CN102140436A - Culture solution for differentiating adipose-derived stromal cells into myocardial cells and preparation method thereof - Google Patents
Culture solution for differentiating adipose-derived stromal cells into myocardial cells and preparation method thereof Download PDFInfo
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- CN102140436A CN102140436A CN2010106104010A CN201010610401A CN102140436A CN 102140436 A CN102140436 A CN 102140436A CN 2010106104010 A CN2010106104010 A CN 2010106104010A CN 201010610401 A CN201010610401 A CN 201010610401A CN 102140436 A CN102140436 A CN 102140436A
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Abstract
The invention discloses a method for preparing culture solution for differentiating adipose-derived stromal cells into myocardial cells. 1L of culture solution comprises the following components: 10g of Gibco DMEM(Dulbecco's Modified Eagle Medium) high glucose medium, 2.3g of NaHCO3, 2.6g of N-2-hydroxyethylpiperazine-N-ethane-sulphonicacid (HEPES), 15 to 25 volume percent of Gibco fetal calf serum, 5*104U of penicillin, 5*104U of streptomycin and 75 to 85 volume percent of deionized water. A simple and definite culture solution system for differentiating the adipose-derived stromal cells into the myocardial cells is obtained, and meets higher application security standard.
Description
Technical field
The present invention relates to biological technical field, especially a kind of adipose stromal cells is divided into nutrient solution of myocardial cell and preparation method thereof.
Background technology
Adipose stromal cells is divided into myocardial cell's nutrient solution, be that tissue-derived stroma cell of a kind of induced lipolysis or adult stem cell are at the in-vitro directed culture system that is divided into the myocardial cell with spontaneous contractile function, because adult stem cell is used for the clinical medicine tissue repair and comes into one's own day by day, so this system also demonstrates its importance day by day in scientific research and using value clinically.Additive in the nutrient solution, the kind of serum and concentration, and the pH value of nutrient solution etc. is all determining stroma cell or the stem cell differentiation degree to functional myocardial cell.In addition, additive also affects the molecular pathways that stroma cell or stem cell directional break up this process to some extent, has greatly limited the research of adipose stromal cells to the molecule mechanism of myocardial cell's differentiation.
External rarely seen 3-5 research institution's report successfully induced adipose stromal cells to having the myocardial cell of spontaneous contractile function external at present, their employed culture system is slightly different, all in all there is following shortcoming in these culture systems: 1. rely on single additive, as: the differentiation of helper-inducers such as demethylation reagent 5-azacytidine or antioxidant 2-Mercaptoethanol; 2. use commercialization nutrient solution MethoCultGF M3534 (additive wherein comprises: rh-Insulin, 2-Mercaptoethanol, rm-Stem Cell Factor, rm-IL-3 and rh-IL-6 etc.) the helper-inducer differentiation that has complicated additive; 3. use and the external evoked differentiation of myocardial cell's co-culture system.
Though above-mentioned culture system all can obtain to have the myocardial cell of spontaneous contractile function, but also there is following problem in they: 1. Fu Za culture system causes being difficult to dividing and effectively induces composition, brings difficulty for further refining the key component that external evoked adipose stromal cells is divided into the myocardial cell; 2. Fu Za additive may directly have influence on the myocardial cell's of generation basic biological characteristics, and then has influence on its clinical application; 3. the external inductor of cultivating altogether with the myocardial cell ties up to difficulty in the clinical application; 4. additive costs an arm and a leg.
Summary of the invention
The present invention is for addressing the above problem, and the nutrient solution that provides a kind of adipose stromal cells to be divided into the myocardial cell solves above several big problems, makes adipose stromal cells simple, clear and definite to the culture system of myocardial cell's differentiation, and reaches higher application safety standard.
Technical solution of the present invention is as follows:
A kind of adipose stromal cells is divided into the preparation method of myocardial cell's nutrient solution,
1., under 120 ℃ of temperature with deionized water sterilization 30 minutes, naturally cool to room temperature; 2., with Gibco DMEM high glucose medium, NaHCO
3, HEPES, Gibco foetal calf serum, penicillin and Streptomycin sulphate pour in 1. the sterilization deionized water, stir with magnetic stirrer; 3., will be 2. successively through the aseptic filtering with microporous membrane of 0.45 μ m and 0.22 μ m; 4., 4 ℃ of preservations are standby.
Described nutrient solution, use HCl or NaOH step 2. in the pH value of adjusting nutrient solution 7.2～7.4.
Described nutrient solution, Gibco foetal calf serum volume percent is 15～25%.
A kind of adipose stromal cells is divided into myocardial cell's nutrient solution, and each component content is as follows:
Gibco DMEM high glucose medium: 10g/L;
NaHCO
3：2.3g/L；
HEPES：2.6g/L；
Gibco foetal calf serum: 15～25% (volume percent);
Penicillin: 5 * 10
4U/L;
Streptomycin sulphate: 5 * 10
4U/L;
Deionized water: 75～85% (volume percent).
Obtain the nutrient solution system that a kind of simple, clear and definite adipose stromal cells breaks up to the myocardial cell, and reach higher application safety standard.
Description of drawings
Fig. 1 is from the morphological change figure of adipose stromal cells to the cell of myocardial cell's differentiation.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.
The present invention realizes that the scheme of above-mentioned purpose is: a kind of adipose stromal cells is divided into the preparation method of myocardial cell's nutrient solution, it is characterized in that: each component content is as follows: Gibco DMEM high glucose medium: 10g/L; NaHCO
3: 2.3g/L; HEPES:2.6g/L; Gibco foetal calf serum: 15～25% (volume percent); Penicillin: 5 * 10
4U/L; Streptomycin sulphate: 5 * 10
4U/L; Deionized water: 75～85% (volume percent).Its preparation method is: 1. under 120 ℃ of temperature deionized water was sterilized 30 minutes, naturally cool to room temperature; 2. with Gibco DMEM high glucose medium, NaHCO
3, HEPES, Gibco foetal calf serum, penicillin and Streptomycin sulphate pour in 1. the sterilization deionized water, stir with magnetic stirrer; 3. will be 2. successively through the aseptic filtering with microporous membrane of 0.45 μ m and 0.22 μ m; 4. 4 ℃ of temperature are preserved standby; 5. need be preheated to 37 ℃ of temperature earlier before using can use.
Culture experiment is as follows:
Get the mouse subcutaneus adipose tissue under the aseptic condition, be cut into 1-mm with eye scissors
3Fritter, place 0.1%I Collagen Type VI enzyme (containing 2%BSA and 1g/L_I Collagen Type VI enzyme) digestion
50-60min, through 30 μ m screen filtrations, to centrifuge tube, 1500rpm * 7min removes supernatant with filtrate collection, with erythrocyte cracked liquid (NH
4Cl 0.15M, KHCO
30.01M EDTA 1.27 * 10
-4M) splitting erythrocyte 10min, 1500rpm * 7min removes supernatant, the resuspended precipitation of PBS, 1500rpm * 7min removes supernatant, and it is resuspended to precipitate this nutrient solution of use, with 1 * 10
4Individual/cm
2Be inoculated in the culture dish.Place 37 ℃, 5%CO
2Cultivate in the incubator of saturated humidity, with the not adherent cell of PBS flush away, changed a deuterzooid nutrient solution later on every 2-3 days behind the 24h.
The result is as follows: 5-7 days begin to occur the various kinds of cell form behind the cell inoculation, comprise that in groups tubule shape cell and spherule cell and minority contain the adipocyte that little fat drips.After 2 days in, the myotube spline structure occurs, and the structure of a part of myotube sample and coupled spherule cell begin independent spontaneous contraction.Be illustrated in figure 1 as from the morphological change figure of adipose stromal cells to the cell of myocardial cell's differentiation.Isolated cells is inoculated in this nutrient solution, the attached cell majority be fibroblast-like cells (the 4th day, A).Approximately 5-7 days, spindle cell occur (the 5th day, B), and in short 1-2 days, form spherule cell rapidly, the myotube of staff cell and elongation, and a part of cell wherein begin spontaneous contraction (the 7th day, C).Nucleus among the D in the arrow indication myotube.Scale 100 μ m.
Should be understood that, for those of ordinary skills, can be improved according to the above description or conversion, and all these improvement and conversion all should belong to the protection domain of claims of the present invention.
Claims (4)
1. an adipose stromal cells is divided into the preparation method of myocardial cell's nutrient solution, it is characterized in that:
1., under 120 ℃ of temperature with deionized water sterilization 30 minutes, naturally cool to room temperature; 2., with Gibco DMEM high glucose medium, NaHCO
3, HEPES, Gibco foetal calf serum, penicillin and Streptomycin sulphate pour in 1. the sterilization deionized water, stir with magnetic stirrer; 3., will be 2. successively through the aseptic filtering with microporous membrane of 0.45 μ m and 0.22 μ m; 4., 4 ℃ of preservations are standby.
2. nutrient solution as claimed in claim 1 is characterized in that: use HCl or NaOH step 2. in the pH value of adjusting nutrient solution 7.2～7.4.
3. nutrient solution as claimed in claim 1 is characterized in that: Gibco foetal calf serum volume percent is 15～25%.
4. an adipose stromal cells is divided into myocardial cell's nutrient solution, it is characterized in that each component content is as follows:
Gibco DMEM high glucose medium: 10g/L;
NaHCO
3：2.3g/L；
HEPES：2.6g/L；
Gibco foetal calf serum: 15～25% (volume percent);
Penicillin: 5 * 10
4U/L;
Streptomycin sulphate: 5 * 10
4U/L;
Deionized water: 75～85% (volume percent).
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559588A (en) * | 2011-12-09 | 2012-07-11 | 姜晓丹 | Method for inducing adipose-derived stromal cells to form neural stem cells by stimulating in vivo environment |
| CN109699632A (en) * | 2018-12-26 | 2019-05-03 | 北京优迅医学检验实验室有限公司 | Cell-preservation liquid, store method and its application |
| CN110499281A (en) * | 2019-08-26 | 2019-11-26 | 首都医科大学附属北京世纪坛医院 | A method of quickly establishing H9c2 cardiac muscle cell's fat model |
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| WO2006017320A2 (en) * | 2004-07-13 | 2006-02-16 | Board Of Regents Of The University Of Texas System | Compositions and methods for myogenesis of fat-derived stem cells expressing telomerase and myocardin |
| CN101705209A (en) * | 2009-11-27 | 2010-05-12 | 中国人民解放军军事医学科学院基础医学研究所 | Method for separating heart stem cells from brown fat and splitting cardioblast |
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| WO2006017320A2 (en) * | 2004-07-13 | 2006-02-16 | Board Of Regents Of The University Of Texas System | Compositions and methods for myogenesis of fat-derived stem cells expressing telomerase and myocardin |
| CN101705209A (en) * | 2009-11-27 | 2010-05-12 | 中国人民解放军军事医学科学院基础医学研究所 | Method for separating heart stem cells from brown fat and splitting cardioblast |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559588A (en) * | 2011-12-09 | 2012-07-11 | 姜晓丹 | Method for inducing adipose-derived stromal cells to form neural stem cells by stimulating in vivo environment |
| CN109699632A (en) * | 2018-12-26 | 2019-05-03 | 北京优迅医学检验实验室有限公司 | Cell-preservation liquid, store method and its application |
| CN110499281A (en) * | 2019-08-26 | 2019-11-26 | 首都医科大学附属北京世纪坛医院 | A method of quickly establishing H9c2 cardiac muscle cell's fat model |
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Application publication date: 20110803 |