CN108220230A - A kind of separation of human adipose-derived stem cell and cultural method - Google Patents

A kind of separation of human adipose-derived stem cell and cultural method Download PDF

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CN108220230A
CN108220230A CN201810102159.2A CN201810102159A CN108220230A CN 108220230 A CN108220230 A CN 108220230A CN 201810102159 A CN201810102159 A CN 201810102159A CN 108220230 A CN108220230 A CN 108220230A
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stem cell
final concentration
separation
derived stem
human adipose
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CN108220230B (en
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李文荣
李新峰
周雁冰
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SHANGHAI LIFE MEDICAL SCIENCE & TECHNOLOGY CO.,LTD.
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Shanghai Laifu Life Science And Biotechnology Co Ltd
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Abstract

Separation and cultural method the invention discloses a kind of human adipose-derived stem cell, include the following steps：Step 1：Using Liberase digestive ferment solution digestion liposuction aspirates, digestive ferment is neutralized after digestion, centrifugation, filtering obtain human adipose-derived stem cell after then further removing heteroproteose cell with lymphocyte separation medium；Step 2：Using serum free medium culture human adipose-derived stem cell, wherein, serum free medium addition following components：Recombinant human epidermal growth factor；Recombination human basic fibroblast growth factor；RhTGF-BETA β；Derivative growth factor of recombined human blood platelet BB；RhMGF；Reduced glutathione；Coacetylase；Biotin；MEM vitamin solutions；MEM amino acid solutions；MEM nonessential amino acid solutions；GlutaMAX additives；Insulin transferrins selenium solution；Gentamicin.The method can significantly improve the yield and vigor of fat stem cell, obtain containing more fat stem cells with proliferative capacity.

Description

A kind of separation of human adipose-derived stem cell and cultural method
Technical field
The present invention relates to the separation and culture of stem cell, and in particular to a kind of separation of human adipose-derived stem cell and culture side Method.
Background technology
Mescenchymal stem cell (Mesenchymal Stem Cells, MSCs) is a branch for stem cell, is a kind of tool Have the cell of self-replacation and Multidirectional Differentiation ability, be widely present in Various Tissues, as marrow, Cord blood and umbilical cord tissue, Placenta tissue and adipose tissue etc..There are three outstanding features for mescenchymal stem cell：1. the mescenchymal stem cell of external culture It is adherent growth；2. mescenchymal stem cell height expresses CD73, CD90 and CD105, CD31, CD34, CD45, HLA- are not expressed The markers such as DR, CD14, CD19 and CD11b；3. under suitable stimulus, it is thin that mescenchymal stem cell can be divided into skeletonization The cell of the Various Tissues such as born of the same parents, adipocyte and nerve cell.
Fat mesenchymal stem cell, it is from fat to be called fat stem cell (Adipose Stem Cells, fat stem cell) A kind of isolated stem cell with multi-lineage potential of fat tissue.Research has shown that fat stem cell in specifically culture item A plurality of types of cells such as lipoblast, cardiac muscle cell and nerve cell can be broken up under part.Fat stem cell has very low Immunogenicity, therefore transplant allosome fat stem cell will not cause strong immunological rejection, be allograft fat Stem cell provides advantage.Fat stem cell derives from a wealth of sources, and fat can be obtained using the method for liposuction or excision fat Fat tissue, the problem of being not present ethically using fat stem cell.Fat stem cell has very strong amplification ability in vitro, can be with A large amount of fat stem cell is obtained by the method for in vitro culture.Fat stem cell is in the row such as the beauty such as chest enlarge, smoothing wrinkle and shaping Industry has to be widely applied very much, and also plays more and more effects in medical field fat stem cell.
Conventional fat stem cell separation method there are problems that：1. clostridiopetidase A comes from bacterial extract, there are residual Stay the possibility of bacterial component, the endotoxin in clostridiopetidase A specific activation stem cell or may generate some other negative shadows It rings；The difference of different batches clostridiopetidase A quality can also impact the yield of stem cell.It is a kind of more preferable it is therefore necessary to find Clostridiopetidase A substitute.2. the concentration and digestion time of digestive ferment all have an impact the yield and activity of fat stem cell, so It is necessary to optimize the method for fractionation of fatty stem cell.3. some scientific researches or medical institutions are still done using animal blood serum culture fat Cell, but the Serology Quality difference of different batches is very big, ingredient and function cannot be consistent；And animal blood serum contains Low-level cytostatic substance and potential virus and mycoplasma contamination.4. fat stem cell is in incubation In there are a degree of differentiation.
Invention content
Separation and cultural method the object of the present invention is to provide a kind of human adipose-derived stem cell, to solve above-mentioned existing fat Method for separating stem cell there are the problem of.
In order to achieve the above objectives, the separation the present invention provides a kind of human adipose-derived stem cell and cultural method, including with Lower step：
Step 1：Disappeared using Liberase (Liberase MNP-S, GMP Grade, purchased from Roche Holding Ag) digestion enzyme solutions Change liposuction aspirates, digestive ferment is neutralized after digestion, then centrifugation, filtering are further gone to clean with lymphocyte separation medium Human adipose-derived stem cell is obtained after cell；
Step 2：Using 1 obtained human adipose-derived stem cell of serum free medium incubation step, wherein, serum free medium Add following components：
The recombinant human epidermal growth factor of final concentration of 10~50ng/ml；
The recombination human basic fibroblast growth factor of final concentration of 10~50ng/ml；
RhTGF-BETA-the β of final concentration of 1~50ng/ml；
The recombinant human Patelet-Derived Growth Factor-BB of final concentration of 10~50ng/ml；
The rhMGF of final concentration of 1~100ng/ml；
The reduced glutathione of final concentration of 1~5mM；
The coacetylase of final concentration of 1~50 μ g/ml；
The biotin of final concentration of 1~50 μ g/ml；
The gentamicin of final concentration of 1~100 μ g/ml and,
The following components of Thermo Fischer Scient Inc.'s production：The minimum essential medium vitamin solution of final concentration of 1x (Minimum Essential Medium (MEM) Vitamin Solution, article No.：11120052)；The MEM of final concentration of 1x Amino acid solution (MEM Amino Acids Solution, article No.：11130051)；The MEM non-essential aminos of final concentration of 1x Acid solution (MEM Non-Essential Amino Acids Solution, 11140050)；The GlutaMAX of final concentration of 1x Additive (GlutaMAX Supplement, article No.：35050061)；Insulin-Transferrin-selenium solution of final concentration of 1x (Insulin-Transferrin-Selenium solution, article No.：41400045).
The separation of above-mentioned human adipose-derived stem cell and cultural method, wherein, in step 1, Liberase digestion enzyme solutions make It is prepared with serum free medium.
The separation of above-mentioned human adipose-derived stem cell and cultural method, wherein, in step 1, digest enzyme solutions and suction lipectomy The volume ratio of object is 1:1.
The separation of above-mentioned human adipose-derived stem cell and cultural method, wherein, in step 1, digestion temperature is 37 DEG C, during digestion Between be 10-30 minutes.
The separation of above-mentioned human adipose-derived stem cell and cultural method, wherein, in step 1, contain 10% using isometric In the serum free medium of fetal calf serum (Fetal Bovine Serum, FBS) and digestive ferment.
The separation of above-mentioned human adipose-derived stem cell and cultural method, wherein, in step 1, diameter is specially successively used in filtering 100 microns and 40 microns of filter screen filtration.
The separation of above-mentioned human adipose-derived stem cell and cultural method, wherein, in step 2, human adipose-derived stem cell is for the first time with 1 The culture bottle culture that the hyaluronic acid of the recombinant human fibronectin polypeptide of~10 μ g/mL and 1~10 μ g/mL were coated with, only uses later The culture bottle culture being coated with by the recombinant human fibronectin polypeptide of 1~10 μ g/mL.
The separation of above-mentioned human adipose-derived stem cell and cultural method, wherein, the culture bottle was coated under conditions of 4 DEG C Night.
The separation of above-mentioned human adipose-derived stem cell and cultural method, wherein, in step 2, serum free medium DMEM- F12。
The separation of above-mentioned human adipose-derived stem cell and cultural method, wherein, in step 2, the passage of human adipose-derived stem cell Journey uses the TrypLE that Thermo Fischer Scient Inc. producesTMSelect vitellophags.
Human adipose-derived stem cell serum free medium provided by the invention growth factor and nutriment containing there are many, this can promote Human adipose-derived stem cell is made normally to grow and be metabolized under serum-free culturing conditions：
Fibronectin is a kind of extracellular matrix protein, can between mediated cell, the adherency of cell and extracellular matrix.With Fibronectin coating culture bottle can promote cell preferably adherent.The present invention is coated with culture bottle using recombinant human fibronectin polypeptide.
Hyaluronic acid, also known as uronic acid, basic structure are by two dissacharide units D-Glucose aldehydic acid and N- Acetylglucos The large-scale polysaccharide of amine composition.Hyaluronic acid can allow stem cell to maintain a undifferentiated state, be used in cell culture Hyaluronic acid is conducive to maintain the dryness of stem cell.
Epidermal growth factor is a kind of growth factor with multiple functions, there is strong rush splitting action to cell.
Basic fibroblast growth factor, transforming growth factor-β, platelet-derived growth factor-BB and stem cell because Son is all the growth factor with promotion cell Proliferation and division, and the combination of the these types of factor is had been demonstrated between can remarkably promoting The proliferation of mesenchymal stem cells and the differentiation capability for enhancing stem cell.
Reduced glutathione is three peptides containing sulfydryl (SH), has active oxidation reduction system in human body System, the important physiological activity such as activation SH enzymes, detoxication.Reduced glutathione also participates in tricarboxylic acid cycle and glycometabolism, rises To the effect of coenzyme.
Coacetylase is the coenzyme of body acetylization reaction, and very important work is played in sugar, lipid and protein metabolism With.
Biotin is also known as biotin, is water soluble vitamin, also belongs to vitamin B complex.It is synthesis it is ascorbic must Substance is wanted, is fat and the indispensable substance of protein eubolism.
GlutaMAX additives are a kind of advanced cell culture additives, it provides the required L- glutamy of cell growth Amine, but it is more more stable than L-Glutamine.
Insulin-Transferrin-selenium solution provides insulin, transferrins and selenium, specially sodium selenite, and effect is such as Under：
Insulin can improve the anabolism ability of cell, stimulate the growth of cell.
Transferrins is the main siderophillin in cell, and transferrins can combine iron ion, reduce its toxicity and be Cell metabolism provides ferro element.
Sodium selenite is the necessary trace element in cell growth, and oxidation resistant effect is played in cell metabolism.
Gentamicin is a kind of antibiotic of wide spectrum.
Relative to the prior art, the invention has the advantages that：
(1) traditional clostridiopetidase A is replaced using Liberase digestive ferments in the present invention, in 37 DEG C and 0.3WU/mL Only need just the cell inside adipose tissue can be all dissociateed under conditions of Liberase within 10 minutes, when saving very much relatively Between, and smaller is damaged caused by cell.Relative to traditional collagenase digestion procedure, the fat stem cell in the present invention divides From the yield and vigor that method can significantly improve stem cell；From CFU-F experimental results it is recognised that the present invention is isolated Contain more fat stem cells with proliferative capacity in cell, it means that the isolated fat of method is done in the present invention Cell might have better therapeutic effect.In addition Liberase between different batches have high consistency and The ingredient of Liberase is more in line with the safety requirements of clinical practice than clostridiopetidase A, so Liberase should be a kind of good The digestive ferment of fractionation of fatty stem cell, the present invention are that good technology changes with the higher Liberase substitutions clostridiopetidase A of safety Into.Fat stem cell after dissociation is further purified with lymphocyte separation medium, and experiment proves the fat stem cell in P1-P10 generations All there is very high purity.
(2) in terms of stem cell culture, the present invention has blood serum medium using serum free medium instead of traditional, more Safely and meet ethics.A variety of growth factors and nutrient are added in the medium, and it is adherent and aobvious to be effectively promoted cell The proliferative capacity of the raising cell of work.The better GlutaMAX of stability in use replaces L- paddy in the serum free medium of the present invention Glutamine, and multivitamin and aminoacid ingredient are added in, the growth for stem cell provides excellent nutritional condition；It and can be very The dryness of good maintenance stem cell, shows into fat heterotopic Osteogenesis, and the fat stem cell in P10 generations is still latent with differentiation well Energy.
(3) the culture bottle cooperation serum free medium that the present invention was coated with using fibronectin uses, and stem cell can be allowed to exist Adherent growth under serum-free condition；Freshly prepared stem cell is put into the culture being coated with by fibronectin and hyaluronic acid Bottle, can allow stem cell promptly to restore from the stress situation in preparation process, faster and preferably stem cell can be promoted adherent.
(4) the cell propagating method in the present invention uses TrypLETMSelect replaces traditional pancreatin.If pancreatin disappears Cell can be caused very big damage, and pancreatin is usually to derive from pig or cattle tissue by changing poor timing, pancreatin Activity is stopped with fetal calf serum, is also the introduction of animal component in cell cultivation process in this way.TrypLETMSelect is A kind of genetic engineering enzyme, the ingredient without animal origin, TrypLETMThe activity of Select does not need to fetal calf serum suspension, only needs It is diluted with physiological saline or culture medium, so using TrypLETMSelect ratios use traditional pancreatin safety. TrypLETMSelect can use TrypLE effectively with milder dissociation attached cellTMSelect carries out cell biography Dai Huirang stem cells grow more preferable.
Description of the drawings
Fig. 1 is the cell yield contrast schematic diagram of method more of the invention and conventional fat stem cell preparation method；
Fig. 2 is the cell viability contrast schematic diagram of method more of the invention and conventional fat stem cell preparation method；
Fig. 3 is the CFU-F contrast schematic diagrams of method more of the invention and conventional fat stem cell preparation method；
Fig. 4 be fat stem cell prepared by the method for the present invention in P10 generations into fat ability schematic diagram；
Fig. 5 is osteogenic ability schematic diagram of the fat stem cell in P10 generations of the method preparation of the present invention.
Specific embodiment
Below in conjunction with attached drawing, by specific embodiment, the invention will be further described, these embodiments are merely to illustrate The present invention is not limiting the scope of the invention.
Embodiment 1
First, digestion enzyme solutions are prepared：
The solution containing 0.01-0.4WU/mL Liberase digestive ferments is prepared with serum free medium (DMEM-F12), and With the filter membrane degerming that aperture is 0.2 μM.
2nd, coated cell culture bottle：
When serum free medium provided by the invention is used, each culture bottle will be coated in advance.Digestion obtains Human adipose-derived stem cell for the first time be coated with the recombinant human fibronectin polypeptide of 1~10 μ g/mL and the hyaluronic acid of 1~10 μ g/mL Culture bottle culture, only use the culture bottle being coated with by the recombinant human fibronectin polypeptide of 1~10 μ g/mL later.T75's 5 milliliters of coating buffers with normal saline dilution are added in culture bottle, before cell is added in, siphon away coating solution.
3rd, detach and cultivate human adipose-derived stem cell：
Liposuction aspirates are added in 250 milliliters of centrifuge tube, then add in isometric physiological saline；Firmly rock Centrifuge tube several times, then allows tube stand a few minutes, and adipose tissue can be floated on above physiological saline；Physiological saline is siphoned away, then Fresh physiological saline is rejoined, washing blood of the fat several times inside fat with same operation repetition is washed off.
With 1:1 volume mixture adipose tissue and Liberase digestion enzyme solutions, disappear in 37 DEG C of water bath with thermostatic control shaking table Change 10 minutes or so, the rotating speed of shaking table is 100rpm.After digestion, add in isometric culture medium containing 10%FBS And digestive ferment.Then it is centrifuged ten minutes with the rotating speed of 400g, collects cell precipitation.
Cell precipitation is resuspended with physiological saline, cell is allowed successively by a diameter of 100 microns and 40 microns of filter screen, to receive Collect individual cells.
20 milliliters of lymphocyte separation medium (Ficoll) is added in a clean centrifuge tube of 50 milliliters, then thin Born of the same parents' suspension is added slowly to above separating liquid.Turn off the deceleration valve of centrifuge, centrifuged 30 minutes with the rotating speed of 400g, in collection White cellular layer between face liquid level and separating liquid.
Cell is resuspended with the physiological saline of 2 times of volumes, is centrifuged 10 minutes with the rotating speed of 300g, loses supernatant.Use physiology salt Water cleans cell again, loses supernatant after centrifugation, and cell is resuspended with fresh serum free medium, and cell is put into culture bottle training It supports.After culture 24 hours, liquid is changed, no adherent cell is washed away with physiological saline.
During present invention culture human adipose-derived stem cell, following nutriment is added into serum free medium：It is dense eventually Spend the recombination human basic fibroblast cell of the recombinant human epidermal growth factor for 10~50ng/ml, final concentration of 10~50ng/ml Growth factor, final concentration of 10~50ng/ml rhTGF-BETA-β, final concentration of 10~50ng/ml recombination Vectors containing human platelet-derived growth-BB, the rhMGF of final concentration of 1~100ng/ml, final concentration of 1~5mM Reduced glutathione, the coacetylase of final concentration of 1~50 μ g/ml, final concentration of 1~50 μ g/ml biotin, final concentration MEM amino acid solutions, the MEM nonessential amino acid of final concentration of 1x of MEM vitamin solutions, final concentration of 1x for 1x are molten Liquid, the GlutaMAX additives of final concentration of 1x, final concentration of 1x Insulin-Transferrin-selenium solution and it is final concentration of 1~ The gentamicin of 100 μ g/ml.The medium component is clear and definite, and without any external source serum composition, it is dry thin significantly to improve fat The adherent ability and proliferative capacity of born of the same parents is conducive to fat stem cell and expands and keep in vitro its dryness.In subsequent culture Practical growing state in journey according to cell changes liquid or passage.When the degrees of fusion of cell reaches 70~80%, use TrypLETMSelect vitellophags pass on, and the passage ratio of cell is 1:3.
4th, method and conventional method more of the invention
For the difference of the method and conventional method of separation in more of the invention and culture fat stem cell, reality of the invention Test by the use of two kinds of collagenase procedures as control：1.5mg/ml Collagenase Is are in 37 DEG C of digestion 30 minutes (C1) and 1mg/ml clostridiopetidase As I digests 60 minutes (C2) at 37 DEG C.The yield and vigor with three kinds of methods dissociated cell from adipose tissue are compared in experiment, From the yield of gained cell after digestion fat it is recognised that the yield of the cell in the present invention is apparently higher than C1 and C2, Fig. 1 is seen. For the vigor of gained cell, method of the invention and C1 are almost without difference, but the cell viability that C2 methods obtain is bright The aobvious cell viability obtained less than separation method of the present invention, is shown in Fig. 2.
The fibroblast cloning obtained to compare these three separation methods forms unit (colony forming Unit-fibroblast, CFU-F) difference, the isolated fat stem cell of three kinds of methods with 2 × 104A/cm2Density It is inoculated into the culture bottle of T25, observes and record under the microscope the colony counts that cell number is more than 10, this hair after 10 days Containing more fat stem cells with proliferative capacity in bright isolated cell, Fig. 3 is seen, it means that side in the present invention The isolated fat stem cell of method might have better therapeutic effect.
5th, the fat stem cell of method of the invention separation keeps good purity and differentiation potential in succeeding generations
The fat stem cell that collection is detached and cultivated with the method for the present invention, flow cytomery experiment display：Fat is dry Cell all keeps good purity in P1, P5 and P10 generation, is shown in Table 1.
1. this patent method of table prepares horizontal with the marker expression of the ASCs of culture
The differentiation potential of the fat stem cell in P10 generations, the oil red O staining method (Red-Oil-O of Fig. 4 are detected simultaneously Staining) result shows that the fat stem cell of P10 still has significantly into fat ability, the silver nitrate method staining (Von of Fig. 5 Kossa staining) result show P10 fat stem cell still have apparent osteogenic ability.
Although present disclosure is discussed in detail by above preferred embodiment, but it should be appreciated that above-mentioned Description is not considered as limitation of the present invention.After those skilled in the art have read the above, for the present invention's A variety of modifications and substitutions all will be apparent.Therefore, protection scope of the present invention should be limited to the appended claims.

Claims (10)

1. separation and the cultural method of a kind of human adipose-derived stem cell, which is characterized in that it includes the following steps：
Step 1：Using Liberase digestive ferment solution digestion liposuction aspirates, digestive ferment, centrifugation, mistake are neutralized after digestion Filter, human adipose-derived stem cell is obtained after then further removing heteroproteose cell with lymphocyte separation medium；
Step 2：Using 1 obtained human adipose-derived stem cell of serum free medium incubation step, wherein, serum free medium addition Following components：
The recombinant human epidermal growth factor of final concentration of 10~50ng/ml；
The recombination human basic fibroblast growth factor of final concentration of 10~50ng/ml；
RhTGF-BETA-the β of final concentration of 1~50ng/ml；
The recombinant human Patelet-Derived Growth Factor-BB of final concentration of 10~50ng/ml；
The rhMGF of final concentration of 1~100ng/ml；
The reduced glutathione of final concentration of 1~5mM；
The coacetylase of final concentration of 1~50 μ g/ml；
The biotin of final concentration of 1~50 μ g/ml；
The gentamicin of final concentration of 1~100 μ g/ml and,
The following components of Thermo Fischer Scient Inc.'s production：The MEM vitamin solutions of final concentration of 1x；
The MEM amino acid solutions of final concentration of 1x；The MEM nonessential amino acid solutions of final concentration of 1x；Final concentration of 1x's GlutaMAX additives；Insulin-Transferrin-selenium solution of final concentration of 1x.
2. separation and the cultural method of human adipose-derived stem cell as described in claim 1, which is characterized in that in step 1, Liberase digestion enzyme solutions are prepared using serum free medium.
3. separation and the cultural method of human adipose-derived stem cell as described in claim 1, which is characterized in that in step 1, digestive ferment The volume ratio of solution and liposuction aspirates is 1:1.
4. separation and the cultural method of human adipose-derived stem cell as described in claim 1, which is characterized in that in step 1, digestion temperature It is 37 DEG C to spend, and digestion time is 10-30 minutes.
5. separation and the cultural method of human adipose-derived stem cell as described in claim 1, which is characterized in that in step 1, using etc. In the serum free medium containing 10%FBS of volume and digestive ferment.
6. separation and the cultural method of human adipose-derived stem cell as described in claim 1, which is characterized in that in step 1, filtering tool Body is the filter screen filtration of 100 microns and 40 microns of priority diameter.
7. separation and the cultural method of human adipose-derived stem cell as described in claim 1, which is characterized in that in step 2, people's fat The stem cell culture bottle that the recombinant human fibronectin polypeptide of 1~10 μ g/mL and the hyaluronic acid of 1~10 μ g/mL were coated with for the first time Culture, only uses the culture bottle culture being coated with by the recombinant human fibronectin polypeptide of 1~10 μ g/mL later.
8. separation and the cultural method of human adipose-derived stem cell as claimed in claim 7, which is characterized in that the culture bottle is 4 It is coated with overnight under conditions of DEG C.
9. separation and the cultural method of human adipose-derived stem cell as described in claim 1, which is characterized in that in step 2, serum-free Culture medium is DMEM-F12.
10. separation and the cultural method of human adipose-derived stem cell as described in claim 1, which is characterized in that in step 2, people's fat The succeeding generations of fat stem cell use TrypLETMSelect vitellophags.
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108893442A (en) * 2018-07-25 2018-11-27 广州赛莱拉干细胞科技股份有限公司 A kind of fat stem cell proliferated culture medium and its Multiplying culture method
CN109055309A (en) * 2018-08-02 2018-12-21 广州白云山拜迪生物医药有限公司 A kind of serum-free cell culture medium and application thereof
CN110343661A (en) * 2019-04-24 2019-10-18 朗姿赛尔生物科技（广州）有限公司 A kind of extraction of body fat stem cell and culture amplification method
CN110373384A (en) * 2019-07-24 2019-10-25 安徽科门生物科技有限公司 A kind of cultural method of serum-free fat stem cell culture medium and fat stem cell
CN113736731A (en) * 2021-11-05 2021-12-03 保信亚太生物科技（深圳）有限公司 Serum-free medium of adipose tissue-derived mesenchymal stem cells and preparation method and application thereof

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102417894A (en) * 2011-10-21 2012-04-18 中国科学院广州生物医药与健康研究院 Method for increasing efficiency of induction of multipotent stem cell generation
CN102533640A (en) * 2010-11-11 2012-07-04 徐善慧 Method for generating adult stem cells into spheroid cell populations
CN105602896A (en) * 2016-02-03 2016-05-25 广西大学 Isolated culture and identification method of porcine adipose-derived stem cells
CN107058219A (en) * 2017-04-13 2017-08-18 上海莱馥生命科学技术有限公司 A kind of method that application stem cell self-characteristic prepares dental pulp stem cell
CN107418930A (en) * 2017-09-04 2017-12-01 上海莱馥医疗科技有限公司 A kind of preparation method purified with amplification human marrow mesenchymal stem cell
CN107475188A (en) * 2017-09-25 2017-12-15 广东颜值科技有限公司 A kind of cultural method of cell culture medium and embryonic stem cell
CN107557331A (en) * 2017-09-15 2018-01-09 上海莱馥医疗科技有限公司 A kind of method for separating and cultivating human adipose-derived stem cell

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102533640A (en) * 2010-11-11 2012-07-04 徐善慧 Method for generating adult stem cells into spheroid cell populations
CN102417894A (en) * 2011-10-21 2012-04-18 中国科学院广州生物医药与健康研究院 Method for increasing efficiency of induction of multipotent stem cell generation
CN105602896A (en) * 2016-02-03 2016-05-25 广西大学 Isolated culture and identification method of porcine adipose-derived stem cells
CN107058219A (en) * 2017-04-13 2017-08-18 上海莱馥生命科学技术有限公司 A kind of method that application stem cell self-characteristic prepares dental pulp stem cell
CN107418930A (en) * 2017-09-04 2017-12-01 上海莱馥医疗科技有限公司 A kind of preparation method purified with amplification human marrow mesenchymal stem cell
CN107557331A (en) * 2017-09-15 2018-01-09 上海莱馥医疗科技有限公司 A kind of method for separating and cultivating human adipose-derived stem cell
CN107475188A (en) * 2017-09-25 2017-12-15 广东颜值科技有限公司 A kind of cultural method of cell culture medium and embryonic stem cell

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
SHUN-CHENG WU，等: "Enhancement of chondrogenesis of human adipose derived stem cells in a hyaluronan-enriched microenvironment", 《BIOMATERIALS》 *
李强，等: "透明质酸应用于干细胞培养的研究进展", 《中国生化药物杂志》 *
杨新建: "《动物细胞培养技术》", 31 August 2013, 中国农业大学出版社 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108893442A (en) * 2018-07-25 2018-11-27 广州赛莱拉干细胞科技股份有限公司 A kind of fat stem cell proliferated culture medium and its Multiplying culture method
CN109055309A (en) * 2018-08-02 2018-12-21 广州白云山拜迪生物医药有限公司 A kind of serum-free cell culture medium and application thereof
CN109055309B (en) * 2018-08-02 2021-07-02 广州白云山拜迪生物医药有限公司 Serum-free cell culture medium and application thereof
CN110343661A (en) * 2019-04-24 2019-10-18 朗姿赛尔生物科技（广州）有限公司 A kind of extraction of body fat stem cell and culture amplification method
CN110343661B (en) * 2019-04-24 2021-08-24 朗姿赛尔生物科技（广州）有限公司 Extraction, culture and amplification method of human adipose-derived stem cells
CN110373384A (en) * 2019-07-24 2019-10-25 安徽科门生物科技有限公司 A kind of cultural method of serum-free fat stem cell culture medium and fat stem cell
CN113736731A (en) * 2021-11-05 2021-12-03 保信亚太生物科技（深圳）有限公司 Serum-free medium of adipose tissue-derived mesenchymal stem cells and preparation method and application thereof

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