CN102127522A - Human umbilical mesenchymal stem cell and preparation method thereof - Google Patents
Human umbilical mesenchymal stem cell and preparation method thereof Download PDFInfo
- Publication number
- CN102127522A CN102127522A CN 201010605542 CN201010605542A CN102127522A CN 102127522 A CN102127522 A CN 102127522A CN 201010605542 CN201010605542 CN 201010605542 CN 201010605542 A CN201010605542 A CN 201010605542A CN 102127522 A CN102127522 A CN 102127522A
- Authority
- CN
- China
- Prior art keywords
- cell
- mesenchymal stem
- stem cell
- human umbilical
- stem cells
- 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.)
- Granted
Links
Images
Abstract
The invention discloses a human umbilical mesenchymal stem cell and a preparation method thereof. The human umbilical mesenchymal stem cell is prepared by carrying out aseptic treatment, mechanical shearing, Wharton jelly exposure, collagenase digestion and isolated culture on an umbilical stalk of a healthy fetus by a term abdominal delivery. After freezing and thawing, the cell activity does not decline dramatically, thereby ensuring that the human umbilical mesenchymal stem cell is an ideal seed cell for cellular therapy. The invention also relates to a primitive mesenchymal stem cell prepared by the method, and the primitive mesenchymal stem cell is confirmed as the mesenchymal stem cell by the identification of morphology, immunophenotype and invitro differentiation experiments in accordance with the mesenchymal stem cell standards established by International Society for Cellular Therapy (ISCT). The cell count, cell activity, purity, doubling time and multiplication capacity of the human umbilical mesenchymal stem cell are apparently higher than those of bone marrow-derived mesenchymal stem cell.
Description
Technical field
The present invention relates to the preparation method of primary mesenchymal stem cells, relating in particular to people's umbilical cord jelly of Wharton is material preparation human umbilical cord mesenchymal stem cells and preparation method thereof.
Technical background
(Mesenchymal stem cell MSC) is the tissue stem cell that a class has multidirectional differentiation potential to mescenchymal stem cell, can obtain from multiple tissue such as fetal blood, liver, marrow, fat.There is the possibility of height virus pollution in bone marrow derived MSC, and obvious downtrending appears in its cell quantity and amplification, differentiation capability with age.Additive method has certain difficulty as cultivate MSC from bleeding of the umbilicus and peripheral blood, and MSC quantity in the peripheral blood of the bleeding of the umbilicus of term birth or mobilization remains in dispute.Come in to have scholar separation and Culture MSC from the rejected material umbilical cord in puerperium, and carry out biology phenotypic evaluation such as morphology, differentiation function and surface marker.Umbilical cord is to be connected in the cord structures that embryo's umbilical region and placenta are asked, outward by amnion, include the mucus reticular tissue of differentiation, except that the yolk sac and allantois of locking, also have Umbilical artery and umbilical vein in the reticular tissue.MSC in umbilical cord source is divided into four kinds: 1. derive from jelly of Wharton (Wharton ' s jelly, WJ), it is the circumvascular Saliva Orthana sample tissue of parcel, apart from the about 3mm of blood vessel periphery, be rich in hyaluronic acid and glycosaminoglycan, formed inoblast hydrogel structure on every side; 2. derive from around the umbilical blood vessels; 3. derive from bleeding of the umbilicus; 4. derive under the umbilical vein blood vessel endothelium.McElreavey etc. find a kind of fibroblast-like cells with high differentiation potential from the WJ tissue of people's umbilical cord.Progenitor cell is rich in discovery WJ zones such as Sarugaser, this regional cell be called as human cord blood pipe peripheral cells (human umbilical cord perivascular cell, HUCPV).The HUCPV cell has higher fibroblast-like cells colony and forms ability, but and bone induce after proliferation and differentiation form the bone tubercle.Employings such as Romanov are cultivated the method for bone marrow MSCs and are cultivated, and are separated to the cell of similar bone MSCs from human umblilical vein endothelial and subendothelial layer.Above-mentioned studies show that, umbilical cord tissue are contained abundant MSCs, can become the good source of MSCs.
The method of separation and cultivator umbilical cord MSC is not quite similar at present, and separating effect is also far from each other.Separation method comprises collagenase digestion substantially, plant piece method or both combinations, umbilical vein inner membrance digestion method, and wherein the collagenase digesting fado is based on collagenase II.
People's umbilical cord MSC can be the ideal tissue engineering seed cell to multiple histocyte differentiation under suitable inductive condition.MSC induces to implant and is expected to become more widely cell therapy in section.Studies show that MSC can implant the muscle deterioration tissue and to myocyte's differentiation, also can promote the implantation of hemopoietic stem cell; MSC also can be applicable to biotechnology such as cartilaginous tissue reconstruction; Also may become the carrier cell that carries suicide gene or anti-cancer adenoviruses medicine in the following anticancer therapy.
Summary of the invention
The technical problem to be solved in the present invention is, provide a kind of material source to be easy to get, human umbilical cord mesenchymal stem cells that cell doubling time is short and preparation method thereof, the mescenchymal stem cell for preparing by the inventive method has stronger self and multidirectional differentiation potential, and is easy to freezing preservation.
Realize the technical scheme of the object of the invention, adopting fetal cord is the raw material preparing mescenchymal stem cell.The preparation method is: after umbilical cord takes out from preservation liquid, cut off bilateral ligation part, remove extravasated blood in the cord vessels, the umbilical cord immersion is contained among antibiotic Hanks ' the Balanced Salt Solution (HBSS) (1 *), wash umbilical cord and umbilical vein inner chamber repeatedly 3 times with D-PBS, reject blood vessel, expose jelly of Wharton, then umbilical cord is cut into 1-3mm
3Put into reagent bottle after the tissue block of size, add 0.1% Digestive system, place to continue digestion 4-10h in 37 ℃ of isothermal vibration instrument, 100 eye mesh screens filter, centrifugal collecting cell.Add HBSS liquid flushing cell 3 times,, adjust cell density 4.8 * 10 with DMEM/F12 culture medium solution re-suspended cell
3～1 * 10
4/ cm
2, being inoculated in 6 orifice plates, 37 ℃, volume fraction are 5%CO
2Cultivate in the incubator, change liquid behind the 24h, changed liquid once every 3 days later on, when treating that cell reaches 80% fusion, the cultivation of going down to posterity, use or freezing preservation are standby immediately.
As optimization, the mescenchymal stem cell of preparation is around umbilical cord jelly of Wharton, the cord vessels, any source under bleeding of the umbilicus and the umbilical vein blood vessel endothelium or all.
The solution that is used to clean umbilical cord and blood vessel remained blood thereof is the D-PBS solution that does not contain calcium ions and magnesium ions; The preferred serum-free DMEM/F12 of the flushing used liquid of jelly of Wharton tissue block culture medium solution.
As optimization, umbilical cord is to be cut into 1-3mm after cleaning blood vessel through D PBS
3Tissue block, enter next separable programming.
The preparation method who is used for primary mesenchymal stem cells, available II Collagen Type VI enzyme digestion, IV Collagen Type VI enzyme digestion, II Collagen Type VI enzyme and pancreatin associating digestion method or II Collagen Type VI enzyme and Unidasa mixed solution digestion umbilical cord tissue, as optimization, first-selected II Collagen Type VI enzyme and Unidasa mixed solution digest.
As optimization, the Digestive system that is used to digest the umbilical cord tissue piece is the mixing solutions of II Collagen Type VI enzyme and Unidasa, and its quality concentration of volume percent is 0.1%.
As optimization, the nutrient solution that inoculating cell uses is to contain volume fraction to be the FBS of 10-20% and the DMEM/F12 of 25mmol/L glutamine.
For mescenchymal stem cell according to method for preparing of the present invention, carry out the biology phenotypic evaluation according to the standard that " international cell therapy association " (ISCT) formulates, show following technical characterictic:
1) cell attachment growth under best culture system condition can form the CFU-F colony, and the relative homogeneous of form being the spindle cell of be arranged in parallel growth or swirl shape growth;
2) expression of adhesion molecules receptor marker thing CD29, CD44, mesenchymal cell mark CD73, CD105, stem cell labeling thing CD90; And do not express endothelial cell marker thing CD31 and hemopoietic stem cell sign CD34, yet expression of HLA-DR not;
3) external scleroblast and the cardiac-like muscle cell of being induced to differentiate into.
Above-mentioned qualification result shows the mescenchymal stem cell non-hematopoietic stem cell that is prepared by invention, and has above-mentioned technical characterictic, meets the mescenchymal stem cell product acceptance criteria.
The mescenchymal stem cell of pressing the inventive method preparation is in 5-6 days culture cycle, cell concentration can be bred 20 times, can be rich in active mescenchymal stem cell in a large number, and can preserve for a long time and do not lose its activity, and operation is simple, and through flow cytometer detection, cell cycle detection and vitro differentiation experimental identification, the mescenchymal stem cell purity and the cytoactive that obtain are higher, has good multiplication potentiality, can set up cell bank fast, with low cost, can be directly used in scientific experiment research and assisting therapy clinically, be rich in application prospect.
Description of drawings
Fig. 1 is the cellular form figure of the adherent back of primary cell to 80% fusion, and wherein, A is the 1st day cellular form; B is the 3rd day cellular form; C is the 7th day cellular form.
Fig. 2 is the immunophenotype cell content figure of the 4th generation mescenchymal stem cell flow cytometer detection.
Fig. 3 A is external evoked scleroblast and the cardiac-like muscle cell aspect graph of being divided into of the 4th generation mescenchymal stem cell--a bone sialoprotein immunohistochemical staining.
Fig. 3 B is that external evoked scleroblast and the cardiac-like muscle cell aspect graph of being divided into of the 4th generation mescenchymal stem cell--RT-PCR detects the expression electrophorogram of myocardium specific gene, swimming lane 1,2-α-muscle rhabdomyosarcoma filamentous actin; Swimming lane 3,4-cardiac muscle sarcoplasmic reticulum calcium atpase; 5,6-people's cardiac muscle transcription factor GATA4; Swimming lane 7,8-α-myoglobulin heavy chain; Swimming lane 9,10 people cardiac muscle transcription factor NKX2.5; Swimming lane 11, the 12-Troponin I; Swimming lane 13,14-confidential reference items GADPH.
Embodiment
The present invention is further illustrated below in conjunction with the drawings and specific embodiments, but the present invention is not limited to following embodiment.
Embodiment 1: the preparation of mesenchymal stem cells of human umbilical cord and placenta
Pluripara's informed consent is gathered mature dose of palace and is produced healthy fetal cord or placenta.The puerpera need do detections such as antibody of AIDS virus, hepatitis B virus antibody, antibody of HCV, syphilis helicoid antibody, gpt, mycoplasma before umbilical cord or the placenta collection, all qualifiedly can gather after the security guaranteeing.
1, umbilical cord or placenta are after operating table takes off, and immersion contains in antibiotic 0.9% physiological saline 4 ℃ of preservations;
2, in the clean platform of behaviour, take out umbilical cord or placenta, wash clean surperficial residual blood with D-PBS;
3, umbilical cord or placenta are cut into 1-3mm
3Put into the blue lid of 200mL reagent bottle after the tissue block of size, add the Digestive system of quality concentration of volume percent 0.1%, place to continue digestion 4～10h in 37 ℃ of constant temperature shakers;
4,100 eye mesh screens filter collecting cell;
5, add D-Hank ' s liquid flushing cell 3 times, with containing the DMEM/F12 re-suspended cell that volume fraction is 10-20% foetal calf serum and glutamine, adjusting cell density is (4.8 * 10
3～1 * 10
4)/cm
2, be inoculated in 6 orifice plates, in 37 ℃, volume fraction 5% CO
2Cultivate in the incubator, change liquid behind the 24-72h, Fig. 1.
Embodiment 2: the cell characteristic of mesenchymal stem cells of human umbilical cord and placenta
1, immunophenotype detects CD29, CD44, and, CD73, CD90, CDl05, CD31, CD34, HLA-DR (seeing Table 1).4-7 is inoculated in for the mescenchymal stem cell in human umbilical cord and placenta source rolls in the bottle, treat that cell reaches when 80-90% merges to use the 10ml trysinization, change over to cell suspension in the 50ml centrifuge tube and centrifugal 3 minutes with 1000rpm.After centrifugal, discard supernatant, PBS washing 2 times is divided into every pipe 1 * 10
6Cell, first pipe is blank (only containing mescenchymal stem cell), is used to regulate the voltage of flow cytometer, second pipe adds homotype control antibodies (PE-MOUSE IgGl/FITC-MOUSE IgG1/APC-Mouse IgG1 κ) 10 μ l, add other corresponding antibodies 10 μ l in all the other pipes, mixing, 4 ℃ of lucifuges were hatched 30 minutes, PBS washing 1 time, abandon supernatant after centrifugal, it is resuspended to add 500 μ l PBS, mixing, (flow cytometer FACSAria, BD company) detection promptly is available on the machine.The cellular immunization phenotype is: CD29, CD44, CD73, CD90, the positive mark of CD105, CD31, CD34, the negative mark of HLA-DR (seeing Table 1,2).
The surface marker of table 1 preferred detection mesenchymal stem cells of human umbilical cord and placenta of the present invention
The detected result of table 2 mesenchymal stem cells of human umbilical cord and placenta surface marker
2, the skeletonization of cell and become myocardium differentiation potential and differentiation back is identified
The 4-6 of digestion amplification in vitro is for mescenchymal stem cell, with 5 * 10
4Total cellular score is inoculated in 6 orifice plates, and every hole adds 2ml DMEM/F12 nutrient solution.When cell reaches the 60%-80% fusion, change corresponding inductive differentiation medium.
2.1 osteogenic induction differentiation
Inductive differentiation medium is the dexamethasone that contains 100nmol/L, the sodium of 10mmol/L, the DMEM/F12 nutrient solution of 50 μ mol/L vitamins Cs and 10%FBS, changed liquid 1 time every 2 days, cultivated 21 days, PBS washed cell 1 time, 70% ethanol is fixed 10 minutes, row bone sialoprotein immunohistochemical staining is identified the nodular generation of calcium.Microscopically is observed, the expression that is positive of the cell after inducing.
Induce differentiation 2.2 become cardiac muscle
Inductive differentiation medium is to contain the U-18496 of 10 μ mol/L and the DMEM/F12 nutrient solution of 10%FBS, adds induced liquid after 24 hours, is replaced by the DMEM/F12 nutrient solution of 10%FBS immediately, changes liquid 1 time every 2 days later on, cultivates for 8 weeks.Extract the RNA of cell, carry out RT-PCR and detect people cardiac muscle transcription factor GATA4 and NKX2.5, myocardium sarcoplasmic reticulum calcium atpase, Troponin I, the expression of α-muscle rhabdomyosarcoma filamentous actin.The result shows that these genes all have expression to a certain degree.Shown in Fig. 3 A, 3B, RT-PCR detects confirmation, does not express above-mentioned myocardial cell's mark before people's umbilical cord MSCs induces, and myocardial cell's mark all has expression to a certain degree after 5-Aza induces.
The mescenchymal stem cell of aforesaid method amplification is in 5-6 days culture cycle, cell concentration can be bred 20 times, can be rich in active umbilical cord and placenta mesenchymal stem cell in a large number, and can preserve for a long time and do not lose its activity, and operation is simple, and detect and the vitro differentiation experimental identification through flow cytometer, the mescenchymal stem cell purity and the cytoactive that obtain are higher, has good multiplication potentiality, can set up cell bank fast, with low cost, can be directly used in scientific experiment research and assisting therapy clinically, be rich in application prospect.
In sum, content of the present invention is not limited in the above-described embodiment, and the knowledgeable people in the same area can propose other embodiment easily within technical director's thought of the present invention, but this embodiment comprises within the scope of the present invention.
Claims (10)
1. preparing human umbilical mesenchymal stem cells, it is characterized in that, it is to adopt following processing method preparation: after preserving liquid taking-up umbilical cord, cut off bilateral ligation part, remove extravasated blood in the cord vessels, the umbilical cord immersion is contained among antibiotic Hanks ' the Balanced Salt Solution, wash umbilical cord and umbilical vein inner chamber repeatedly 3 times with D-PBS, reject blood vessel, expose jelly of Wharton, then umbilical cord is cut into 1-3mm
3Put into reagent bottle after the tissue block of size, adding quality concentration of volume percent is 0.1% Digestive system, places to continue digestion 4～10h in 37 ℃ of isothermal vibration instrument, and 100 eye mesh screens filter, centrifugal collecting cell; Add HBSS liquid flushing cell 3 times,, adjust cell density 4.8 * 10 with DMEM/F12 culture medium solution re-suspended cell
3～1 * 10
4/ cm
2, being inoculated in 6 orifice plates, 37 ℃, volume fraction are 5%CO
2Cultivate in the incubator, change liquid behind the 24h, changed liquid once every 3 days later on, when treating that cell reaches 80% fusion, the cultivation of going down to posterity, use or freezing preservation are standby immediately.
2. preparing human umbilical mesenchymal stem cells according to claim 1 is characterized in that, the mescenchymal stem cell of preparation is around umbilical cord jelly of Wharton, the cord vessels, any source under bleeding of the umbilicus and the umbilical vein blood vessel endothelium or all.
3. preparing human umbilical mesenchymal stem cells according to claim 1, it is characterized in that, described umbilical cord adopts mature dose of palace to produce healthy fetal cord, and the puerpera has done antibody of AIDS virus, hepatitis B virus antibody, antibody of HCV, syphilis helicoid antibody, gpt, detection of mycoplasma before gathering, and all.
4. preparing human umbilical mesenchymal stem cells according to claim 4 is characterized in that, the umbilical cord after the collection places 4 ℃ of preservations, handles within the 6h.
5. preparing human umbilical mesenchymal stem cells according to claim 1 is characterized in that, described D-PBS solution is calcium ions and magnesium ion not.
6. preparing human umbilical mesenchymal stem cells according to claim 1, it is characterized in that, add a kind of in II Collagen Type VI enzyme, IV Collagen Type VI enzyme, II Collagen Type VI enzyme and pancreatin mixed solution or II Collagen Type VI enzyme and the Unidasa mixed solution in the described Digestive system.
7. preparing human umbilical mesenchymal stem cells according to claim 1 is characterized in that, the time of digestion umbilical cord tissue piece is 6h.
8. preparing human umbilical mesenchymal stem cells according to claim 1 is characterized in that, described culture medium solution is a serum-free DMEM/F12 culture medium solution.
9. preparing human umbilical mesenchymal stem cells according to claim 8 is characterized in that, described culture medium solution is to contain the FBS that volume fraction is 10-20% and the DMEM/F12 of 25mmol/L glutamine.
10. as the mescenchymal stem cell of claim 1～9 method preparation as described in each, it is characterized in that in immunological phenotype is identified, having following technical characterictic:
1. adherent growth becomes the fiber-like growth in plastics system cultivation vessel under the type culture condition;
2. expression of adhesion molecules receptor marker thing CD29/CD44, mesenchymal cell mark CD73 (SH3)/CD105 (SH2), and stem cell labeling thing CD90, express or do not express endothelial cell marker thing CD31, hemopoietic stem cell sign CD34 and histocompatibility II class antigen HLA-DR and hang down;
3. external evoked scleroblast and the cardiac-like muscle cell of being divided into.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010605542 CN102127522B (en) | 2010-12-27 | 2010-12-27 | Human umbilical mesenchymal stem cell and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010605542 CN102127522B (en) | 2010-12-27 | 2010-12-27 | Human umbilical mesenchymal stem cell and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102127522A true CN102127522A (en) | 2011-07-20 |
| CN102127522B CN102127522B (en) | 2012-11-21 |
Family
ID=44265797
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201010605542 Active CN102127522B (en) | 2010-12-27 | 2010-12-27 | Human umbilical mesenchymal stem cell and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102127522B (en) |
Cited By (33)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102321575A (en) * | 2011-10-08 | 2012-01-18 | 南方医科大学珠江医院 | Separation and culture method of human umbilical mesenchymal cell |
| CN102365933A (en) * | 2011-10-24 | 2012-03-07 | 广州市天河诺亚生物工程有限公司 | Mesenchymal stem cell preservation liquid, and preparation method and application thereof |
| CN102660497A (en) * | 2012-05-21 | 2012-09-12 | 博雅干细胞科技有限公司 | Method for freezing and reviving umbilical cord tissues and for separating and increasing stem cells |
| CN102660501A (en) * | 2012-05-21 | 2012-09-12 | 博雅干细胞科技有限公司 | Method for separating and amplifying mesenchymal stem cell from fresh tissue of umbilical cord |
| 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 |
| CN102876631A (en) * | 2012-10-09 | 2013-01-16 | 博雅干细胞科技有限公司 | Method for separating immune cells from blood and application of method to disease treatment |
| CN103266081A (en) * | 2012-01-21 | 2013-08-28 | 中国人民解放军军事医学科学院附属医院 | Efficient method for isolating and culturing mesenchymal stem cells from umbilical cord |
| CN104212764A (en) * | 2014-09-22 | 2014-12-17 | 上海华颜干细胞科技有限公司 | Preparation method of clinical mesenchymal stem cells |
| CN104450609A (en) * | 2013-09-25 | 2015-03-25 | 深圳华大基因科技有限公司 | Method of separating and cultivating umbilical cord mesenchymal stem cells |
| CN104531614A (en) * | 2014-12-26 | 2015-04-22 | 哈尔滨壹加壹再生医学科技有限公司 | Preparation method for obtaining mesenchymal stem cells by retaining umbilical blood vessels |
| CN104830751A (en) * | 2015-05-29 | 2015-08-12 | 广州赛莱拉干细胞科技股份有限公司 | Primary separation culture method of umbilical vein endothelial cells and reagent box of primary separation culture method |
| CN104862274A (en) * | 2015-05-27 | 2015-08-26 | 贵州北科泛特尔生物科技有限公司 | Method for efficiently separating umbilical cord mesenchymal stem cells |
| CN104988119A (en) * | 2015-07-08 | 2015-10-21 | 深圳爱生再生医学科技有限公司 | Wharton jelly digestive juice |
| CN104988118A (en) * | 2015-07-08 | 2015-10-21 | 深圳爱生再生医学科技有限公司 | Method for preparing umbilical cord mesenchymal stem cells |
| CN105238751A (en) * | 2015-11-30 | 2016-01-13 | 深圳市合一康生物科技股份有限公司 | Umbilical cord tissue mesenchymal stem cell isolated culture method |
| CN105891512A (en) * | 2016-05-11 | 2016-08-24 | 天津普瑞赛尔生物科技有限公司 | Kit for identifying umbilical cord mesenchymal stem cells |
| CN105985930A (en) * | 2016-06-15 | 2016-10-05 | 王仲 | Mesenchymal stem cells combined with traditional Chinese medicines for activating amplification and preparation method and application of mesenchymal stem cells |
| CN106635974A (en) * | 2016-10-19 | 2017-05-10 | 浙江译美生物科技有限公司 | Method for separating and culturing human umbilical cord mesenchymal stem cells |
| CN106867981A (en) * | 2017-04-14 | 2017-06-20 | 青岛青春派生物科技有限公司 | A kind of human umbilical tissue digestive ferment and preparation method thereof |
| CN107502587A (en) * | 2017-09-28 | 2017-12-22 | 吉林省拓华生物科技有限公司 | CD29+People's Mesenchymal Stem Cells from Umbilical Cord and its purposes in the Seeding Cells in Bone Tissue Engineering for preparing treatment bone injury |
| CN107557332A (en) * | 2017-09-28 | 2018-01-09 | 吉林省拓华生物科技有限公司 | CD29+People's Mesenchymal Stem Cells from Umbilical Cord and its purposes in skeletal muscle atrophy medicine under preparing treatment high glucose and high fat environment |
| CN108743618A (en) * | 2018-04-25 | 2018-11-06 | 中国人民解放军成都军区总医院 | A kind of preparation method and applications of syringeability stem cell aqueogel |
| CN108865984A (en) * | 2018-06-22 | 2018-11-23 | 安徽 | A kind of preparation and extracting method of umbilical cord mesenchymal stem cells |
| CN109385396A (en) * | 2017-08-04 | 2019-02-26 | 上海赛傲生物技术有限公司 | Clinical application grade umbilical cord mesenchymal stem cells and its method for separating and preparing |
| CN109486756A (en) * | 2019-01-21 | 2019-03-19 | 陕西森凯组织工程与再生医学科技有限公司 | The cultural method of umbilical cord mesenchymal stem cells |
| CN109628393A (en) * | 2019-01-22 | 2019-04-16 | 汇麟生物科技(北京)有限公司 | A kind of method and its digestive juice for separating funicle mesenchyme stem cell |
| CN110157665A (en) * | 2019-05-24 | 2019-08-23 | 吉林省农业科学院 | A kind of separation of human umbilical cord mesenchymal stem cells and cultural method |
| CN110964693A (en) * | 2019-12-27 | 2020-04-07 | 河北银丰鼎诚生物技术有限公司 | Separation method of umbilical cord mesenchymal stem cells |
| CN111793599A (en) * | 2020-06-05 | 2020-10-20 | 天津市康婷生物工程集团有限公司 | Osteogenic differentiation method by combining umbilical cord mesenchymal stem cells with bone scaffold material |
| CN111893092A (en) * | 2020-06-28 | 2020-11-06 | 国大生命科学产业集团(深圳)有限公司 | Human umbilical cord-derived mesenchymal stem cells and preparation method thereof |
| WO2022103129A1 (en) | 2020-11-11 | 2022-05-19 | 이엔셀 주식회사 | Early mesenchymal stem cells with reduced aging and preserved stem cell ability, and culturing method therefor |
| CN114940970A (en) * | 2022-06-15 | 2022-08-26 | 玺瑞生命科学(深圳)有限公司 | Preparation method of human umbilical cord mesenchymal stem cells |
| CN116004528A (en) * | 2021-11-25 | 2023-04-25 | 瑞因细胞工程科技(广州)有限公司 | Preparation method of umbilical cord mesenchymal stem cells |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101298606A (en) * | 2008-02-14 | 2008-11-05 | 天津环宇商桥商务信息咨询有限公司 | Preparation, storage and use of umbilical cord and placenta mesenchymal stem cell for clinical therapy |
| CN101492654A (en) * | 2008-03-17 | 2009-07-29 | 协和干细胞基因工程有限公司 | Method for using umbilical stalk placenta to prepare mesenchyma stem cell |
-
2010
- 2010-12-27 CN CN 201010605542 patent/CN102127522B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101298606A (en) * | 2008-02-14 | 2008-11-05 | 天津环宇商桥商务信息咨询有限公司 | Preparation, storage and use of umbilical cord and placenta mesenchymal stem cell for clinical therapy |
| CN101492654A (en) * | 2008-03-17 | 2009-07-29 | 协和干细胞基因工程有限公司 | Method for using umbilical stalk placenta to prepare mesenchyma stem cell |
Non-Patent Citations (1)
| Title |
|---|
| 《中国组织工程研究与临床康复》 20100305 蒋洁，等 脐带沃顿胶间充质干细胞的分离培养及其诱导分化 第1734-1738页 1-10 第14卷, 第10期 2 * |
Cited By (43)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102321575B (en) * | 2011-10-08 | 2012-11-21 | 南方医科大学珠江医院 | Separation and culture method of human umbilical mesenchymal cell |
| CN102321575A (en) * | 2011-10-08 | 2012-01-18 | 南方医科大学珠江医院 | Separation and culture method of human umbilical mesenchymal cell |
| CN102365933A (en) * | 2011-10-24 | 2012-03-07 | 广州市天河诺亚生物工程有限公司 | Mesenchymal stem cell preservation liquid, and preparation method and application thereof |
| CN102365933B (en) * | 2011-10-24 | 2016-04-27 | 广州市天河诺亚生物工程有限公司 | A kind of mesenchyme stem cell preserving fluid and preparation method thereof and application |
| CN103266081A (en) * | 2012-01-21 | 2013-08-28 | 中国人民解放军军事医学科学院附属医院 | Efficient method for isolating and culturing mesenchymal stem cells from umbilical cord |
| CN103266081B (en) * | 2012-01-21 | 2015-05-13 | 中国人民解放军军事医学科学院附属医院 | Efficient method for isolating and culturing mesenchymal stem cells from umbilical cord |
| 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 |
| CN102676452B (en) * | 2012-04-26 | 2014-09-24 | 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 |
| CN102660501A (en) * | 2012-05-21 | 2012-09-12 | 博雅干细胞科技有限公司 | Method for separating and amplifying mesenchymal stem cell from fresh tissue of umbilical cord |
| CN102660497B (en) * | 2012-05-21 | 2013-06-12 | 博雅干细胞科技有限公司 | Method for freezing and reviving umbilical cord tissues and for separating and increasing stem cells |
| CN102660497A (en) * | 2012-05-21 | 2012-09-12 | 博雅干细胞科技有限公司 | Method for freezing and reviving umbilical cord tissues and for separating and increasing stem cells |
| CN102876631A (en) * | 2012-10-09 | 2013-01-16 | 博雅干细胞科技有限公司 | Method for separating immune cells from blood and application of method to disease treatment |
| CN104450609B (en) * | 2013-09-25 | 2017-07-14 | 深圳华大基因科技有限公司 | A kind of method for separating and cultivating umbilical cord mesenchymal stem cells |
| CN104450609A (en) * | 2013-09-25 | 2015-03-25 | 深圳华大基因科技有限公司 | Method of separating and cultivating umbilical cord mesenchymal stem cells |
| CN104212764A (en) * | 2014-09-22 | 2014-12-17 | 上海华颜干细胞科技有限公司 | Preparation method of clinical mesenchymal stem cells |
| CN104531614A (en) * | 2014-12-26 | 2015-04-22 | 哈尔滨壹加壹再生医学科技有限公司 | Preparation method for obtaining mesenchymal stem cells by retaining umbilical blood vessels |
| CN104862274A (en) * | 2015-05-27 | 2015-08-26 | 贵州北科泛特尔生物科技有限公司 | Method for efficiently separating umbilical cord mesenchymal stem cells |
| CN104830751A (en) * | 2015-05-29 | 2015-08-12 | 广州赛莱拉干细胞科技股份有限公司 | Primary separation culture method of umbilical vein endothelial cells and reagent box of primary separation culture method |
| CN104988119A (en) * | 2015-07-08 | 2015-10-21 | 深圳爱生再生医学科技有限公司 | Wharton jelly digestive juice |
| CN104988118A (en) * | 2015-07-08 | 2015-10-21 | 深圳爱生再生医学科技有限公司 | Method for preparing umbilical cord mesenchymal stem cells |
| CN105238751A (en) * | 2015-11-30 | 2016-01-13 | 深圳市合一康生物科技股份有限公司 | Umbilical cord tissue mesenchymal stem cell isolated culture method |
| CN105238751B (en) * | 2015-11-30 | 2021-07-23 | 深圳市合一康生物科技股份有限公司 | Isolated culture method of umbilical cord tissue mesenchymal stem cells |
| CN105891512A (en) * | 2016-05-11 | 2016-08-24 | 天津普瑞赛尔生物科技有限公司 | Kit for identifying umbilical cord mesenchymal stem cells |
| CN105985930A (en) * | 2016-06-15 | 2016-10-05 | 王仲 | Mesenchymal stem cells combined with traditional Chinese medicines for activating amplification and preparation method and application of mesenchymal stem cells |
| CN105985930B (en) * | 2016-06-15 | 2019-12-13 | 辛普森国际生命科技(天津)有限公司 | Mesenchymal stem cell combined with traditional Chinese medicine activation and amplification and preparation method and application thereof |
| CN106635974A (en) * | 2016-10-19 | 2017-05-10 | 浙江译美生物科技有限公司 | Method for separating and culturing human umbilical cord mesenchymal stem cells |
| CN106867981A (en) * | 2017-04-14 | 2017-06-20 | 青岛青春派生物科技有限公司 | A kind of human umbilical tissue digestive ferment and preparation method thereof |
| CN109385396A (en) * | 2017-08-04 | 2019-02-26 | 上海赛傲生物技术有限公司 | Clinical application grade umbilical cord mesenchymal stem cells and its method for separating and preparing |
| CN107502587A (en) * | 2017-09-28 | 2017-12-22 | 吉林省拓华生物科技有限公司 | CD29+People's Mesenchymal Stem Cells from Umbilical Cord and its purposes in the Seeding Cells in Bone Tissue Engineering for preparing treatment bone injury |
| CN107557332A (en) * | 2017-09-28 | 2018-01-09 | 吉林省拓华生物科技有限公司 | CD29+People's Mesenchymal Stem Cells from Umbilical Cord and its purposes in skeletal muscle atrophy medicine under preparing treatment high glucose and high fat environment |
| CN107557332B (en) * | 2017-09-28 | 2021-02-26 | 吉林省拓华生物科技有限公司 | CD29+Human umbilical cord-derived mesenchymal stem cells and application thereof in preparation of medicine for treating skeletal muscle atrophy in high-sugar and high-fat environment |
| CN108743618A (en) * | 2018-04-25 | 2018-11-06 | 中国人民解放军成都军区总医院 | A kind of preparation method and applications of syringeability stem cell aqueogel |
| CN108865984A (en) * | 2018-06-22 | 2018-11-23 | 安徽 | A kind of preparation and extracting method of umbilical cord mesenchymal stem cells |
| CN109486756A (en) * | 2019-01-21 | 2019-03-19 | 陕西森凯组织工程与再生医学科技有限公司 | The cultural method of umbilical cord mesenchymal stem cells |
| CN109628393A (en) * | 2019-01-22 | 2019-04-16 | 汇麟生物科技(北京)有限公司 | A kind of method and its digestive juice for separating funicle mesenchyme stem cell |
| CN110157665A (en) * | 2019-05-24 | 2019-08-23 | 吉林省农业科学院 | A kind of separation of human umbilical cord mesenchymal stem cells and cultural method |
| CN110964693A (en) * | 2019-12-27 | 2020-04-07 | 河北银丰鼎诚生物技术有限公司 | Separation method of umbilical cord mesenchymal stem cells |
| CN111793599A (en) * | 2020-06-05 | 2020-10-20 | 天津市康婷生物工程集团有限公司 | Osteogenic differentiation method by combining umbilical cord mesenchymal stem cells with bone scaffold material |
| CN111793599B (en) * | 2020-06-05 | 2023-08-08 | 天津市康婷生物工程集团有限公司 | Method for osteogenic differentiation of umbilical cord mesenchymal stem cells combined with bone scaffold material |
| CN111893092A (en) * | 2020-06-28 | 2020-11-06 | 国大生命科学产业集团(深圳)有限公司 | Human umbilical cord-derived mesenchymal stem cells and preparation method thereof |
| WO2022103129A1 (en) | 2020-11-11 | 2022-05-19 | 이엔셀 주식회사 | Early mesenchymal stem cells with reduced aging and preserved stem cell ability, and culturing method therefor |
| CN116004528A (en) * | 2021-11-25 | 2023-04-25 | 瑞因细胞工程科技(广州)有限公司 | Preparation method of umbilical cord mesenchymal stem cells |
| CN114940970A (en) * | 2022-06-15 | 2022-08-26 | 玺瑞生命科学(深圳)有限公司 | Preparation method of human umbilical cord mesenchymal stem cells |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102127522B (en) | 2012-11-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102127522B (en) | Human umbilical mesenchymal stem cell and preparation method thereof | |
| CN105238751B (en) | Isolated culture method of umbilical cord tissue mesenchymal stem cells | |
| CN103667182B (en) | A kind of mesenchymal stem cells MSCs is in vitro to induction method and the inducing culture of osteoblast differentiation | |
| CN104164403B (en) | Method for extracting and culturing adipose-derived stem cells | |
| CN102443566B (en) | Acquisition method of adipose-derived stem cells | |
| CN102367435B (en) | Preparation of human platelet-rich plasma and application of same in isolation and culture of human mesenchymal stem cells | |
| CN107022521A (en) | Decidua vera tissue freezing, the method recovered and be separately cultured mescenchymal stem cell | |
| CN109234229B (en) | Method for separating mesenchymal stem cells from placental blood vessels and digestive enzyme composition used in same | |
| CN106801032B (en) | Construction method of human amniotic epithelial stem cell bank | |
| CN104762259A (en) | Culture medium for mesenchymal stem cells and large-scale culture method thereof | |
| CN105950550A (en) | Mesenchymal stem cell serum-free medium and cell isolation and cultivation methods | |
| CN102978156A (en) | Expansion in vitro purification culture method of mesenchymal stem cells and culture medium | |
| CN107299082A (en) | Placenta interstitial cell and the method for being trained mescenchymal stem cell are separated from tissue | |
| CN101591644A (en) | The preparation of umbilical cord-mesenchymal stem cells (UC-MSCs) used in clinical therapy and storage | |
| CN104450611A (en) | Primary separation and culture method of human amniotic mesenchymal stem cells | |
| CN108795855A (en) | A kind of serum free medium of mescenchymal stem cell | |
| CN108220229A (en) | A kind of preparation method for improving umbilical cord derived mesenchymal stem cell primary cell yield | |
| CN109735490A (en) | A kind of fat stem cell extracting method | |
| CN104651305A (en) | Method for acquiring bioactive proteins by utilizing umbilical cord mesenchymal stem cells | |
| CN104762257A (en) | Method for preparing mesenchymal stem cell from umbilical cord | |
| CN101831403B (en) | Method for amplifying mesenchymal stem cells of human umbilical cord and placenta in vitro | |
| CN110475855A (en) | For the method that cell prepares dental pulp stem cell derived from pulp tissue | |
| CN103087977A (en) | Culture solution for in vitro efficient amplification of animal cells and application of culture solution | |
| CN104531617A (en) | Method for preparing dendritic cells and obtained dendritic cells | |
| CN102191229A (en) | Method for rapidly and effectively acquiring umbilical cord mesenchymal stem cell (MSC) |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |