CN104232573A - Growth medium and use thereof, and method for cultivating umbilical cord mesenchymal stem cells - Google Patents
Growth medium and use thereof, and method for cultivating umbilical cord mesenchymal stem cells Download PDFInfo
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- CN104232573A CN104232573A CN201410459791.4A CN201410459791A CN104232573A CN 104232573 A CN104232573 A CN 104232573A CN 201410459791 A CN201410459791 A CN 201410459791A CN 104232573 A CN104232573 A CN 104232573A
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Abstract
The invention discloses a growth medium for cultivating stem cells. The growth medium contains a basal culture medium and an additive, wherein the additive contains an antibody for resisting a human vascular endothelial growth factor, an antibody for resisting a human epidermal growth factor receptor 1, an antibody for resisting a human epidermal growth factor receptor 2 and vernine-5-sodium triphosphate. The invention also provides use of the growth medium in cultivation of umbilical cord mesenchymal stem cells, and also discloses a method for cultivating the umbilical cord mesenchymal stem cells. According to the method, the umbilical cord mesenchymal stem cells are inoculated and cultivated in the growth medium. Through the technical scheme, the in vitro amplification capability of the umbilical cord mesenchymal stem cells can be greatly improved.
Description
Technical field
The present invention relates to technical field of cell biology, particularly, relate to a kind of for the growth medium of culturing stem cells, the purposes of this growth medium and the method for cultivating umbilical cord mesenchymal stem cells.
Background technology
Stem cell is that a class has not breaking up or PD cell of the of self-replication capacity and multi-lineage potential.Under certain condition, it can be divided into several functions cell.Potentiality of development according to stem cell is divided three classes: myeloid-lymphoid stem cell, multipotential stem cell and unipotent stem cell.
Mescenchymal stem cell, with a kind of multipotential stem cell, is the important member of stem cell line, derives from and grows early stage mesoderm and ectoderm.Mescenchymal stem cell has the features such as multi-lineage potential, hematopoiesis support and the implantation of promotion stem cell, immunoregulation and self-replacation because of it and day by day receives the concern of people.If mescenchymal stem cell is in vivo or under external specific inductive condition, the Various Tissues cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelium can be divided into, still there is multi-lineage potential after continuous passage cultivation and freezen protective, can be used as the injuries of tissues and organs reparation that desirable seed cell causes for old and feeble and pathology.Mescenchymal stem cell, due to its wide material sources, is easy to separation and Culture, and has stronger differentiation potential and can the advantage such as autotransplantation, has higher clinical value.
Umbilical cord mesenchymal stem cells refers to a kind of multipotential stem cell be present in neonatal umbilical cord tissue.Isolated umbilical cord mesenchymal stem cells from people's umbilical cord, its cell content and multiplication capacity are all better than mesenchymal stem cells MSCs, and immunogenicity is lower than mesenchymal stem cells MSCs, have in addition and draw materials conveniently, without advantages such as ethics disputes, therefore there is in mescenchymal stem cell higher clinical value.
In practical clinical, the sufficient amount of umbilical cord mesenchymal stem cells is the necessary factor ensureing stem-cell therapy.The necessary ways of the quantity being increase umbilical cord mesenchymal stem cells that in vitro umbilical cord mesenchymal stem cells increased.But current umbilical cord mesenchymal stem cells can only go down to posterity 15-20 generation in vitro, and go down to posterity within latter more than 3 days, just can reach more than 80% degrees of fusion, also there will be after these external 15 generations of going down to posterity and lose the phenomenon of differentiation potential.Therefore, there is the defect of amplification ability in the method for existing cultivation umbilical cord mesenchymal stem cells.
Summary of the invention
There is the defect of amplification ability in the method that the object of the invention is to overcome existing cultivation umbilical cord mesenchymal stem cells, provides a kind of for the growth medium of culturing stem cells, the purposes of this growth medium and the method for cultivating umbilical cord mesenchymal stem cells.
The present inventor finds, by adding specific antibody and ATP in basic medium, can improve the amplification ability of umbilical cord mesenchymal stem cells significantly, resulting in the present invention.
On the one hand, the invention provides a kind of growth medium for culturing stem cells, this growth medium contains basic medium and additive, and described additive contains the antibody of human vessel endothelium growth factor resisting, the antibody of anti-human ErbB1, the antibody of anti-human epidermal growth factor acceptor 2 and guanosine-5-triphosphoric acid trisodium salt.
On the other hand, present invention also offers a kind of method of cultivating umbilical cord mesenchymal stem cells, the method comprises: be inoculated in growth medium as above by umbilical cord mesenchymal stem cells and cultivate.
Again on the one hand, present invention also offers described growth medium and cultivate the purposes in umbilical cord mesenchymal stem cells.
By technique scheme, the present invention can promote umbilical cord mesenchymal stem cells amplification ability in vitro significantly.Such as, the subculture in vitro separately number of umbilical cord mesenchymal stem cells can be brought up to 25-40 generation by the present invention, and can reach the degrees of fusion of more than 80% in 40-60 hour after going down to posterity, and also there will not be the phenomenon of losing differentiation potential after these external 20 generations of going down to posterity.
Other features and advantages of the present invention are described in detail in embodiment part subsequently.
Accompanying drawing explanation
Accompanying drawing is used to provide a further understanding of the present invention, and forms a part for specification sheets, is used from explanation the present invention, but is not construed as limiting the invention with embodiment one below.In the accompanying drawings:
Fig. 1 is the growth curve chart of the umbilical cord mesenchymal stem cells grown in growth medium different in testing example 1.In Fig. 1,1 and 2 represent that preparation embodiment 1 and 2,3-6 represent preparation comparative example 1-4 respectively, and X-coordinate represents growth time, and ordinate zou represents that 550nm place measures each hole absorption photometric value (D value).
Embodiment
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in detail.Should be understood that, embodiment described herein, only for instruction and explanation of the present invention, is not limited to the present invention.
In the present invention, when not doing contrary explanation, the volumetric quantities of the liquid of use is the numerical value at 20 DEG C.
The invention provides a kind of growth medium for culturing stem cells, this substratum contains basic medium and additive, and described additive contains the antibody of human vessel endothelium growth factor resisting, the antibody of anti-human ErbB1, the antibody of anti-human epidermal growth factor acceptor 2 and guanosine-5-triphosphoric acid trisodium salt.
Wherein, human vascular endothelial growth factor (human vascular endothelial growth factor, hVEGF) refer to that gene accession number in NCBI (NCBI Gene ID) is the translation product of the gene of 7422, its official's full name (Official Full Name) is VEGF-A (vascular endothelial growth factor A, VEGFA), its common another name also comprises VPF, VEGF or MVCD1.
Wherein, human epidermal growth factor acceptor 1 (human epidermal growth factor receptor 1, HER1) refer to that gene accession number in NCBI (NCBI Gene ID) is the translation product of the gene of 1956, its official's full name (Official Full Name) is EGF-R ELISA (epidermal growth factor receptor, EGFR), its common another name also comprises EGFR, ERBB, mENA, ERBB1 or PIG61.
Wherein, ErbB-2 (human epidermal growth factor receptor2, HER2) refer to that gene accession number in NCBI (NCBI Gene ID) is the translation product of the gene of 2064, its official's full name (Official Full Name) is v-erb-b2 bird EBL viral oncogene autoploid 2 (v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2), its common another name also comprises NEU, NGL, TKR1, CD340, HER-2, MLN 19 or HER-2/neu.
Wherein, guanosine-5-triphosphoric acid trisodium salt (guanosine-5 '-triphosphate trisodium salt) refers to the compound shown in formula (1), and its No. CAS is 36051-31-7.
Wherein, the content of the antibody of described human vessel endothelium growth factor resisting can be 0.01-0.2ng/mL, the content of the antibody of described anti-human ErbB1 can be 0.01-0.1ng/mL, and the content of the antibody of described anti-human epidermal growth factor acceptor 2 can be 0.05-0.4ng/mL; The content of guanosine-5-triphosphoric acid trisodium salt can be 0.2-4ng/mL.
Wherein, according to the preferred embodiment of the present invention, the content of the antibody of human vessel endothelium growth factor resisting is 0.05-0.1ng/mL, the content of the antibody of anti-human ErbB1 is 0.03-0.08ng/mL, and the content of the antibody of anti-human epidermal growth factor acceptor 2 is 0.1-0.2ng/mL; The content of guanosine-5-triphosphoric acid trisodium salt is 0.5-2ng/mL.In this preferred implementation, growth medium of the present invention can strengthen the expanding effect to umbilical cord mesenchymal stem cells further.
Wherein, as long as the antibody of described human vessel endothelium growth factor resisting can be combined with human vascular endothelial growth factor specifically and hinder the enforcement of its biological function; As long as the antibody of described anti-human ErbB1 can be combined with human epidermal growth factor acceptor 1 specifically and hinder the enforcement of its biological function; As long as the antibody of described anti-human epidermal growth factor acceptor 2 can be combined with ErbB-2 specifically and hinder the enforcement of its biological function.Above-mentioned antibody is by being commercially available.According to the preferred embodiment of the present invention, the antibody of described human vessel endothelium growth factor resisting is rhuMAb-VEGF; The antibody of described anti-human ErbB1 is Cetuximab, and the antibody of described anti-human epidermal growth factor acceptor 2 is Herceptin.
Wherein, the English of described rhuMAb-VEGF is called Bevacizumab, and commodity are called Arastin (Avastin); The English of described Cetuximab is called Cetuximab, and commodity are called Erbitux (Erbitux); Described Herceptin English Trastuzumab by name, commodity are called Trastuzumab (Herceptin).
Wherein, the not special requirement of described basic medium, can can cultivate mammiferous substratum for conventional the various of use, such as, described basic medium includes but not limited at least one in DMEM substratum, MEM substratum, IMEM substratum and RPMI1640 substratum.Above-mentioned basic medium can be commercially available, such as, can buy from Gibco or Hyclone company and obtain.Such as, according to the goods catalogue of Gibco, the article number of the article number of DMEM substratum to be the article number of 11965, MEM substratum be 11095, IMEM substratum is the article number of 21700, RPMI1640 substratum is 11875.
Wherein, growth medium of the present invention can, for the substratum containing serum, also can be not containing the substratum of serum.In order to accelerate the adherent of umbilical cord mesenchymal stem cells, preferably, the serum of described growth medium also containing 5-20 volume %.Wherein, described serum can comprise at least one in foetal calf serum, new-born calf serum and calf serum, is more preferably foetal calf serum.
Wherein, the preparation method of above-mentioned growth medium of the present invention can comprise: mixed with described additive by basic medium.
Present invention also offers growth medium as above and cultivate the purposes in umbilical cord mesenchymal stem cells.
Present invention also offers a kind of method of cultivating umbilical cord mesenchymal stem cells, the method comprises: be inoculated in growth medium as above by umbilical cord mesenchymal stem cells and cultivate.
Wherein, the temperature of cultivation and CO
2concentration can be the selection of umbilical cord mesenchymal stem cells field routine; Such as, the temperature of cultivation can be 36-38 DEG C; The CO cultivated
2concentration can be 4-6 volume %.
Wherein, the present invention's growth medium as above can promote that umbilical cord mesenchymal stem cells increases quickly, and correspondingly shorten and go down to posterity the cycle, therefore under preferable case, the cycle of going down to posterity of cultivation is 40-60 hour.
Wherein, the present invention's growth medium as above can promote that umbilical cord mesenchymal stem cells extends passage number, and under preferable case, preferably, the algebraically gone down to posterity of cultivation is 25-40 generation.
Wherein, described umbilical cord mesenchymal stem cells can be obtained by tissue block adherent culture method or homogenate collagenase digestion.
Below, the present invention is further described by embodiment.
Preparation embodiment 1
At DMEM substratum (purchased from Gibco, article number is 11965) in add foetal calf serum (purchased from Gibco), rhuMAb-VEGF (purchased from Roche Holding Ag), Cetuximab (purchased from Merck & Co., Inc.), Herceptin (purchased from Roche Holding Ag) and guanosine-5-triphosphoric acid trisodium salt (purchased from Sigma, identical below); The amount of adding makes final concentration to be: foetal calf serum 10 volume %, rhuMAb-VEGF 0.08ng/mL, Cetuximab 0.06ng/mL, Herceptin 0.15ng/mL and guanosine-5-triphosphoric acid trisodium salt 1ng/mL; Obtain the growth medium that this prepares embodiment.
Preparation embodiment 2
Foetal calf serum (purchased from Gibco), rhuMAb-VEGF (purchased from Roche Holding Ag), Cetuximab (purchased from Merck & Co., Inc.), Herceptin (purchased from Roche Holding Ag) and guanosine-5-triphosphoric acid trisodium salt is added in DMEM substratum (purchased from Gibco, article number is 11965); The amount of adding makes final concentration to be: foetal calf serum 10 volume %, rhuMAb-VEGF 0.01ng/mL, Cetuximab 0.1ng/mL, Herceptin 0.05ng/mL and guanosine-5-triphosphoric acid trisodium salt 300 μ g/mL; Obtain the growth medium that this prepares embodiment.
Preparation comparative example 1
At DMEM substratum (purchased from Gibco, article number is 11965) in add foetal calf serum (purchased from Gibco), Cetuximab (purchased from Merck & Co., Inc.), Herceptin (purchased from Roche Holding Ag) and guanosine-5-triphosphoric acid trisodium salt (purchased from Sigma, identical below); The amount of adding makes final concentration to be: foetal calf serum 10 volume %, Cetuximab 0.06ng/mL, Herceptin 0.15ng/mL and guanosine-5-triphosphoric acid trisodium salt 1ng/mL; Obtain the growth medium that this prepares comparative example.
Preparation comparative example 2
At DMEM substratum (purchased from Gibco, article number is 11965) in add foetal calf serum (purchased from Gibco), rhuMAb-VEGF (purchased from Roche Holding Ag), Herceptin (purchased from Roche Holding Ag) and guanosine-5-triphosphoric acid trisodium salt (purchased from Sigma, identical below); The amount of adding makes final concentration to be: foetal calf serum 10 volume %, rhuMAb-VEGF 0.08ng/mL, Herceptin 0.15ng/mL and guanosine-5-triphosphoric acid trisodium salt 1ng/mL; Obtain the growth medium that this prepares comparative example.
Preparation comparative example 3
At DMEM substratum (purchased from Gibco, article number is 11965) in add foetal calf serum (purchased from Gibco), rhuMAb-VEGF (purchased from Roche Holding Ag), Cetuximab (purchased from Merck & Co., Inc.) and guanosine-5-triphosphoric acid trisodium salt (purchased from Sigma, identical below); The amount of adding makes final concentration to be: foetal calf serum 10 volume %, rhuMAb-VEGF 0.08ng/mL, Cetuximab 0.06ng/mL and guanosine-5-triphosphoric acid trisodium salt 1ng/mL; Obtain the growth medium that this prepares comparative example.
Preparation comparative example 4
At DMEM substratum (purchased from Gibco, article number is 11965) in add foetal calf serum (purchased from Gibco), rhuMAb-VEGF (purchased from Roche Holding Ag), Cetuximab (purchased from Merck & Co., Inc.), Herceptin (purchased from Roche Holding Ag) and guanosine-5-triphosphoric acid trisodium salt (purchased from Sigma, identical below); The amount of adding makes final concentration to be: foetal calf serum 10 volume %, rhuMAb-VEGF 0.08ng/mL, Cetuximab 0.06ng/mL and Herceptin 0.15ng/mL; Obtain the growth medium that this prepares comparative example.
Testing example 1
After c-section fetal birth, intercept umbilical cord, peel off amnion, peel off Wal Tong Shi glue (Wharton ' s jelly), shred into 0.5-1.5mm
3tissue block, add IV Collagenase Type (10g/L), after putting 37 DEG C of constant-temperature shaking incubators vibration digestion 3h, filter with 80 mesh filter screens, by the cell suspension centrifuge washing of filtration.Cell is mixed, with 1.0 × 10 with serum free medium (purchased from Cambrex, UItraCULTURE)
6/ cm
2inoculum density be inoculated in T-75cm
2in culturing bottle, be placed in 37 DEG C, saturated humidity, 5 volume % CO
2be cultured to the degrees of fusion of 80% in incubator, obtain 1st generation umbilical cord mesenchymal stem cells.
Adherent culture obtained above is had in the culturing bottle of 1st generation umbilical cord mesenchymal stem cells and add 2.5g/L trysinization, and being passaged in Tissue Culture Plate in the ratio of 1: 3, the growth medium used that goes down to posterity is preparation embodiment 1-2 and the growth medium that obtains of preparation comparative example 1-4.When reaching for the 3rd generation, use tetrazolium bromide (MTT) method to detect cytoactive and draw the growth curve of cell, concrete operations comprise: will grow to the 3rd generation umbilical cord mesenchymal stem cells enzymolysis suspension of 80% degrees of fusion, adjustment cell concn to 1 × 10
4individual/mL, adds 24 orifice plates, every hole 800 μ L, be placed in 37 DEG C, saturated humidity, 5 volume % CO
2cultivate in incubator, in 5 days, a 24 orifice plates are taken out every 8 hours, after every hole adds the MTT solution (5g/L) of 80 μ L, 4h is placed at 37 DEG C, the dimethyl sulfoxide (DMSO) (DMSO) of 600 μ L is added in every hole, each hole absorption photometric value (D value) is measured at 550nm place by microplate reader, the relative populations of cell is represented with D value, draw growth curve, result as shown in Figure 1, in Fig. 1, 1 and 2 represent preparation embodiment 1 and 2, 3-6 represents preparation comparative example 1-4 respectively, X-coordinate represents growth time, ordinate zou represents that 550nm place measures each hole absorption photometric value (D value).
Can find out according to growth curve, the umbilical cord mesenchymal stem cells in the growth medium that preparation embodiment 1 and 2 obtains can grow to plateau before 56 hours.But the umbilical cord mesenchymal stem cells in the growth medium that preparation comparative example 1-4 obtains can only can grow to plateau after 72 hours.This shows, growth medium of the present invention can accelerate the speed of growth of umbilical cord mesenchymal stem cells effectively.
Testing example 2
After c-section fetal birth, intercept umbilical cord, peel off amnion, peel off Wal Tong Shi glue (Wharton ' s jelly), shred into 0.5-1.5mm
3tissue block, add IV Collagenase Type (10g/L), after putting 37 DEG C of constant-temperature shaking incubators vibration digestion 3h, filter with 80 mesh filter screens, by the cell suspension centrifuge washing of filtration.Cell is mixed, with 1.0 × 10 with serum free medium (purchased from Cambrex, UItraCULTURE)
5/ cm
2inoculum density be inoculated in T-75cm
2in culturing bottle, be placed in 37 DEG C, saturated humidity, 5 volume % CO
2be cultured to the degrees of fusion of 80% in incubator, obtain 1st generation umbilical cord mesenchymal stem cells.
Adherent culture obtained above is had in the culturing bottle of 1st generation umbilical cord mesenchymal stem cells and add 2.5g/L trysinization, and be passaged in Tissue Culture Plate in the ratio of 1: 3, the growth medium that the growth medium going down to posterity use obtains for preparation embodiment 1-2, when being cultured to the degrees of fusion of 80%, by 1
:the ratio of 3 goes down to posterity, the growth medium that the growth medium going down to posterity use obtains for preparation embodiment 1-2, after carrying out and so forth being passaged to and obtaining the 30th generation umbilical cord mesenchymal stem cells, to the 30th generation umbilical cord mesenchymal stem cells carry out stem cell surface mark mensuration, the detection to osteoblast differentiation potential, the detection to Adipocyte Differentiation potential, the detection to quasi-liver cell differentiation potential, the detection to myoblast differentiation potential, the detection of differentiating into nerve cells potential and the detection to hematopoetic cell differentiation potential.Concrete detection method comprises: stem cell surface mark mensuration, the detection to osteoblast differentiation potential and the detection to Adipocyte Differentiation potential are according to document (Wang Juan etc., human umbilical cord mesenchymal stem cells in-vitro separation, Isolation and characterization, Ji'nan University's journal, 30th volume, 2009) method in is carried out; Detection to quasi-liver cell differentiation potential is carried out according to document (bear China etc., cryopreserved human umbilical cord blood-derived mesenchymal stem cells into hepatocyte-like cells differentiation-inducing, Chinese Tissue Engineering Study and clinical rehabilitation, the 7th phase in 2007); To myoblast differentiation potential detection according to document (Liu Dai, mesenchymal stem cells derived from human umbilical blood to the in vitro study of myoblast differentiation, master thesis, 2009) carry out; The detection of differentiating into nerve cells potential is carried out according to document (Sun Hongtao etc., umbilical cord mesenchymal stem cells separation, qualification and Neural Differentiation, Chinese neurosurgery related disease research magazine, in April, 2008); Detection to hematopoetic cell differentiation potential is carried out according to document (Hu Kaimeng etc., the research that human umbilical cord mesenchymal stem cells breaks up to hematopoietic cell direction, Ph.D. Dissertation, 2012).
The result of above-mentioned detection shows, the growth medium that the Umbilical cord blood mesenchymal stem cells of separation and Culture uses preparation embodiment 1-2 to obtain carries out subculture, when carry out 30 generations go down to posterity, also there is the specific surfaces mark of stem cell, to osteoblast differentiation potential, to Adipocyte Differentiation potential, to quasi-liver cell differentiation potential, to myoblast differentiation potential, differentiating into nerve cells potential and to hematopoetic cell differentiation potential.
As can be seen here, the subculture in vitro separately number of umbilical cord mesenchymal stem cells can improve by the present invention, and the speed of growth after going down to posterity is accelerated, this is external repeatedly go down to posterity after also there will not be the phenomenon of loss differentiation potential.
Below the preferred embodiment of the present invention is described in detail by reference to the accompanying drawings; but; the present invention is not limited to the detail in above-mentioned embodiment; within the scope of technical conceive of the present invention; can carry out multiple simple variant to technical scheme of the present invention, these simple variant all belong to protection scope of the present invention.
It should be noted that in addition, each concrete technical characteristic described in above-mentioned embodiment, in reconcilable situation, can be combined by any suitable mode, in order to avoid unnecessary repetition, the present invention illustrates no longer separately to various possible array mode.
In addition, also can carry out arbitrary combination between various different embodiment of the present invention, as long as it is without prejudice to thought of the present invention, it should be considered as content disclosed in this invention equally.
Claims (10)
1. the growth medium for culturing stem cells, this growth medium contains basic medium and additive, it is characterized in that: described additive contains the antibody of human vessel endothelium growth factor resisting, the antibody of anti-human ErbB1, the antibody of anti-human epidermal growth factor acceptor 2 and guanosine-5-triphosphoric acid trisodium salt.
2. growth medium according to claim 1, it is characterized in that: the content of the antibody of human vessel endothelium growth factor resisting is 0.01-0.2ng/mL, the content of the antibody of anti-human ErbB1 is 0.01-0.1ng/mL, and the content of the antibody of anti-human epidermal growth factor acceptor 2 is 0.05-0.4ng/mL; The content of guanosine-5-triphosphoric acid trisodium salt is 0.2-4ng/mL.
3. growth medium according to claim 2, it is characterized in that: the content of the antibody of human vessel endothelium growth factor resisting is 0.05-0.1ng/mL, the content of the antibody of anti-human ErbB1 is 0.03-0.08ng/mL, and the content of the antibody of anti-human epidermal growth factor acceptor 2 is 0.1-0.2ng/mL; The content of guanosine-5-triphosphoric acid trisodium salt is 0.5-2ng/mL.
4. according to the growth medium in claim 1-3 described in any, it is characterized in that: the antibody of described human vessel endothelium growth factor resisting is rhuMAb-VEGF; The antibody of described anti-human ErbB1 is Cetuximab, and the antibody of described anti-human epidermal growth factor acceptor 2 is Herceptin.
5. according to growth medium according to claim 1, it is characterized in that: described basic medium comprises at least one in DMEM substratum, MEM substratum, IMEM substratum and RPMI1640 substratum.
6. growth medium according to claim 1, is characterized in that: the serum of described growth medium also containing 5-20 volume %.
7. the growth medium in claim 1-6 described in any one is cultivating the purposes in umbilical cord mesenchymal stem cells.
8. cultivate a method for umbilical cord mesenchymal stem cells, it is characterized in that: the method comprises: umbilical cord mesenchymal stem cells is inoculated in the growth medium in claim 1-6 described in any one and cultivates.
9. method according to claim 8, is characterized in that: the cycle of going down to posterity of cultivation is 40-60 hour.
10. method according to claim 8 or claim 9, is characterized in that: the algebraically gone down to posterity of cultivation is 25-40 generation.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104774807A (en) * | 2015-04-28 | 2015-07-15 | 安沂华 | Method for inductively differentiating umbilical cord mesenchymal stem cells into oligodendroglia cells |
| CN108192861A (en) * | 2017-12-27 | 2018-06-22 | 重庆斯德姆生物技术有限公司 | A kind of preparation method of mescenchymal stem cell applied to climacteric syndrome |
| WO2020000452A1 (en) * | 2018-06-29 | 2020-01-02 | 深圳市博奥康生物科技有限公司 | Novel rnai interference fragment targeting human ngl gene, rnai vector,preparation method therefor and application thereof |
| WO2020000458A1 (en) * | 2018-06-29 | 2020-01-02 | 深圳市博奥康生物科技有限公司 | Grna targeting sequence for specifically targeting human ngl gene and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101525594A (en) * | 2009-04-17 | 2009-09-09 | 中国医学科学院血液学研究所 | Complete medium with low serum concentration for cultivating mesenchymal stem cells and method for cultivating mesenchymal stem cells using same |
| CN102634482A (en) * | 2012-01-13 | 2012-08-15 | 成都美进生物科技有限公司 | Serum-free complete medium for mesenchymal stem cell |
| CN102676452A (en) * | 2012-04-26 | 2012-09-19 | 天津美德太平洋科技有限公司 | Culture medium containing human umbilical cord mesenchymal stem cell exudates and preparation method and applications thereof |
| CN102978156A (en) * | 2012-11-13 | 2013-03-20 | 湖州市中心医院 | Expansion in vitro purification culture method of mesenchymal stem cells and culture medium |
| WO2013082543A1 (en) * | 2011-11-30 | 2013-06-06 | Advanced Cell Technology, Inc. | Mesenchymal stromal cells and uses related thereto |
| CN103421740A (en) * | 2013-07-25 | 2013-12-04 | 江苏奥思达干细胞有限公司 | In-vitro culture and proliferation method for human mesenchymal stem cells |
-
2014
- 2014-09-11 CN CN201410459791.4A patent/CN104232573B/en not_active Expired - Fee Related
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101525594A (en) * | 2009-04-17 | 2009-09-09 | 中国医学科学院血液学研究所 | Complete medium with low serum concentration for cultivating mesenchymal stem cells and method for cultivating mesenchymal stem cells using same |
| WO2013082543A1 (en) * | 2011-11-30 | 2013-06-06 | Advanced Cell Technology, Inc. | Mesenchymal stromal cells and uses related thereto |
| CN102634482A (en) * | 2012-01-13 | 2012-08-15 | 成都美进生物科技有限公司 | Serum-free complete medium for mesenchymal stem cell |
| CN102676452A (en) * | 2012-04-26 | 2012-09-19 | 天津美德太平洋科技有限公司 | Culture medium containing human umbilical cord mesenchymal stem cell exudates and preparation method and applications thereof |
| CN102978156A (en) * | 2012-11-13 | 2013-03-20 | 湖州市中心医院 | Expansion in vitro purification culture method of mesenchymal stem cells and culture medium |
| CN103421740A (en) * | 2013-07-25 | 2013-12-04 | 江苏奥思达干细胞有限公司 | In-vitro culture and proliferation method for human mesenchymal stem cells |
Non-Patent Citations (1)
| Title |
|---|
| 刘岱 等: "人脐带血间充质干细胞分离培养体系的优化筛选", 《中国组织工程研究与临床康复》, no. 10, 5 March 2009 (2009-03-05), pages 1839 - 1843 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104774807A (en) * | 2015-04-28 | 2015-07-15 | 安沂华 | Method for inductively differentiating umbilical cord mesenchymal stem cells into oligodendroglia cells |
| CN104774807B (en) * | 2015-04-28 | 2018-05-08 | 安沂华 | The method that umbilical cord mesenchymal stem cells are induced differentiation into oligodendroglia |
| CN108192861A (en) * | 2017-12-27 | 2018-06-22 | 重庆斯德姆生物技术有限公司 | A kind of preparation method of mescenchymal stem cell applied to climacteric syndrome |
| WO2020000452A1 (en) * | 2018-06-29 | 2020-01-02 | 深圳市博奥康生物科技有限公司 | Novel rnai interference fragment targeting human ngl gene, rnai vector,preparation method therefor and application thereof |
| WO2020000458A1 (en) * | 2018-06-29 | 2020-01-02 | 深圳市博奥康生物科技有限公司 | Grna targeting sequence for specifically targeting human ngl gene and application thereof |
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