CN101575590B - Method for preparing human umbilical cord mesenchymal stem cells - Google Patents
Method for preparing human umbilical cord mesenchymal stem cells Download PDFInfo
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Abstract
The invention discloses a method for preparing human umbilical mesenchymal stem cells, which comprises the following steps: (1) removing residual blood from the human umbilical cord, shearing the umbilical cord and digesting the umbilical cord by collagenase; (2) adding pancreatin into the umbilical cord to digest the umbilical cord; (3) filtering the digestive solution by using a screen, and removing tissues which are not digested; (4) diluting the filtered digestive solution by using phosphate buffer solution; (5) centrifugating the solution to obtain cells; and (6) inoculating the cells to a culture medium to culture so as to obtain large amount of human umbilical cord mesenchymal stem cells. The obtained cells can meet the requirements of clinical and experimental study, and lay a foundation for the future application.
Description
Technical field
The present invention relates to a kind of preparation method of umbilical cord mesenchymal stem cells.
Background technology
Mescenchymal stem cell (mesenchymal stem cells; MSCs) be to derive to grow early stage mesoderm and ectodermic one type of multipotential stem cell; In marrow, find at first, receive people's attention day by day because of it has following characteristics: (1) multidirectional differentiation potential.In vivo/can be divided into multiple histocytes such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelium under the outer specific inductive condition; Still have multidirectional differentiation potential after continuous passage cultivation and the freezing preservation, can be used as the ideal seed cell and be used for organizational project.(2) hematopoiesis support and short the implantation.As the main cellular constituent of hematopoieticmicroenviron-ment---the ancestral cells of stromal cell lines, MSCs realizes the finely regulating to hematopoiesis, the growth of hematopoiesis support stem/progenitor cells through direct effect, secretory cell epimatrix and the various kinds of cell factor of cell pair cell.Can promote the implantation of hematopoietic stem/progenitor cells with the hemopoietic stem cell co-transplantation, for improving the umbilical cord blood transplantation success ratio, it is significant to widen the umbilical cord blood transplantation scope of application.(3) immunoregulation.MSCs expresses the MHC-I quasi-molecule; Do not express CD80, CD86, collaborative stimulation molecule such as HLA-DR; Externally suppress mixed lymphocyte reacion; Inducing immune tolerance in the body for the graft versus host disease that causes behind prevention and the treatment recessive allele hematopoietic cell transplantation, induces the tolerance of organ transplantation immunity that application promise in clinical practice is arranged.(4) self-replacation.MSCs can self-replacation as stem cell, and amplification in vitro, multiplication capacity are strong, can be used as the stem cell platform of genetic manipulation, and very tempting prospect is arranged in gene therapy.
The main source of MSCs is adult's marrow at present; But adult's bone marrow MSCs cell quantity and proliferation and differentiation potential descend with the increase at age; Viral infection rate is higher; And the collection of donor MSCs palpus row bone marrow puncture, the source is restricted, and seeking new MSCs source is the focus of at present domestic and international stem-cell research.Discover that MSCs extensively exists at human fetal and the multiple tissue in living back, comprises periosteum, fat, amniotic fluid, corium, tooth, Skelettmuskel, tire lung, tire liver, bleeding of the umbilicus and umbilical cord etc.There is more dispute in report about bleeding of the umbilicus MSCs, and hematopoietic stem/progenitor cells in the branch defection of the bleeding of the umbilicus MSCs loss bleeding of the umbilicus, and it is frozen to influence bleeding of the umbilicus.The umbilical cord wide material sources are convenient to draw materials, and donor is had no adverse effect; The restriction of amoral ethics problem; And umbilical cord is expected to improve the umbilical cord blood transplantation success ratio as derive from the bleeding of the umbilicus co-transplantation from body MSCs, and therefore, umbilical cord MSCs is expected to become the alternative source of ideal of bone marrow MSCs.Recently in the proposition umbilical cord such as Yuri A MSCs be present in endothelium with in subcutaneous, can adopt the endothelium digestion method from fetal cord, to isolate MSCs.But adopt the endothelium digestion method, have only 30% umbilical cord sample can obtain MSCs, 70% sample can not obtain the MSCs that can repeatedly go down to posterity.
Summary of the invention
The objective of the invention is to overcome the deficiency of prior art, a kind of preparing human umbilical mesenchymal stem cells efficiently is provided.
Technical scheme of the present invention is summarized as follows:
A kind of preparing human umbilical mesenchymal stem cells is characterized in that being made up of following step:
(1) people's umbilical cord is removed remained blood, shred, use collagenase digesting; (2) add trysinization again; (3) Digestive system is used screen filtration, removes not digestion tissue; (4) Digestive system after will filtering dilutes with phosphoric acid buffer; (5) centrifugal, obtain cell; (6) cell inoculation is cultivated in substratum, obtained a large amount of human umbilical cord mesenchymal stem cells.
People's umbilical cord shreds preferably 0.05-1.5mm
3Fritter.
The weight that adds collagenase is the 0.05%-0.2% of raw material weight, preferably 0.1%, and the digestion temperature is 37 ℃, digestion time is 30-60 minute, preferably 45 minutes.
Add pancreatin (Sigma, weight USA) is the 0.05%-0.2% of raw material weight, preferably 0.125%, the digestion temperature is 37 ℃, digestion time is 30-60 minute, preferably 45 minutes, in digestive process, can select for use rotor to stir.
Screen cloth can before and after with 100 orders, 200 eye mesh screens.
With phosphoric acid buffer (PBS) dilution, the volume ratio of Digestive system and phosphoric acid buffer is 1:8-12, preferably 1:10 with the Digestive system after filtering.
When centrifugal, the rotating speed of whizzer is-2000 rev/mins of 1000 commentaries on classics, changes 10-20 minute, preferred 1500 rev/mins, changes 15 minutes.
The substratum of cell inoculation is a serum free medium.
Advantage of the present invention:
A kind of preparing human umbilical mesenchymal stem cells of the present invention, operation is simple.90% mature umbilical cord can be isolated abundant MSCs, and can amplification obtain 1 * 10 in 5 weeks
9The umbilical cord MSCs that MSCs cell, present method obtain can satisfy clinical and needs experimental study in external a large amount of amplifications.
Description of drawings
Fig. 1 is the cellular form of MSCs;
Fig. 2 is the cell growth curve of the MSCs in umbilical cord source;
Fig. 3 is the cell cycle analysis of the MSCs in umbilical cord source;
Fig. 4 is the MSCs in the umbilical cord source differentiation of inducing to scleroblast and adipocyte;
Fig. 5 analyzes the MSCs cytokine expression situation in umbilical cord source for RT-PCR;
Fig. 6 is the MSCs in the umbilical cord source support effect to hematopoiesis CD34+ cell.
Embodiment
Below in conjunction with specific embodiment the present invention is further described.
The separation of cell and cultivation
(1) people's umbilical cord is washed by phosphoric acid buffer (PBS) repeatedly, remove residual blood, shred to about 1mm
3Fritter, add the collagenase of raw material weight 0.1%, 37 ℃ of digestion 45 minutes; (2) add raw material weight 0.125% pancreatin (Sigma, USA) 37 ℃ of digestion is 45 minutes, in digestive process, stirs with rotor, adds the effect of an amount of serum termination pancreatin; (3) Digestive system is used screen filtration, successively uses 100 orders and 200 purpose strainer filterings, removes indigested tissue; (4) Digestive system after will filtering dilutes with phosphoric acid buffer, and Digestive system and phosphoric acid buffer liquid proportional are the 1:10 ratio; (5) centrifugal, obtain cell, the rotating speed of whizzer is the centrifugal 15min of 1500rpm/min, obtains cell; (6) cell is by 1～2 * 10
4/ cm
2Be inoculated in 25ml plastic culture bottle,, put 37 ℃, 5%CO with 10ml serum free medium (Jiangxi consonance stem cell company)
2Incubator is cultivated, and changes liquid after 4-5 days, discards non-adherent cell, and half amount was changed liquid in later every 3-4 days.Treat that cell 80% merges, 0.25% trysinization is gone down to posterity by 1:3.
(1) people's umbilical cord is washed by phosphoric acid buffer (PBS) repeatedly, remove residual blood, shred to about 0.05mm
3Fritter, add the collagenase of raw material weight 0.05%, 37 ℃ of digestion 60 minutes; (2) add raw material weight 0.05% pancreatin (Sigma, USA) 37 ℃ of digestion is 60 minutes, in digestive process, stirs with rotor, adds the effect of an amount of serum termination pancreatin; (3) Digestive system is used screen filtration, successively uses 100 orders and 200 purpose strainer filterings, removes indigested tissue; (4) Digestive system after will filtering dilutes with phosphoric acid buffer, and Digestive system and phosphoric acid buffer liquid proportional are the 1:8 ratio; (5) centrifugal, obtain cell, the rotating speed of whizzer is the centrifugal 20min of 1000rpm/min, obtains cell; (6) cell is by 1～2 * 10
6/ cm
2Be inoculated in 75ml plastic culture bottle, (Stemcell company Germany), puts 37 ℃, 5%CO with the 30ml serum free medium
2Incubator is cultivated, and changes liquid after 4-5 days, discards non-adherent cell, and half amount was changed liquid in later every 3-4 days.Treat that cell 80% merges, 0.25% trysinization is gone down to posterity by 1:3.Cell count that present method obtains and success ratio are apparently higher than the usual way according to the digestion of document endothelium.
(1) people's umbilical cord is washed by phosphoric acid buffer (PBS) repeatedly, remove residual blood, shred to about 1.5mm
3Fritter, add the collagenase of raw material weight 0.2%, 37 ℃ of digestion 30 minutes; (2) add raw material weight 0.2% pancreatin (Sigma, USA) 37 ℃ of digestion is 30 minutes, in digestive process, stirs with rotor, adds the effect of an amount of serum termination pancreatin; (3) Digestive system is used screen filtration, successively uses 100 orders and 200 purpose strainer filterings, removes indigested tissue; (4) Digestive system after will filtering dilutes with phosphoric acid buffer, and Digestive system and phosphoric acid buffer liquid proportional are the 1:12 ratio; (5) centrifugal, obtain cell, the rotating speed of whizzer is the centrifugal 10min of 2000rpm/min, obtains cell; (6) cell is by 1～2 * 10
6/ cm
2Be inoculated in 25ml plastic culture bottle, (Gibco company USA), puts 37 ℃, 5%CO with the 15ml serum free medium
2Incubator is cultivated, and changes liquid after 4-5 days, discards non-adherent cell, and half amount was changed liquid in later every 3-4 days.Treat that cell 80% merges, 0.25% trysinization is gone down to posterity by 1:3.Cell count that present method obtains and success ratio are apparently higher than the usual way according to the digestion of document endothelium.
Morphological observation
Adopt the isolating primary cell of present method more than adherent in 24-48 hour; Cultivate 1-2 week; The visible attached cell of inverted microscope is fusiformis, polygon, and rate of growth is very fast after 2 weeks, becomes the spindle cell of the relative homogeneous of form; Be be arranged in parallel growth or swirl shape growth, merge the attached cell layer in forming 80% 2-3 week.Passed for 20 generations continuously, cellular form does not have obvious change.And adopt the endothelium digestion method of bibliographical information to have only 30% sample can isolate MSCs, 70% sample attached cell is worn for not surpassing for 2 generations.(Fig. 1)
Fig. 1 is the cellular form of the MSCs in umbilical cord source.(A-C), the MSCs that adopts present method to obtain cultivates the cellular form after 7 days; (D, E), MSCs passes the cellular form of the first-generation after 14-20 days; (H) MSCs passes the 1st, 3,5 days cellular form of the s-generation for F, G; (I-L), traditional endothelium digestion method obtains MSCs cultured cells form, passed for two generations after cellular form change.
Embodiment 5
The mensuration of cell growth curve
With attached cell with 0.25% trysinization after, by 2 * 10
4Cells/well is seeded in 24 orifice plates, and is every at a distance from 3 holes of 24h digestion, collecting cell, and expect blue living cell counting, the drafting growth curve with 0.4% tire.
Cell proliferation is very fast, and at logarithmic phase, cell doubling time all is about 30h (Fig. 2).The every biography generation of cell, cell count increases by 2 times approximately.
Fig. 2 is the cell growth curve of the MSCs in umbilical cord source.Third generation MSCs is by 2 * 10
4Cells/well is seeded in 24 orifice plates, and is every at a distance from 3 holes of 24h digestion, collecting cell, and expect blue living cell counting with 0.4% tire, get the MV of 5 experimental results, the drafting growth curve.
The mensuration of cell cycle
In the logarithmic phase of cell growth, peptic cell, film 24h is fixed and passed through to cold 70% ethanol for 4 ℃; Hatch 30min for the RNaseA37 of 10 μ g/mL ℃, add 4 ℃ of lucifuges of 50 μ g/mL propidium iodides and hatch 5min, flow cytometer (FACA Calibur type; USA) detect ModiFIT software analysis result.
The flow cytometry analysis Cell cycles showed, the cell above 80% is in G
0/ G
1Phase, S+G
2The cell of+M phase only accounts for 14% (Fig. 3)
Fig. 3 is the cell cycle analysis of the MSCs in umbilical cord source.78.84% cell at G0-G1 phase and 11.16% cell in the S+G2+M phase.
Embodiment 7
External evoked differentiation
1. induce noble cells with 2 * 10 to scleroblast
4The density in/hole is inoculated in 25cm
2Plastic culture bottle and six orifice plates induce differentiated system for containing 1.0 * 10
-8The M DEXAMETHASONE BP98,2.0 * 10
-4The M xitix, 7.0 * 10
-3The IMDM substratum of M β-phospho-glycerol, amount was changed liquid once in per 3 days half.Detecting calcification matrix with Von Kossa dyeing on the 14th day precipitates.Be covered with black particle between osteogenic induction test group Von-Kossa dyeing in the 14th day visible cell, the particle size heterogeneity, prompting has the mineralising apposition, and black mineralizer deposition (Fig. 4 A) is not seen in the cellular control unit Von-Kossa dyeing that does not add the skeletonization inducing culture.
2. induce noble cells with 2 * 10 to adipocyte
4The density in/hole is seeded in 25cm
2Plastic culture bottle and six orifice plates induce differentiated system for containing 10%FCS, 10
-6The IMDM substratum of M DEXAMETHASONE BP98,100 μ g/mL1-methyl-3-isobutyl--xanthine (IBMX), 50 μ g/mL xitix.Amount was changed liquid once in per 3 days half.Inverted microscope is observed the formation of fat granule down, adopts oil red 0 dyeing to detect fat granule on the 14th day.Fat induces 7 days promptly visible down endochylema lactones of inverted microscope to drip formation, and oil red-O dyeing in the 14th day is strong positive, and the cellular control unit that does not add fatty inducing culture does not have fat and drips formation, oil red-0 dyeing negative (Fig. 4 B).
Detect with RT-PCR through inductive cell and contrast (without inducing) cell and cultivate the 14th day lipoproteinesterase (LPL) and SPP1 (OPN) genetic expression 3.RT-PCR detect differentiated result, identify the fatty and Osteoblast Differentiation potential of attached cell one-tenth.Trizol test kit (Sangon company) extracts total RNA.The primer is respectively: lipoproteinesterase (lipoproteinlipase, LPL; 298bp): 5 '-ATG GAG AGC AAA GCC CTG CTC-3 '; 6 '-TAC AGG GCG GCC ACAAGT TTT; SPP1 (osteopontin, OPN; 330bp): 5 '-CTA GGC ATC ACC TGT GC CAT ACC-3 '; 5 '-CAG TGA CCA GTT CAT CAG ATT CAT C-3 '; B2M (β 2-microglobulin, β
2M; 115bp): 5 '-ACC CCC ACT GAA AAA GAT GA-3 '; 5 '-ATC TTC AAA CCT CCA TGA TG-3 '.The PCR condition is: 95 ℃ of 10min, and 94 ℃ of sex change 50s, 58 ℃ of annealing 30s, 72 ℃ are extended 1min, circulate 30 times, and last 72 ℃ are extended 10min.
RT-PCR result confirms that above-mentioned derivative attached cell expresses lipogenesis and bone forming specific gene lipoproteinesterase and SPP1 gene respectively, and cellular control unit does not have expression (Fig. 4 C).
Fig. 4 is the MSCs in the umbilical cord source differentiation of inducing to scleroblast and adipocyte.A: the MSCs in umbilical cord source is to osteoblastic differentiation; B: the MSCs in umbilical cord source is to the differentiation of adipocyte; C: the abduction delivering of lipoproteinesterase and SPP1 gene.
Hematopoiesis is supported
1. Hemopoietic factor secretion
Adopt the RT-PCR method to detect umbilical cord MSC cytokine secretion.The following .PCR condition of design of primers is: 95 ℃ of 10min, and 94 ℃ of sex change 50s, 58 ℃ of annealing 30s, 72 ℃ are extended 1min, circulate 30 times, and last 72 ℃ are extended 10min.RT-PCR result shows that umbilical cord MSC expresses SCF, TPO, and FL, IL-6, IL-11, SDF-1, VEGF does not express IL-3 (Fig. 5).
Gene Primer?sequence
lipoprotein?lipase S：5’-ATGGAGAGCAAAGCCCTGCTC-3’
A：5’-TACAGGGCGGCCACAAGTTTT-3’
Osteopontin S：5’-CTAGGCATCACCTGTGCCATACC-3’
A：5’-CAG?TGACCAGTTCATCAGATTCATC-3’
SCF S：5’-CTCCTATTTAATCCTCTCGTC-3’
A：5’-TACTACCATCTCGCTTATCCA-3’
TPO S：5’-TGGAGCCCAACAACCTATCTC-3’
A：5’-GGGCTGAAAGGCACATTTGGT-3’
FL S：5’-CCTCCTTGGGGCCCTGCAGAGCCT-3’
A：5’-GGGTGGAGCTGGACCACAGGG-3’
M-CSF S：5’-TGA?GAG?GCA?GTC?CGA?GGG?AT-3’
A：5’-GAA?TTC?CCT?CTA?CAC?TGG?CA-3’
IL-3 S：5’-ATGAGCCGCCTGCCCGTCCTG-3’
A：5’-GCGAGGTCAAAGTCGTCTGTTG-3’
IL-6 S：5’-GTAGCCGCCCCACACAGACAGCC-3’
A：5’-GCCATCTTTGGAAGGTTCAGG-3’
GM-CSF S：5’-GTCTCCTGAACCTGAGTAGAGACA-3’
A：5’-AAGGGGATGACAAGCAGAAAGTCC-3’
G-CSF S：5’-AGCTTCCTGCTCAAGTGCTTAGAG-3’
A：5’-TTCTTCCATCTGCTGCCAGATGGT-3’
SDF-1 S：5’-CCCTTCAGATTGTAGCCCGG-3’
A：5’-CGATCCCAGATCAATGTGCC-3’
VEGF S：5’-TCGGGCCTCCGAAACCATGA-3’
A：5’-CCTGGTGAGAGATCTGGTTC-3’
β
2-MG S：5’-TCTGGCCTTGAGGCTATCCAGCGT-3’
A：5’-GTGGTTCACACGGCAGGCATACTC-3’
Fig. 5 analyzes the MSCs cytokine expression situation in umbilical cord source for RT-PCR.
2. hematopoiesis support
Third generation MSCs2.0 * 10
4/ hole kind is in 24 orifice plates,
60Bleeding of the umbilicus CD34+ cell 2.0 * 10 is planted in C020Gy irradiation back
4, in 8 weeks of co-cultivation, half amount is changed liquid weekly, and the cell kind that swaps out is cultivated two all backs counting colonies in the methylcellulose gum perfect medium, is a colony more than or equal to 50 cells.Control group is the independent bleeding of the umbilicus CD34+ of paving ancient piece of jade, round, flat and with a hole in its centre cell cell cultures group not and adult derived from bone marrow MSC and bleeding of the umbilicus CD34+ cell co-cultivation group.
Cell cultures colony count result shows: promptly do not have colony separately bleeding of the umbilicus CD34+ cell cultures two weeks of group and form; And umbilical cord MSC and bleeding of the umbilicus CD34+ cell co-cultivation group can be kept 6 Zhou Shangke and have colony to form; Each all colony forming efficiency and marrow and bleeding of the umbilicus CD34+ cell co-cultivation group indifference, prompting umbilical cord MSC has the ability (Fig. 6) of supporting long-term hematopoiesis.
Fig. 6 is the MSCs in the umbilical cord source support effect to hematopoiesis CD34+ cell.The MSCs and the hematopoiesis CD34+ co-culture of cells in umbilical cord source are got suspension cell weekly and are carried out the semisolid cultivation again, and numeration is drawn to colony.
Conclusion:
1. the method for from the mature umbilical cord of people, isolating mescenchymal stem cell has been set up in this research, and operation is simple, can from 90% umbilical cord sample, isolate abundant MSCs, and 5 weeks can obtain 1 * 10
9MSCs can satisfy clinical and experiment is studied umbilical cord mesenchymal stem cells.
2. the external easy amplification of umbilical cord mesenchymal stem cells; Multiplication capacity is strong; Immunophenotype and derived from bone marrow mesenchymal stem cells, the multidirectional differentiation function of external tool, cytokine secretion enrich, have the function that long-term hematopoiesis is supported, are that the mesenchymal stem cells MSCs ideal substitutes the source.
Claims (6)
1. preparing human umbilical mesenchymal stem cells is characterized in that being made up of following step:
(1) people's umbilical cord is removed remained blood, shred, use collagenase digesting; (2) add trysinization again; (3) Digestive system is used screen filtration, removes not digestion tissue; (4) Digestive system after will filtering dilutes with phosphoric acid buffer; (5) centrifugal, obtain cell; (6) cell inoculation is cultivated in substratum, obtained a large amount of human umbilical cord mesenchymal stem cells, the weight of said adding collagenase is the 0.05%-0.2% of raw material weight, and the digestion temperature is 37 ℃, and digestion time is 30-60 minute; The weight of said adding pancreatin is the 0.05%-0.2% of raw material weight, and the digestion temperature is 37 ℃, and digestion time is 30-60 minute; The volume ratio of said Digestive system and phosphoric acid buffer is 1: 8-12; The rotating speed of whizzer is-2000 rev/mins of 1000 commentaries on classics, changes 10-20 minute; The substratum of said cell inoculation is a serum free medium.
2. a kind of preparing human umbilical mesenchymal stem cells according to claim 1 is characterized in that said people's umbilical cord being shredded 1mm
3Fritter.
3. a kind of preparing human umbilical mesenchymal stem cells according to claim 1, the weight that it is characterized in that said adding collagenase is 0.1% of raw material weight, said digestion time is 45 minutes.
4. a kind of preparing human umbilical mesenchymal stem cells according to claim 1, the weight that it is characterized in that said adding pancreatin is 0.125% of raw material weight, said digestion time is 45 minutes, in said digestive process, stirs with rotor.
5. a kind of preparing human umbilical mesenchymal stem cells according to claim 1, the volume ratio that it is characterized in that said Digestive system and phosphoric acid buffer is 1: 10.
6. a kind of preparing human umbilical mesenchymal stem cells according to claim 1, the rotating speed that it is characterized in that said whizzer is 1500 rev/mins, changes 15 minutes.
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| JP6169316B2 (en) | 2008-08-20 | 2017-07-26 | アンスロジェネシス コーポレーション | Improved cell composition and method for producing the same |
| CN101864395B (en) * | 2010-05-25 | 2011-11-02 | 中国人民解放军总医院第一附属医院 | In-vitro inducing differentiation of umbilical cord mesenchymal stem cells into tissue engineering skin seed cells |
| CA2801009C (en) * | 2010-06-01 | 2021-10-26 | Auxocell Laboratories, Inc. | Native wharton's jelly stem cells and their purification |
| CN102250767B (en) * | 2011-06-28 | 2013-06-19 | 江苏省北科生物科技有限公司 | Digitalized automatic production system for mesenchymal stem cells of umbilical cord |
| CN103451151B (en) * | 2013-06-19 | 2016-01-20 | 武汉道培胎盘干细胞生物技术有限公司 | A kind of method of cultivator 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 |
| CN104694469A (en) * | 2015-03-26 | 2015-06-10 | 江苏希瑞干细胞技术有限公司 | Preparation method for mesenchymal stem cells from placental decidua basalis |
| CN104988117B (en) * | 2015-07-03 | 2016-06-15 | 深圳中基恒润投资有限公司 | Separate and cultivate mescenchymal stem cell from umbilical cord and to the method for chondrogenic differentiation |
| CN107043746A (en) * | 2017-04-14 | 2017-08-15 | 青岛青春派生物科技有限公司 | A kind of human umbilical tissue digestion method |
| CN107496455A (en) * | 2017-08-30 | 2017-12-22 | 北京百奥泰生物科技有限公司 | A kind of injection for treating degenerative osteoarthropathy and preparation method thereof |
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Effective date of registration: 20170918 Address after: 300457 No. fourth, 80 Avenue, Tianjin economic and Technological Development Zone Patentee after: Tianjin AmCellGene Engineering Co., Ltd. Address before: B3-B4 Science Park, Fourth Avenue, TEDA Development Zone, Tianjin Patentee before: Taida Life Science and Technology Research Center, Hematology Inst., China Medic |