial cell osteogenesis induction culture solution, subsequently changing every 3 days, and ending 21 days to form osteoblasts.
Step six, detection: after 48 hours of induction, the morphological change of the cells and the expression of the stem cell-related characteristic proteins vWF, VE-cadherin, FSP-1 and alpha-SMA are observed by using a fluorescence microscope, and the results are shown in FIG. 1 and FIG. 2; after 7 days of induction, the microvascular endothelial cells formed osteoblast surface marker ALP, and the result is shown in FIG. 3; after 21 days of induction, microvascular endothelial cells formed osteoblast specific mineralized nodules and osteoblasts had formed, the results are shown in fig. 4.
The above-mentioned embodiments, objects, technical solutions and advantages of the present invention are further described in detail, it should be understood that the above-mentioned embodiments are only illustrative of the present invention and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (7)
1. An osteogenic differentiation induction medium for endothelial cells, comprising: is prepared from the following components in percentage by weight,
alpha-MEM culture medium, fetal bovine serum 5-50% by volume percentage, Insulin-Transferrin-Selenium (ITS-G) 0.1-5% by volume percentage, bFGF 1-10 ng/ml, EGF 1-10 ng/ml, VEGF 5-50 ng/ml, TGF-beta 5-50 ng/ml concentration, MSCGS 0.1-5% by volume percentage, sodium pyruvate 0.1-10mM, mercaptoethanol 0.01-1mM, Dexamethane 10-200nM concentration, Vc 10-150 ug/ml concentration, beta-sodium glycerophosphate 1-100mM concentration, L-glutamine 10-50mM concentration, and streptomycin 0.1-5% by volume percentage.
2. The osteogenic differentiation medium for endothelial cells according to claim 1, wherein: is prepared from the following components in percentage by weight,
alpha-MEM medium, fetal bovine serum 20% volume percent, Insulin-Transferrin-Selenium (ITS-G) 1% volume percent, bFGF 5 ng/ml, EGF 1 ng/ml, VEGF 50 ng/ml, TGF-beta 25 ng/ml concentration, MSCGS 1% volume percent, sodium pyruvate 0.5mM, mercaptoethanol 0.05mM, Dexamethane 80nM concentration, Vc 70 ug/ml concentration, beta-sodium glycerophosphate 10mM concentration, L-glutamine 20mM concentration, streptomycin 1% volume percent.
3. The osteogenic differentiation medium for endothelial cells according to claim 1 or 2, wherein the TGF- β is TGF- β 2.
4. A preparation method of an endothelial cell osteogenesis induced differentiation medium is characterized by comprising the following steps:
step one, component preparation:
preparing VEGF mother liquor, TGF-beta mother liquor, sodium pyruvate mother liquor, mercaptoethanol mother liquor, Dexamethasone mother liquor, Vc mother liquor, beta-sodium glycerophosphate mother liquor, L-glutamine mother liquor and streptomycin mother liquor according to reagent requirements, and filtering and sterilizing the mother liquor by using a 0.22 mu m filter membrane;
step two, preparing a working concentration culture solution of the endothelial cells of the skin microvascular in a super clean bench according to the formula of the invention, wherein the working concentration culture solution comprises an alpha-MEM culture medium, 20% volume percentage of fetal bovine serum, 5 ng/ml of bFGF, 1 ng/ml of EGF, 50 ng/ml of VEGF, 1% volume ratio of Insulin-Transferrin-Selenium (ITS-G), 25 ng/ml of TGF-beta, 1% volume ratio of MSCGS, 0.5mM of sodium pyruvate, 0.05mM of mercaptoethanol, 80nM concentration of Dexamethasone, 70 ug/ml of Vc, 10mM of beta-sodium glycerophosphate, 20mM of L-glutamine and 1% volume percentage of streptomycin;
step three, pretreatment:
reviving the endothelial cells of the skin microvasculature from a liquid nitrogen tank, adjusting the cell state to reach the logarithmic phase of growth, digesting the cells by using 0.25 percent of pancreatin, and resuspending the cells by using an endothelial cell osteogenesis induced differentiation culture solution to ensure that the concentration of the cells is 1X10^ 6/mL;
step four, performing grouped culture, namely adding 100ul of cell suspension in the step three, namely adding 1x10^5 cells into a 12-hole plate, supplementing 900ul of endothelial cell osteogenesis induced differentiation culture medium into the 12-hole plate, placing the cells in an incubator at 37 ℃ under the condition of 5% CO2 for culture;
step five, liquid changing: removing the culture solution in the 12-hole plate every 3 days, adding a new endothelial cell osteogenesis induction culture solution, replacing every 3 days, and forming osteoblasts after 21 days;
and step six, detection.
5. The method of claim 4, wherein the skin microvascular endothelial cells are selected from the group consisting of epidermal microvascular endothelium and dermal microvascular endothelium.
6. The method according to claim 5, wherein in step two, the TGF- β is TGF- β 2.
7. The method of claim 6, further comprising 1% by volume of a non-essential amino acid, or 1% by volume of an antibiotic.
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