CN102154200B - Preparation and storage of mesenchymal stem cells for clinical treatment - Google Patents
Preparation and storage of mesenchymal stem cells for clinical treatment Download PDFInfo
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Abstract
The invention relates to preparation and storage of mesenchymal stem cells for clinical treatment. The preparation method comprises the following steps: storing a physiological saline umbilical cord containing 0.2% of genramicin and 0.54% of anticoagulant; shearing the umbilical cord to 2-3cm; centrifuging, suspending, and centrifuging; directly adhering and carrying out explant-culture; adding an alpha-minimum essential medium (MEM) containing 15% of fetal calf serum and 0.2% of genramicin, and culturing; after cell fusion, digesting the cells with digestive juice containing 0.05% of pancreatin/0.2mM ethylene diamine tetraacetic acid (EDTA); carrying out passage in a ratio of 1:3 to 4-5 generations, so as to obtain a large amount of mesenchymal stem cells; and putting a frozen storing liquid (the ratio of medium to fetal calf serum to dimethylsulfoxide is 5:4:1) in the mesenchymal stem cells, placing in a refrigerator of minus 70 DEG C overnight and then storing in liquid nitrogen, wherein the freezing temperature of the liquid nitrogen is minus 196 DEG C.
Description
Technical field
The present invention relates to a kind of preparation of medical treatment product, specifically is preparation and the preservation that is applied to the human umbilical cord mesenchymal stem cells that clinical treatment uses.
Technical background
Mescenchymal stem cell (mesenchymal stem cells; MSCs) be a type of stem cell; Gaining the name because of being divided into stroma, is to belong to mesoblastic one type of multipotential stem cell, the potential that has very strong self and grow to multiple tissue differentiation.MSCs mainly is present between reticular tissue and organ in the matter; The abundantest with content in the marrow; Can be to the histocyte differentiation in multiple mesoderm and neuroderm source; As can be divided into scleroblast, chondrocyte, adipocyte, Tenocyte cell, smooth muscle cell, marrow stromal cell, inoblast and multiple vascular endothelial cell; Even neural neurone and neurogliocyte, also have functions such as immunomodulatory and promotion hematopoietic reconstitution simultaneously, make it to become " the practical stem cell " of treatment multisystem disease.Multidirectional differentiation, tissue repair and the immunoloregulation function of MSCs make it possess application prospects clinically.At present, MSCs is applied to clinical treatment graft versus host disease (GVHD), cardiovascular reparation, Spinal injury, bone and repair of cartilage, burn, rheumatoid arthritis, Parkinson's disease etc. both at home and abroad.People are carrying on the trial of using MSCs treatment other diseases, believe to have future more disease to treat with MSCs.
Umbilical cord, is convenient to draw materials because of wide material sources as the important source of mescenchymal stem cell, and donor is had no adverse effect, and no ethics restriction is accepted extensively by people.The MSCs immunogenicity in umbilical cord source is low simultaneously, transplants between allosome can not cause rejection, has avoided using the side reaction that is brought because of immunosuppressor, has strengthened its clinical scope of application.Therefore, umbilical cord MSCs is expected to by large-scale application in clinical study.
At present, the biological characteristics and the function of mescenchymal stem cell are known by people, and how safety and effective external preservation, acquisition, amplification and storage mescenchymal stem cell have significant effects for its extensive clinical application.The mescenchymal stem cell research aspect that present clinical treatment is used remains defectiveness.Mainly comprise:
1 umbilical cord collection posterior umbilicus is quiet, arterial blood solidifies, and has strengthened the difficulty that umbilical cord is handled, and hypercytosis increases the chance that hemopoietic stem cell pollutes simultaneously.
2 enzymolysis processs digestion tissues has increased the security risk with cross infection that causes allergic reaction of animal-based protein in the process of clinical application.
The selection of substratum has influenced the amplification efficiency of cell in the 3 mescenchymal stem cell culturing process.
The protein concentration of 4 attached cells digestion enzymolysis influences the existing state of cell.
The objective of the invention is to improve foregoing, set up store method and the mescenchymal stem cell economy of external umbilical cord, amplification method efficiently, for the research of clinical stem cell provides powerful guarantee.
Summary of the invention
It is the preparation method of the mescenchymal stem cell in source with the umbilical cord that the object of the invention is to provide a kind of; Use the risk that heterologous protein digestion methods such as collagenase and trypsinase possibly bring before having overcome; Reduce preparation cost, increase the yield and the security of mescenchymal stem cell.
Preparing human umbilical mesenchymal stem cells of the present invention is following:
1. aseptic condition is gathered normal natural labor or c-section person's fetal cord down, preserves umbilical cord tissue with the saline water that contains 0.2% qingfengmeisu qiong, 0.54% sodium citrate anticoagulant.Carry out pathogeny microbiology and virusology simultaneously and detect, qualified fully after, carry out following steps.
2. umbilical cord is placed the saline water that contains 0.2% qingfengmeisu qiong, wash 2-3 time repeatedly, cut off quiet, the arteries of navel, reject macroscopic vascular tissue and the blood clot that possibly exist with sterile scissors.With scissors umbilical cord is trimmed to the 2-3cm size, collection organization's piece places in the 50ml centrifuge tube, high speed centrifugation; Centrifuge speed is 2000 rev/mins, centrifugal 10 minutes, discards supernatant; Again add the saline water that contains 0.2% qingfengmeisu qiong, sedimentary tissue block is fully vibrated, make it loose; High speed centrifugation, centrifuge speed are 2000 rev/mins, centrifugal 10 minutes.
3. the tissue block that step 2 obtained is suspended with α-MEM (the Alpha Minimum Essential Medium) substratum that contains 15% foetal calf serum, 0.2% qingfengmeisu qiong; The back high speed centrifugation fully vibrates; Centrifuge speed is 2000 rev/mins; Centrifugal 10 minutes, this step was in order to remove a large amount of tissue block mucus.
4. the umbilical cord tissue piece that step 3 obtained is tiled in the plastic culture bottle, retains the 1-2cm space between each tissue block, place 37 ℃ of 5%CO
2In the incubator 60 minutes, treat that tissue block is adherent fully after, add the α-MEM substratum that contains 15% foetal calf serum, 0.2% qingfengmeisu qiong.
5. every cultivation was carried out half amount in 3-5 days and is changed the liquid processing.
6. under inverted microscope, observed in about 10-12 days when occurring adherent, shuttle type or multiangular cell around the tissue block in cultivating,, add the α-MEM substratum that contains 15% foetal calf serum, 0.2% qingfengmeisu qiong, place 5%CO the whole sucking-offs of tissue block
2Continue in the incubator to cultivate.After this 1 pericyte increases rapidly, becomes the comparatively long shuttle type cell of homogeneous of form, is the vortex shape growth.
7. after treating cell 80% fusion of vortex shape growth; Earlier wash culturing bottle repeatedly 3 times with saline water; Digestive system peptic cell with containing 0.05% pancreatin/0.2mM EDTA went down to posterity by 1: 3, and culture condition is replaced by the α-MEM substratum that contains 10% foetal calf serum, 0.2% qingfengmeisu qiong.
8. after amplification in 20-25 days, cell reached for 4～5 generations, can obtain a large amount of mescenchymal stem cells.Cellular form is as shown in Figure 1.
9. step 7 amplification back cell being carried out Pathogen Biology detects; Carry out phenotype simultaneously and detect, phenotype result is as shown in Figure 2, and qualified back adds frozen storing liquid (substratum: foetal calf serum: DMSO=5: 4: 1); Put into the cell cryopreservation box;-70 ℃ of refrigerators are placed overnight being placed in the liquid nitrogen and are preserved, and place liquid nitrogen to preserve, and the liquid nitrogen freezing temperature is-196 ℃.Record umbilical cord source-information, cell algebraically, cell quantity and preparation date, get final product.This umbilical cord mesenchymal stem cells can be used for clinical treatment.
Aforesaid operations all carries out under the Strict aseptic condition, and the probability that umbilical cord intractability that blood coagulation brought and hemopoietic stem cell pollute has effectively been avoided in the use of antithrombotics after the umbilical cord collection.Big area is planted piece and is cultivated workload and the opportunities for contamination that has reduced the umbilical cord repeated shear.Owing in the tissue block treating processes, do not adopt the processing of animal-based proteins such as collagenase and trypsinase, reduced and taken place to pollute and anaphylactoid chance, and the earth has reduced preparation cost simultaneously.α-MEM substratum pair cell amplification efficiency is higher.The degree of injury of the enzymolysis, digestion scheme pair cell of 0.05% pancreatin/0.2mM EDTA is lower.
Human umbilical cord mesenchymal stem cells preparation method effect of the present invention is good, can from people's umbilical cord, obtain a large amount of MSCs, and simple, and cost is low, and security is good, has a good application prospect.
Description of drawings:
The form of the MSC in Fig. 1 umbilical cord source: the MSC cell in umbilical cord source passes the form (HE dyeing) in the 2nd day the 5th generation, and A is 10 times of forms under the object lens, and B is 40 times of forms under the object lens.
The MSC phenotype in Fig. 2 umbilical cord source: its phenotypic characteristic is that CD31, HLA-DR, CD34 and CD45 are negative, and CD29, CD44, CD73, CD90 and CD105 are positive.
Embodiment:
Below further specify the present invention through instance, but not as limitation of the present invention.
Embodiment 1:
1. aseptic condition is gathered normal natural labor or c-section person's fetal cord down, and about 30CM is long, preserves umbilical cord tissue with the saline water that contains 0.2% qingfengmeisu qiong, 0.54% antithrombotics.Carry out pathogeny microbiology and virusology simultaneously and detect, qualified fully after, carry out following steps.
2. umbilical cord is placed the saline water that contains 0.2% qingfengmeisu qiong, wash 2-3 time repeatedly, cut off quiet, the arteries of navel, reject macroscopic vascular tissue and the blood clot that possibly exist with sterile scissors.With scissors umbilical cord is trimmed to the 2-3cm size, collection organization's piece places in the 50ml centrifuge tube, high speed centrifugation; Centrifuge speed is 2000 rev/mins, centrifugal 10 minutes, discards supernatant; Again add the saline water that contains 0.2% qingfengmeisu qiong, sedimentary tissue block is fully vibrated, make it loose; High speed centrifugation, centrifuge speed are 2000 rev/mins, centrifugal 10 minutes.
3. the tissue block that step 2 obtained is used the α-MEM substratum inner suspension that contains 15% foetal calf serum, 0.2% qingfengmeisu qiong, the back high speed centrifugation that fully vibrates, centrifuge speed is 2000 rev/mins, centrifugal 10 minutes, this step was in order to remove a large amount of tissue block mucus.
4. the umbilical cord tissue piece that step 3 obtained is tiled in the plastic culture bottle, retains the 1-2cm space between each tissue block, place 37 ℃ of 5%CO
2In the incubator 60 minutes, treat that tissue block is adherent fully after, add the α-MEM substratum that contains 15% foetal calf serum, 0.2% qingfengmeisu qiong.
5. every cultivation was carried out half amount in 3-5 days and is changed the liquid processing.
6. under inverted microscope, observed in about 10-12 days when occurring adherent, shuttle type or multiangular cell around the tissue block in cultivating,, add the α-MEM substratum that contains 15% foetal calf serum, 0.2% qingfengmeisu qiong, place 5%CO the whole sucking-offs of tissue block
2Continue in the incubator to cultivate.After this 1 pericyte increases rapidly, becomes the comparatively long shuttle type cell of homogeneous of form, is the vortex shape growth.
7. after treating cell 80% fusion of vortex shape growth; Earlier wash culturing bottle repeatedly 3 times with saline water; Digestive system peptic cell with containing 0.05% pancreatin/0.2mMEDTA went down to posterity by 1: 3, and culture condition is replaced by the α-MEM substratum that contains 10% foetal calf serum, 0.2% qingfengmeisu qiong.
8. after amplification in 20-25 days, cell reached for 4～5 generations, can obtain a large amount of mescenchymal stem cells.
9. step 7 amplification back cell being carried out Pathogen Biology detects; Carrying out phenotype simultaneously detects; Phenotype result is as shown in Figure 2, and qualified back adds frozen storing liquid (substratum: foetal calf serum: DMSO=5: 4: 1), puts into the cell cryopreservation box;-70 ℃ of refrigerators are placed overnight being placed in the liquid nitrogen and are preserved, and the liquid nitrogen freezing temperature is-196 ℃.Record umbilical cord source-information, cell algebraically, cell quantity and preparation date, get final product.This umbilical cord mesenchymal stem cells can be used for clinical treatment.
Embodiment 2
1. aseptic condition is gathered normal natural labor or c-section person's fetal cord down, and about 40CM is long, preserves umbilical cord tissue with the saline water that contains 0.2% qingfengmeisu qiong, 0.54% antithrombotics.Carry out pathogeny microbiology and virusology simultaneously and detect, qualified fully after, carry out following steps.
2. umbilical cord is placed the saline water that contains 0.2% qingfengmeisu qiong, wash 2-3 time repeatedly, cut off quiet, the arteries of navel, reject macroscopic vascular tissue and the blood clot that possibly exist with sterile scissors.With scissors umbilical cord is trimmed to the 2-3cm size, collection organization's piece places in the 50ml centrifuge tube, high speed centrifugation; Centrifuge speed is 2000 rev/mins, centrifugal 10 minutes, discards supernatant; Again add the saline water that contains 0.2% qingfengmeisu qiong, sedimentary tissue block is fully vibrated, make it loose; High speed centrifugation, centrifuge speed are 2000 rev/mins, centrifugal 10 minutes.
3. the tissue block that step 2 obtained is used the α-MEM substratum inner suspension that contains 15% foetal calf serum, 0.2% qingfengmeisu qiong, the back high speed centrifugation that fully vibrates, centrifuge speed is 2000 rev/mins, centrifugal 10 minutes, this step was in order to remove a large amount of tissue block mucus.
4. the umbilical cord tissue piece that step 3 obtained is tiled in the plastic culture bottle, retains the 1-2cm space between each tissue block, place 37 ℃ of 5%CO
2In the incubator 60 minutes, treat that tissue block is adherent fully after, add the α-MEM substratum that contains 15% foetal calf serum, 0.2% qingfengmeisu qiong.
5. every cultivation was carried out half amount in 3-5 days and is changed the liquid processing.
6. under inverted microscope, observed in about 8-10 days when occurring adherent, shuttle type or multiangular cell around the tissue block in cultivating,, add the α-MEM substratum that contains 15% foetal calf serum, 0.2% qingfengmeisu qiong, place 5%CO the whole sucking-offs of tissue block
2Continue in the incubator to cultivate.After this 1 pericyte increases rapidly, becomes the comparatively long shuttle type cell of homogeneous of form, is the vortex shape growth.
7. after treating cell 80% fusion of vortex shape growth; Earlier wash culturing bottle repeatedly 3 times with saline water; Digestive system peptic cell with containing 0.05% pancreatin/0.2mMEDTA went down to posterity by 1: 3, and culture condition is replaced by the α-MEM substratum that contains 10% foetal calf serum, 0.2% qingfengmeisu qiong.
8. after amplification in 20-25 days, cell reached for 4～5 generations, can obtain a large amount of mescenchymal stem cells.
9. step 7 amplification back being obtained to carry out Pathogen Biology detects; Carrying out phenotype simultaneously detects; Phenotype result is as shown in Figure 2, and qualified back adds frozen storing liquid (substratum: foetal calf serum: DMSO=5: 4: 1), puts into the cell cryopreservation box;-70 ℃ of refrigerators are placed overnight being placed in the liquid nitrogen and are preserved, and the liquid nitrogen freezing temperature is-196 ℃.Record umbilical cord source-information, cell algebraically, cell quantity and preparation date, promptly get.This umbilical cord mesenchymal stem cells can be used for clinical treatment.
Claims (3)
1. one kind is the preparation method of the mescenchymal stem cell in source with the umbilical cord, it is characterized in that the process following steps:
1). aseptic condition is gathered normal natural labor or c-section person's fetal cord down, preserves umbilical cord tissue with the saline water that contains 0.2% qingfengmeisu qiong, 0.54% sodium citrate anticoagulant;
2). umbilical cord is placed the saline water that contains 0.2% qingfengmeisu qiong, wash 2-3 time repeatedly, cut off quiet, the arteries of navel, reject macroscopic vascular tissue and the blood clot that possibly exist with sterile scissors; With scissors umbilical cord is trimmed to the 2-3cm size, collection organization's piece places in the 50ml centrifuge tube, high speed centrifugation; Centrifuge speed is 2000 rev/mins, centrifugal 10 minutes, discards supernatant; Again add the saline water that contains 0.2% qingfengmeisu qiong, sedimentary tissue block is fully vibrated, make it loose; High speed centrifugation, centrifuge speed are 2000 rev/mins, centrifugal 10 minutes;
3). the tissue block that step 2 obtained is suspended with the α that contains 15% foetal calf serum, 0.2% qingfengmeisu qiong-MEM substratum, the back high speed centrifugation that fully vibrates, centrifuge speed is 2000 rev/mins, centrifugal 10 minutes;
4). the umbilical cord tissue piece that step 3 obtained is tiled in the plastic culture bottle, retains the 1-2cm space between each tissue block, place 37 ℃ of 5%CO
2In the incubator 60 minutes, treat that tissue block is adherent fully after, add the α-MEM substratum that contains 15% foetal calf serum, 0.2% qingfengmeisu qiong;
5). every cultivation was carried out half amount in 3-5 days and is changed the liquid processing;
6). under inverted microscope, observed in about 10-12 days when occurring adherent, shuttle type or multiangular cell around the tissue block in cultivating,, add the α-MEM substratum that contains 15% foetal calf serum, 0.2% qingfengmeisu qiong, place 5%CO the whole sucking-offs of tissue block
2Continue in the incubator to cultivate, become the comparatively long shuttle type cell of homogeneous of form, be the vortex shape growth;
7). after treating cell 80% fusion of vortex shape growth; Earlier wash culturing bottle repeatedly 3 times with saline water; Again with the Digestive system peptic cell that contains 0.05% pancreatin/0.2mM EDTA; Go down to posterity in 1: 3 ratio, culture condition is replaced by the α-MEM substratum that contains 10% foetal calf serum, 0.2% qingfengmeisu qiong;
8). after amplification in 20-25 days, cell reached for 4～5 generations, obtained a large amount of mescenchymal stem cells;
9). the back cell that will increase is earlier resuspended with the substratum of precooling; Drip the foetal calf serum that contains DMSO of precooling subsequently; Wherein the ratio of substratum, foetal calf serum and DMSO is 5: 4: 1, and the cell after resuspended is put into freezing storing box ,-70 ℃ of refrigerators place overnight be placed on to preserve in the liquid nitrogen get final product.
2. one kind is the preparation method of the mescenchymal stem cell in source with the umbilical cord, it is characterized in that the process following steps:
1). aseptic condition is gathered normal natural labor or c-section person's fetal cord down, and 30cm is long, preserves umbilical cord tissue with the saline water that contains 0.2% qingfengmeisu qiong, 0.54% antithrombotics;
2). umbilical cord is placed the saline water that contains 0.2% qingfengmeisu qiong, wash 2-3 time repeatedly, cut off quiet, the arteries of navel, reject macroscopic vascular tissue and the blood clot that possibly exist with sterile scissors; With scissors umbilical cord is trimmed to the 2-3cm size, collection organization's piece places in the 50ml centrifuge tube, high speed centrifugation; Centrifuge speed is 2000 rev/mins, centrifugal 10 minutes, discards supernatant; Again add the saline water that contains 0.2% qingfengmeisu qiong, sedimentary tissue block is fully vibrated, make it loose; High speed centrifugation, centrifuge speed are 2000 rev/mins, centrifugal 10 minutes;
3). the tissue block that step 2 obtained is used the α-MEM substratum inner suspension that contains 15% foetal calf serum, 0.2% qingfengmeisu qiong, the back high speed centrifugation that fully vibrates, centrifuge speed is 2000 rev/mins, centrifugal 10 minutes;
4). the umbilical cord tissue piece that step 3 obtained is tiled in the plastic culture bottle, retains the 1-2cm space between each tissue block, place 37 ℃ of 5%CO
2In the incubator 60 minutes, treat that tissue block is adherent fully after, add the α-MEM substratum that contains 15% foetal calf serum, 0.2% qingfengmeisu qiong;
5). every cultivation was carried out half amount in 3-5 days and is changed the liquid processing;
6). under inverted microscope, observed in about 10-12 days when occurring adherent, shuttle type or multiangular cell around the tissue block in cultivating,, add the α-MEM substratum that contains 15% foetal calf serum, 0.2% qingfengmeisu qiong, place 5%CO the whole sucking-offs of tissue block
2Continue in the incubator to cultivate, become the comparatively long shuttle type cell of homogeneous of form, be the vortex shape growth;
7). after treating cell 80% fusion of vortex shape growth; Earlier wash culturing bottle repeatedly 3 times with saline water; Digestive system peptic cell with containing 0.05% pancreatin/0.2mMEDTA goes down to posterity in 1: 3 ratio, and culture condition is replaced by the α-MEM substratum that contains 10% foetal calf serum, 0.2% qingfengmeisu qiong;
8). after amplification in 20-25 days, cell reached for 4～5 generations, obtained a large amount of mescenchymal stem cells;
9). the back cell that will increase is earlier resuspended with the substratum of precooling; Drip the foetal calf serum that contains DMSO of precooling subsequently; Wherein the ratio of substratum, foetal calf serum and DMSO is 5: 4: 1, and the cell after resuspended is put into freezing storing box ,-70 ℃ of refrigerators place overnight be placed on to preserve in the liquid nitrogen get final product.
3. one kind is the preparation method of the mescenchymal stem cell in source with the umbilical cord, it is characterized in that the process following steps:
1). aseptic condition is gathered normal natural labor or c-section person's fetal cord down, and 40cm is long, preserves umbilical cord tissue with the saline water that contains 0.2% qingfengmeisu qiong, 0.54% antithrombotics;
2). umbilical cord is placed the saline water that contains 0.2% qingfengmeisu qiong, wash 2-3 time repeatedly, cut off quiet, the arteries of navel, reject macroscopic vascular tissue and the blood clot that possibly exist with sterile scissors; With scissors umbilical cord is trimmed to the 2-3cm size, collection organization's piece places in the 50ml centrifuge tube, high speed centrifugation; Centrifuge speed is 2000 rev/mins, centrifugal 10 minutes, discards supernatant; Again add the saline water that contains 0.2% qingfengmeisu qiong, sedimentary tissue block is fully vibrated, make it loose; High speed centrifugation, centrifuge speed are 2000 rev/mins, centrifugal 10 minutes;
3). the tissue block that step 2 obtained is used the α-MEM substratum inner suspension that contains 15% foetal calf serum, 0.2% qingfengmeisu qiong, the back high speed centrifugation that fully vibrates, centrifuge speed is 2000 rev/mins, centrifugal 10 minutes;
4). the umbilical cord tissue piece that step 3 obtained is tiled in the plastic culture bottle, retains the 1-2cm space between each tissue block, place 37 ℃ of 5%CO
2In the incubator 60 minutes, treat that tissue block is adherent fully after, add the α-MEM substratum that contains 15% foetal calf serum, 0.2% qingfengmeisu qiong;
5). every cultivation was carried out half amount in 3-5 days and is changed the liquid processing;
6). under inverted microscope, observed in about 8-10 days when occurring adherent, shuttle type or multiangular cell around the tissue block in cultivating,, add the α-MEM substratum that contains 15% foetal calf serum, 0.2% qingfengmeisu qiong, place 5%CO the whole sucking-offs of tissue block
2Continue in the incubator to cultivate, become the comparatively long shuttle type cell of homogeneous of form, be the vortex shape growth;
7). after treating cell 80% fusion of vortex shape growth; Earlier wash culturing bottle repeatedly 3 times with saline water; Digestive system peptic cell with containing 0.05% pancreatin/0.2mMEDTA goes down to posterity in 1: 3 ratio, and culture condition is replaced by the α-MEM substratum that contains 10% foetal calf serum, 0.2% qingfengmeisu qiong;
8). after amplification in 20-25 days, cell reached for 4～5 generations, obtained a large amount of mescenchymal stem cells;
9). the back cell that will increase is earlier resuspended with the substratum of precooling; Drip the foetal calf serum that contains DMSO of precooling subsequently; Wherein the ratio of substratum, foetal calf serum and DMSO is 5: 4: 1, and the cell after resuspended is put into freezing storing box ,-70 ℃ of refrigerators place overnight be placed on to preserve in the liquid nitrogen get final product.
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| CN102511471B (en) * | 2011-12-15 | 2013-08-28 | 成都清科生物科技有限公司 | Mesenchymal stem cell frozen stock solution and preparation method thereof |
| CN104839146B (en) * | 2015-05-29 | 2018-02-09 | 广州赛莱拉干细胞科技股份有限公司 | Composition and application thereof, placenta preserve preparation and preparation method thereof |
| CN107114356A (en) * | 2016-08-01 | 2017-09-01 | 北京世纪劲得生物技术有限公司 | A kind of placenta and umbilical cord cells protect liquid |
| CN106719601A (en) * | 2016-11-30 | 2017-05-31 | 广州赛莱拉干细胞科技股份有限公司 | A kind of umbilical cord preserving fluid and its application |
| CN107653226A (en) * | 2017-11-15 | 2018-02-02 | 南京三生生物技术股份有限公司 | A kind of human umbilical cord mesenchymal stem cells isolated culture method |
| CN110540959A (en) * | 2019-10-08 | 2019-12-06 | 孟明耀 | Umbilical cord mesenchymal stem cell isolation culture amplification method |
| CN111955454A (en) * | 2020-05-14 | 2020-11-20 | 领航干细胞再生医学工程有限公司 | Human in-vitro tooth preservative fluid and preparation method thereof |
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| CN101591644A (en) * | 2009-04-13 | 2009-12-02 | 中国人民解放军第三○二医院 | The preparation of umbilical cord-mesenchymal stem cells (UC-MSCs) used in clinical therapy and storage |
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| CN1878860A (en) * | 2003-11-11 | 2006-12-13 | 韩薰 | Method of isolating and culturing mesenchymal stem cell derived from umbilical cord blood |
| CN101591644A (en) * | 2009-04-13 | 2009-12-02 | 中国人民解放军第三○二医院 | The preparation of umbilical cord-mesenchymal stem cells (UC-MSCs) used in clinical therapy and storage |
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| Title |
|---|
| Ikuo Ishige et. al.Comparison of mesenchymal stem cells derived from arterial, venous, and Wharton’s jelly explants of human umbilical cord.《Int J Hematol》.2009,第90卷261-269. * |
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