CN105434468A - Preparation method of skin cell damage repairing reagent - Google Patents
Preparation method of skin cell damage repairing reagent Download PDFInfo
- Publication number
- CN105434468A CN105434468A CN201510945652.7A CN201510945652A CN105434468A CN 105434468 A CN105434468 A CN 105434468A CN 201510945652 A CN201510945652 A CN 201510945652A CN 105434468 A CN105434468 A CN 105434468A
- Authority
- CN
- China
- Prior art keywords
- cell
- umbilical cord
- stem cell
- culture
- preparation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
- A61K31/728—Hyaluronic acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
Abstract
The invention relates to a preparation method of a skin cell damage repairing reagent; P4-generation umbilical cord mesenchymal stem cells have a large number of cells, phenotypes are stable, a lot of mixed cytokines can be secreted, and the requirements of mass production of the cell damage repairing reagent can be met, so that the purpose that the cell damage repairing reagent is applied in clinical treatment of skin damage repairing is realized.
Description
Technical field
The invention belongs to biological technical field, be mainly used in the separation and Culture of attached cell, particularly a kind of preparation method of Skin Cell injury repairing reagent.
Background technology
Mescenchymal stem cell (mesenchymalstemcells, MSC) be the special stem cell of a class, derive from and grow early stage mesoderm and ectoderm, classification belongs to pluripotent stem cell, in vivo or under external specific inductive condition, MSC can be divided into adipose cell, osteocyte, chondrocyte, neurocyte, hepatocyte, myocardial cell, the Various Tissues cells such as endotheliocyte, 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 pathological changes, because it has the characteristic of multi-lineage potential, various diseases can be widely used in clinical and treat such as hepatic fibrosis, nerve injury, cerebral palsy, many difficult and complicated illness such as myocardial infarction, MSC does not express MHC (MHC) II class antigen, and does not express or the multiple transplantation immunity of low expression repels relevant costimulating factor, as CD80, CD40, CD46, CD40L is a para-immunity deficient cells, so there is very large treatment advantage in immunological diseases, can be used for graft versus host disease (GVHD), rheumatoid, lupus erythematosus, the immunodeficiency such as multiple sclerosis, simple hUC-MSC conditioned medium has hemopoiesis-supporting founction, and promote that CD34+ cell is to myeloid differentiation, so MSC has good treatment prospect in hematologic disease, MSC is one of focus of current stem-cell research.
MSC finds at first in bone marrow, passed through research deep in a large number afterwards, find that MSC is distributed widely in the bone marrow of fetus and adult, periosteum, spongy bone, fat, synovial membrane, skeletal muscle, tire liver, deciduous teeth, umbilical cord, Cord blood, wherein the mescenchymal stem cell purity in umbilical cord source is high, quantity is many, and drawing materials conveniently of umbilical cord, there is not ethical issues.
Umbilical cord is connected in the cord structures between embryo's umbilical part and Placenta Hominis, covers amniotic membrane outward, includes vein, umbilical artery and namely irrigate Dun Shi glue from mesoblastic glue sample mucus connective tissue.Large quantity research confirms that umbilical cord is irrigated in Dun Shi glue and is rich in abundant mescenchymal stem cell.
The current separation method to umbilical cord mesenchymal stem cells is a lot, separation method main at present utilizes collagenase digestion and piece of tissue creep plate method separating funicle mesenchyme stem cell, all there is certain drawback in two kinds of methods, 1. digestion method because umbilical cord individual variation and can not fully digest, the primary cell quantity obtained very little, can only obtain 3-5 × 10 after a umbilical cord digestion
5individual cell., there is disengaging time long in digestion method separating funicle mesenchyme stem cell, the problem that cost is high.2. although creep plate method does not use collagenase to reduce costs, cell climbs out of overlong time, and general 13-15 talent can have a small amount of cell to climb out of, the research progress of impact experiment.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of Skin Cell injury repairing reagent, in the present invention, hyaluronic acid is as one of composition in cell injury reparation reagent, obtained by Production by Microorganism Fermentation, there is purity high, output advantages of higher, can large-scale production be realized, commercial demand can be met.
The technical solution adopted for the present invention to solve the technical problems is:
1, a preparation method for Skin Cell injury repairing reagent, is characterized in that: be made up of the following step:
1. take fully the healthy fetal cord of the moon, the method combined with gold colloidal, quantitative fluorescent PCR detects cord serum HbsAg, anti-HCV, HCV-RNA, anti-HDV, anti-HEV, anti-HIV-1/2, HIV-1-RNA, CMV-DNA, testing result is negative umbilical cord and can uses;
2. umbilical cord is taken out from collecting bottle, be placed in sterilized petri dishes, cut the segment being cut into about 5cm with aseptic operation;
3. umbilical cord section is immersed in sterile beaker, repeatedly cleans with PBS and remain in endovascular blood, till the PBS after cleaning is substantially colourless;
4. the umbilical cord after cleaning is transferred in kidney basin, use operating scissors or scalpel to be cut into the fritter of 1-5mm, evenly bottom tiling to T75 culture bottle;
5. the culture bottle of the umbilical cord tissue block that tiled is put into CO2 gas incubator, dry 30 minutes;
6. in culture bottle, slowly add the DMEM/F-12 culture medium containing 10% hyclone additive, the consumption of culture medium is advisable with just submergence piece of tissue;
7. within every 6 days, change liquid 1 time, period tissues observed paste block border cell growing state, when most of piece of tissue cell climbs out of and merges about Growth of Cells to 80% around each block organization block, carry out the primary of umbilical cord mesenchymal stem cells and go down to posterity;
8. mescenchymal stem cell cell is reached P4 for time, by 8000cells/cm
2density be inoculated in T75 culture bottle, add 10mlDF12 complete medium;
9. until cell grow to about 80% merge time, with 5mLPBS wash buffer cell surface 3 times;
10. exhaust PBS buffer, add 10mLDF12 culture medium and continue cultivation 72 hours;
collect the umbilical cord mesenchymal stem cells culture supernatant of cultivation after 72 hours, supernatant is collected in 50mL centrifuge tube;
300g, centrifugal 5 minutes of room temperature;
abandon precipitation, collect stem cell culture supernatant, with the super filter tube of 3KD, stem cell supernatant is concentrated, concentrated 10 times, obtain the effective stem cell secretion hybrid cytokine containing more than 3KD molecular weight;
the filter membrane of stem cell secretion hybrid cytokine after concentrated with 0.22 μm is filtered, is placed in aseptic bottle ,-20 DEG C of preservations;
5g trehalose is added at stem cell secretion hybrid cytokine;
add 1mg hyaluronic acid at stem cell secretion hybrid cytokine, obtain Skin Cell injury repairing reagent.
And described trehalose adds concentration and is: 5%.
And described hyaluronic acid adds concentration and is: 1mg/mL.
The invention has the beneficial effects as follows:
1, in the present invention P4 to have cell quantity for umbilical cord mesenchymal stem cells many, phenotype is stablized, and a large amount of cell mixing factors can be secreted, the requirement that large-scale production cell injury repairs reagent can be met, thus realize cell injury being repaired the object that reagent is applied to the reparation of clinical treatment skin injury.
2, the present invention's super filter tube carries out ultrafiltration and concentration to the stem cell secretion factor collected, not only retain the effective cell mixing factor, the concentration of the cell mixing factor can be increased simultaneously, greatly can shorten the time that skin injury is repaired, improve the effect that skin injury is repaired.
3, in the present invention, stem cell mixing secrete cytokines obtains from stem cell incubation, obtain by having bioactive emiocytosis, compared with the cytokine obtained with chemosynthesis, biological activity is stronger, and be applied to clinical, there is lower immunogenicity.
4, in the present invention, the propagation of skin relevant cell and migration are the important steps in injured skin repair process, the cell injury prepared by this invention repairs reagent, propagation and the migration of skin relevant cell HSF cell can be promoted, can solve the core link in injured skin repair process, be serious skin injured patient relieving the pain caused by diseases.
Accompanying drawing explanation
Fig. 1 is the propagation that the stem cell secretion hybrid cytokine of variable concentrations can promote Hacat (Fig. 1-1), HSF (Fig. 1-2) cell;
Fig. 2 is the propagation that the hyaluronic acid of variable concentrations can promote Hacat (Fig. 2-1), HSF (Fig. 2-2) cell to a certain extent;
Fig. 3 is the propagation that Skin Cell injury repairing reagent can promote Hacat (Fig. 3-1), HSF (Fig. 3-2) cell;
Fig. 4 is that Skin Cell injury repairing reagent can reverse the damage Hacat (Fig. 4-1) of part because of hydrogen peroxide induction, the propagation of HSF (Fig. 4-2) cell;
Fig. 5 is Hacat repair time (Fig. 5-1), the HSF (Fig. 5-2) that Skin Cell injury repairing reagent significantly can shorten cut.
Detailed description of the invention
Below in conjunction with accompanying drawing, also by specific embodiment, the invention will be further described, and following examples are descriptive, are not determinate, can not limit protection scope of the present invention with this.
The invention provides a kind of preparation method of Skin Cell injury repairing reagent, the method is made up of the following step:
1. take fully the healthy fetal cord of the moon, the method combined with gold colloidal, quantitative fluorescent PCR detects cord serum HbsAg, anti-HCV, HCV-RNA, anti-HDV, anti-HEV, anti-HIV-1/2, HIV-1-RNA, CMV-DNA, testing result is negative umbilical cord and can uses;
2. umbilical cord is taken out from collecting bottle, be placed in sterilized petri dishes, cut the segment being cut into about 5cm with aseptic operation;
3. umbilical cord section is immersed in sterile beaker, repeatedly cleans with PBS and remain in endovascular blood, till the PBS after cleaning is substantially colourless;
4. the umbilical cord after cleaning is transferred in kidney basin, use operating scissors or scalpel to be cut into the fritter of 1-5mm, evenly bottom tiling to T75 culture bottle;
5. the culture bottle of the umbilical cord tissue block that tiled is put into CO2 gas incubator, dry 30 minutes;
6. in culture bottle, slowly add the DMEM/F-12 culture medium containing 10% hyclone additive, the consumption of culture medium is advisable with just submergence piece of tissue;
7. within every 6 days, change liquid 1 time, period tissues observed paste block border cell growing state, when most of piece of tissue cell climbs out of and merges about Growth of Cells to 80% around each block organization block, carry out the primary of umbilical cord mesenchymal stem cells and go down to posterity;
8. mescenchymal stem cell cell is reached P4 for time, by 8000cells/cm
2density be inoculated in T75 culture bottle, add 10mlDF12 complete medium;
9. until cell grow to about 80% merge time, with 5mLPBS wash buffer cell surface 3 times;
10. exhaust PBS buffer, add 10mLDF12 culture medium and continue cultivation 72 hours;
collect the umbilical cord mesenchymal stem cells culture supernatant of cultivation after 72 hours, supernatant is collected in 50mL centrifuge tube;
300g, centrifugal 5 minutes of room temperature;
abandon precipitation, collect stem cell culture supernatant, with the super filter tube of 3KD, stem cell supernatant is concentrated, concentrated 10 times, obtain the effective stem cell secretion hybrid cytokine containing more than 3KD molecular weight;
the filter membrane of stem cell secretion hybrid cytokine after concentrated with 0.22 μm is filtered, is placed in aseptic bottle ,-20 DEG C of preservations;
the cells and supernatant reagent of preparation 10%, 20%, the 50% different content stem cell secretion cell mixing factor, adds 5% trehalose simultaneously;
get sterilizing deionized water, add 0.05,0.1,0.2,0.5,1,2mg hyaluronic acid, prepare 0.05 respectively, 0.1,0.2,0.5, the hyaluronic acid aqueous solution of 1mg/mL, 2mg/mL, be mixed with the hyaluronic acid reagent of variable concentrations;
cell proliferation experiment: Hacat (application on human skin cutin horn cell), HSF (human skin fibroblast) cell are spread 96 orifice plates by 1000 cells/well;
bed board, after 24 hours, discards the former culture medium in 96 orifice plates, and every hole adds the cell reagent of preparation in 100 μ L steps 15 and 16, continues cultivation 72 hours;
discard the former culture medium in 96 orifice plates, every hole adds the serum-free medium of 100 μ L containing 10%CCK-8 reagent, hatches 2 hours for 37 DEG C;
detect the absorbance at OD450nm place by microplate reader, determine the best stem cell secretion cell mixing factor and hyaluronic acid contents;
the stem cell secretion cell mixing factor and hyaluronic compound method: get sterilizing deionized water, add a certain amount of hyaluronic acid, the hyaluronic acid aqueous solution that preparation is best, add the best stem cell secretion cell mixing factor more respectively, be mixed with hyaluronic acid and repair reagent containing the cell injury of the stem cell secretion cell mixing factor;
cell proliferation experiment: Hacat (application on human skin cutin horn cell), HSF (human skin fibroblast) cell are spread 96 orifice plates by 1000 cells/well;
bed board, after 24 hours, discards the former culture medium in 96 orifice plates, and the cell injury that every hole adds preparation in 100 μ L steps 21 repairs reagent, continues cultivation 72 hours;
discard the former culture medium in 96 orifice plates, every hole adds the serum-free medium of 100 μ L containing 10%CCK-8 reagent, hatches 2 hours for 37 DEG C;
the absorbance at OD450nm place is detected by microplate reader;
cell scratch experiment: get 6 orifice plates, carries out equal division line at the bottom of the plate of 6 orifice plates, and 5 lines are drawn in every hole, and spacing is 0.5cm;
by HSF (human skin fibroblast) by 4-6*10
5individual cells/well spreads 6 orifice plates, and culture medium is DMEM in high glucose+5%-10%FBS, and every hole adds 2-3mL culture medium culturing;
cell scoring devices within second day, is used to carry out scratching to cell;
with PBS buffer solution for cleaning cell surface twice, thoroughly cleaning is because of the cell debris of cut generation;
exhaust PBS buffer, the cell injury that every hole adds preparation in 2mL step 21 repairs reagent;
after cut, 0h, 6h, the 9h ad-hoc location to line is taken pictures respectively, carries out label record;
with Image software analysis cut photo, on cell migration result processes, and calculates cell migration rate.
Claims (3)
1. a preparation method for Skin Cell injury repairing reagent, is characterized in that: be made up of the following step:
1. take fully the healthy fetal cord of the moon, the method combined with gold colloidal, quantitative fluorescent PCR detects cord serum HbsAg, anti-HCV, HCV-RNA, anti-HDV, anti-HEV, anti-HIV-1/2, HIV-1-RNA, CMV-DNA, testing result is negative umbilical cord and can uses;
2. umbilical cord is taken out from collecting bottle, be placed in sterilized petri dishes, cut the segment being cut into about 5cm with aseptic operation;
3. umbilical cord section is immersed in sterile beaker, repeatedly cleans with PBS and remain in endovascular blood, till the PBS after cleaning is substantially colourless;
4. the umbilical cord after cleaning is transferred in kidney basin, use operating scissors or scalpel to be cut into the fritter of 1-5mm, evenly bottom tiling to T75 culture bottle;
5. the culture bottle of the umbilical cord tissue block that tiled is put into CO2 gas incubator, dry 30 minutes;
6. in culture bottle, slowly add the DMEM/F-12 culture medium containing 10% hyclone additive, the consumption of culture medium is advisable with just submergence piece of tissue;
7. within every 6 days, change liquid 1 time, period tissues observed paste block border cell growing state, when most of piece of tissue cell climbs out of and merges about Growth of Cells to 80% around each block organization block, carry out the primary of umbilical cord mesenchymal stem cells and go down to posterity;
8. mescenchymal stem cell cell is reached P4 for time, by 8000cells/cm
2density be inoculated in T75 culture bottle, add 10mlDF12 complete medium;
9. until cell grow to about 80% merge time, with 5mLPBS wash buffer cell surface 3 times;
10. exhaust PBS buffer, add 10mLDF12 culture medium and continue cultivation 72 hours;
collect the umbilical cord mesenchymal stem cells culture supernatant of cultivation after 72 hours, supernatant is collected in 50mL centrifuge tube;
300g, centrifugal 5 minutes of room temperature;
abandon precipitation, collect stem cell culture supernatant, with the super filter tube of 3KD, stem cell supernatant is concentrated, concentrated 10 times, obtain the effective stem cell secretion hybrid cytokine containing more than 3KD molecular weight;
the filter membrane of stem cell secretion hybrid cytokine after concentrated with 0.22 μm is filtered, is placed in aseptic bottle ,-20 DEG C of preservations;
5g trehalose is added at stem cell secretion hybrid cytokine;
add 1mg hyaluronic acid at stem cell secretion hybrid cytokine, obtain Skin Cell injury repairing reagent.
2. the preparation method of Skin Cell injury repairing reagent according to claim 1, is characterized in that: described trehalose adds concentration and is: 5wt%.
3. the preparation method of Skin Cell injury repairing reagent according to claim 1, is characterized in that: described hyaluronic acid adds concentration and is: 1mg/mL.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510945652.7A CN105434468A (en) | 2015-12-15 | 2015-12-15 | Preparation method of skin cell damage repairing reagent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510945652.7A CN105434468A (en) | 2015-12-15 | 2015-12-15 | Preparation method of skin cell damage repairing reagent |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105434468A true CN105434468A (en) | 2016-03-30 |
Family
ID=55545402
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510945652.7A Pending CN105434468A (en) | 2015-12-15 | 2015-12-15 | Preparation method of skin cell damage repairing reagent |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105434468A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106367388A (en) * | 2016-11-14 | 2017-02-01 | 天津市康婷生物工程有限公司 | Method for protecting umbilical cord mesenchymal stem cell culture supernatant at 4 DEG C |
| CN107261213A (en) * | 2017-06-13 | 2017-10-20 | 北京恒峰铭成生物科技有限公司 | A kind of active hydrogel and preparation method thereof and the application in operation wound reparation |
| CN107779431A (en) * | 2016-08-30 | 2018-03-09 | 天津市康婷生物工程有限公司 | The preparation method of stem cell medicine |
| CN107779430A (en) * | 2016-08-30 | 2018-03-09 | 天津市康婷生物工程有限公司 | The collection method of umbilical cord mesenchymal stem cells supernatant |
| CN110368356A (en) * | 2019-08-08 | 2019-10-25 | 北京京蒙细胞生物科技股份有限公司 | Skin injury reparative factor composition and preparation method thereof |
| CN112334144A (en) * | 2018-08-03 | 2021-02-05 | 株式会社细胞应用技术研究所 | Method for producing culture supernatant preparation |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101864471A (en) * | 2010-02-02 | 2010-10-20 | 天津市康婷生物工程有限公司 | Microbial fermentation method for producing hyaluronic acid |
| CN104027794A (en) * | 2014-06-19 | 2014-09-10 | 徐妍 | Application of human umbilical cord mesenchymal stem cell complex cell factor in preparing biological agent for repairing skin injury |
| CN104946585A (en) * | 2015-07-07 | 2015-09-30 | 美天妮（天津）生物科技有限公司 | Production and freeze-drying method and application for supernate of culture solution of mesenchymal stem cell |
| CN105126087A (en) * | 2015-09-23 | 2015-12-09 | 福建龙生生物科技有限公司 | External solution preparation for repairing skin wounds, preparation method thereof and application |
-
2015
- 2015-12-15 CN CN201510945652.7A patent/CN105434468A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101864471A (en) * | 2010-02-02 | 2010-10-20 | 天津市康婷生物工程有限公司 | Microbial fermentation method for producing hyaluronic acid |
| CN104027794A (en) * | 2014-06-19 | 2014-09-10 | 徐妍 | Application of human umbilical cord mesenchymal stem cell complex cell factor in preparing biological agent for repairing skin injury |
| CN104946585A (en) * | 2015-07-07 | 2015-09-30 | 美天妮（天津）生物科技有限公司 | Production and freeze-drying method and application for supernate of culture solution of mesenchymal stem cell |
| CN105126087A (en) * | 2015-09-23 | 2015-12-09 | 福建龙生生物科技有限公司 | External solution preparation for repairing skin wounds, preparation method thereof and application |
Non-Patent Citations (1)
| Title |
|---|
| 胡克苏: "人脐带间充质干细胞移植对大鼠烧伤治疗作用及其机制", 《中国博士学位论文全文数据库》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107779431A (en) * | 2016-08-30 | 2018-03-09 | 天津市康婷生物工程有限公司 | The preparation method of stem cell medicine |
| CN107779430A (en) * | 2016-08-30 | 2018-03-09 | 天津市康婷生物工程有限公司 | The collection method of umbilical cord mesenchymal stem cells supernatant |
| CN106367388A (en) * | 2016-11-14 | 2017-02-01 | 天津市康婷生物工程有限公司 | Method for protecting umbilical cord mesenchymal stem cell culture supernatant at 4 DEG C |
| CN106367388B (en) * | 2016-11-14 | 2021-01-01 | 天津市康婷生物工程集团有限公司 | Umbilical cord mesenchymal stem cell culture supernatant 4 ℃ protection method |
| CN107261213A (en) * | 2017-06-13 | 2017-10-20 | 北京恒峰铭成生物科技有限公司 | A kind of active hydrogel and preparation method thereof and the application in operation wound reparation |
| CN112334144A (en) * | 2018-08-03 | 2021-02-05 | 株式会社细胞应用技术研究所 | Method for producing culture supernatant preparation |
| CN110368356A (en) * | 2019-08-08 | 2019-10-25 | 北京京蒙细胞生物科技股份有限公司 | Skin injury reparative factor composition and preparation method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105434468A (en) | Preparation method of skin cell damage repairing reagent | |
| CN106754674B (en) | The method and its application of amnion mesenchymal stem cell are prepared from Human plactnta amnion | |
| CN103266081B (en) | Efficient method for isolating and culturing mesenchymal stem cells from umbilical cord | |
| CN102676452B (en) | Culture medium containing human umbilical cord mesenchymal stem cell exudates and preparation method and applications thereof | |
| KR100908481B1 (en) | Mesenchymal stem cell culture medium and culture method of mesenchymal stem cells using the same | |
| KR100871984B1 (en) | Multipotent Stem Cell Derived from Placenta Tissue and Cellular Therapeutic Agents Comprising the Same | |
| CN105154395A (en) | Preparing method for clinic level cells for enhancing immune modulating function of MSCs | |
| CN102127522A (en) | Human umbilical mesenchymal stem cell and preparation method thereof | |
| CN108220229B (en) | Preparation method for improving output of umbilical cord source mesenchymal stem cell primary cells | |
| CN105238750B (en) | A kind of method of recovery umbilical cord mesenchymal stem cells | |
| CN104762257B (en) | A kind of method preparing mescenchymal stem cell from umbilical cord | |
| CN104099294A (en) | Medium based on stem cell-excreted factor and its preparation method and use method | |
| CN108300690A (en) | A kind of isolated culture method and serum free medium of fat mesenchymal stem cell | |
| CN104818264A (en) | Digestive enzyme composition, and preparation and application thereof | |
| CN104651305A (en) | Method for acquiring bioactive proteins by utilizing umbilical cord mesenchymal stem cells | |
| CN109260227A (en) | It is a kind of for treating the autologous fat mesenchymal stem cell injection preparation method of Alzheimer's disease | |
| CN104673748A (en) | Method for extracting mesenchymal stem cells from human umbilical cord | |
| CN104983742A (en) | Stem cell preparation for treating degenerative osteoarthropathy and preparation method of stem cell preparation | |
| CN105505865A (en) | Separation method for umbilical cord mesenchymal stem cells | |
| CN109251890A (en) | A kind of method and its application of high efficiency extraction endometrium mescenchymal stem cell | |
| CN108504628A (en) | The cultural method of human umbilical cord mesenchymal stem cells | |
| CN106434543A (en) | Culture medium and cell cultural method | |
| CN112280735B (en) | Umbilical cord-derived mesenchymal stem cells and preparation method and application thereof | |
| CN109266605A (en) | A kind of method of eye pouch fat stem cell separation and in vitro culture | |
| CN101914496B (en) | Simplified method for preparing umbilical mesenchymal stem cells in large scale |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160330 |
|
| RJ01 | Rejection of invention patent application after publication |