CN102586184B - Method for establishing placental mesenchyme stem cell library - Google Patents
Method for establishing placental mesenchyme stem cell library Download PDFInfo
- Publication number
- CN102586184B CN102586184B CN 201210044638 CN201210044638A CN102586184B CN 102586184 B CN102586184 B CN 102586184B CN 201210044638 CN201210044638 CN 201210044638 CN 201210044638 A CN201210044638 A CN 201210044638A CN 102586184 B CN102586184 B CN 102586184B
- Authority
- CN
- China
- Prior art keywords
- cell
- placenta
- cells
- placental
- stem cell
- 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.)
- Active
Links
Images
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a method for establishing a placental mesenchyme stem cell library. The method comprises the steps of digesting placental lobules with PBS buffer solution containing tissue digestive enzyme to separate mononuclear cells; suspending the obtained cells with an MSC (mesenchyme stem cell) culture medium; culturing the cells in a 5% CO2 incubator at 37 DEG C; after disseminated cells form clones, respectively culturing each cloned cells with the MSC culture medium; after cell infusion, digesting passages with pancreatin to obtain placental mesenchyme stem cells; detecting the obtained placental mesenchyme stem cells in at least one of the following items: cell viability, cell contamination, hereditary diseases, HLA-ABC/DR type matching; freezing the passaged mesenchymestem cells in liquid nitrogen; establishing a data base of the mesenchyme stem cells comprising the information mentioned above; and associating the data base with the freezed cells. The mesenchyme stem cells involved in the method of the invention has high purity.
Description
Technical field
Thereby the present invention relates to from placenta the separating mesenchymal stem cell and set up placenta mesenchyma stem cell
The method in storehouse.
Background technology
Mescenchymal stem cell (mesenchymal stem cell, MSC) for example the mankind's mescenchymal stem cell is separated from marrow the earliest, derive from the tissue stem cell that a mesoblastic class has multi-lineage potential and self-renewal capacity, have to scleroblast in vivo with under the external specified conditions, the chondrocyte, adipocyte, endotheliocyte, neurocyte, the myocyte, the ability of the multiple adult cytodifferentiation such as liver cell (Caplan AI.Mesenchymal stem cells.J Orthop Res.1991,9:641-650.Pittenger MF, Mackay AM, Beck SC, et al.Multilineage potential of adult human mesenchymal stem cells.Science.1999; 284:143-147).The up-to-date mescenchymal stem cell that studies show that has immunomodulatory and Hematopoiesis Support affect, and is easy to foreign gene importing expression.Therefore the seed cell of mescenchymal stem cell during still tissue-engineered bone, cartilage and cardiac muscle make up, important carrier cell in the gene therapy, and because mescenchymal stem cell promotes hematopoietic reconstitution and the anti-host response function of inhibition of transplant, in hematopoietic stem cell transplantation and organ transplantation, be with a wide range of applications.Mescenchymal stem cell has the characteristic of external adherent growth, utilizes this specific character, people successfully from the Various Tissues such as liver, kidney, pancreas, muscle, cartilage, skin, peripheral blood separation and Culture go out mescenchymal stem cell.
The mescenchymal stem cell of reporting at present is mainly derived from marrow, adopts density gradient centrifugation to obtain.Although separation method is easy, donor is got marrow need to experience a relatively more painful operation, and has very high infection chance in the process of drawing materials and after drawing materials; Because the content of MSC is extremely rare among the human bone marrow, per 10
5～10
6Approximately only have 1 in the individual mononuclearcell, and along with the increase at age, the quantity of mescenchymal stem cell, propagation and differentiation capability descend significantly all in the marrow, make it in research and use in the especially clinical application to be restricted.The placenta that originates from the embryonic development period extraembryonic mesoderm is comprised of a matter, blood vessel and nurse cell, contains a large amount of mesenchyme compositions.Up-to-date studies show that contains abundant stem cell in the placenta, separation and Culture goes out these multipotential stem cells and will open up a brand-new and abundant source for experimental study and clinical application from placenta.
The existing method that thereby separate stem cells is set up the placenta stem-cell storehouse from placenta still has shortcomings, and for example purity is not enough and/or quantity is not high, and then demonstrates the expectation that these methods still can not satisfy people.The for example invention of CN 101270349A (Chinese patent application numbers 200810061267.6, open day on September 24th, 2008) disclosed being entitled as " placenta mesenchyma stem cell separates and the amplification in vitro cultural method "; The invention of CN 101693884A (Chinese patent application numbers 200910117522.9, open day on April 14th, 2010) disclosed being entitled as method of separating and extracting stem cells " a kind of from placenta, umbilical cord or fatty tissue "; CN 102146359A (Chinese patent application numbers 201110005964.1, open day on August 10th, 2011) disclosed being entitled as " extracted the method for primary mesenchymal stem cells and serum-free amplification " from placenta invention.These methods are remaining to be further improved aspect the purity of extract and/or the rate of recovery.
Thereby this area still needs to have new separate stem cells from placenta to set up the method in placenta stem-cell storehouse.
Summary of the invention
The objective of the invention is to solve the existing defective of obtaining the placenta mesenchyma stem cell method, thus provide a kind of practicality simply from placenta a large amount of separating mesenchymal stem cells set up the method in placenta stem-cell storehouse.The inventor finds to adopt special working method, and the cell purity that obtains is high and/or the cell rate of recovery is high.The present invention is based on this discovery and be accomplished.
Therefore, first aspect present invention provides a kind of method of setting up placental mesenchyme stem cell library, and the method may further comprise the steps:
(a) get placental lobules, fully wash with the PBS damping fluid, to remove residual blood in the placenta;
(b) placental lobules is cut into bulk, adds the PBS damping fluid that contains the tissue digestion enzyme, hatch digestion at 37 ℃ again;
(c) tissue block is filtered with copper mesh, grind in case of necessity to impel filtration;
(d) filtered liquid of collecting is centrifugal, separate mononuclearcell, the cell that suspends and obtain with the MSC substratum again is then at 37 ℃, 5%CO
2Cultivate in the incubator;
(e) after disseminated cell formed the clone, each clone cell of picking was cultivated respectively with the MSC substratum, after cytogamy, goes down to posterity with trysinization, namely gets placenta mesenchyma stem cell;
(f) for step (e) gained placenta mesenchyma stem cell, detect at least one item of following items: cytoactive, cell contamination, inherited disease, HLA-ABC/DR join type;
(g) placenta mesenchyma stem cell after step (e) gained is gone down to posterity is frozen in liquid nitrogen;
(h) set up the database of the placenta stem-cell that comprises above information, and make this database carry out related with the freeze-stored cell of step (g).
According to the method for first aspect present invention, wherein said step (a) is under aseptic condition placental lobules to be cut, and fully washes placental lobules with the PBS damping fluid and removes residual blood in the placenta.
Method according to first aspect present invention, wherein said step (a) is within four hours postpartum, under aseptic condition, placental lobules is cut, fully washed placental lobules with the PBS damping fluid that contains 10% volume foetal calf serum (FBS) and remove residual blood in the placental lobules.
According to the method for first aspect present invention, the tissue digestion enzyme in the wherein said step (b) is to be selected from following one or more: dispase, pancreatin, deoxyribonuclease I (DNase I), collagenase IV, Unidasa.In one embodiment, the tissue digestion enzyme in the described step (b) comprises: dispase, pancreatin, deoxyribonuclease I (DNase I), collagenase IV, Unidasa.In one embodiment, in step (b), comprise approximately 0.1mg/mLdispase, approximately 0.25mg/mL pancreatin, approximately 0.25mg/mL DNase I, approximately 1mg/mL collagenase IV and about 1mg/mL Unidasa in the described PBS damping fluid.
According to the method for first aspect present invention, in step (b), hatched 10～30 minutes at 37 ℃, preferred 10～20 minutes, for example 10 minutes, 15 minutes or 20 minutes.
According to the method for first aspect present invention, in step (b), placental lobules is cut into approximately 1cm
3The tissue block of size.
According to the method for first aspect present invention, in step (c), tissue block is ground with syringe simultaneously with the copper mesh filtration.In one embodiment, in step (c), tissue block and cell suspension one are reinstated 160～240 orders (preferred 200 orders) copper mesh filter and use simultaneously syringe piston tissue abrasion piece.
According to the method for first aspect present invention, in step (d), described MSC substratum is the PBS damping fluid that contains 10%FBS.In an embodiment of either side of the present invention, described MSC substratum is the PBS damping fluid that contains 10%FBS.In one embodiment, in step (d), the filtered liquid of collecting is separated mononuclearcell with density gradient centrifugation, washing, the cell that suspends and obtain with the MSC substratum again is then at 37 ℃, 5%CO
2Cultivate in the incubator.In one embodiment, in step (d), the cell suspension of collecting after filtering is joined in the centrifuge tube, centrifugal 10 minutes of 1000rpm outwells supernatant, with the PBS damping fluid re-suspended cell that contains 10%FBS.
According to the method for first aspect present invention, in step (e), after disseminated cell formed the clone, each clone cell of picking was cultivated respectively with the MSC substratum, after cell 60～90% merges, went down to posterity with trysinization, namely got placenta mesenchyma stem cell.In one embodiment, in step (e), after being dispersed in attached cell formation clone, with 0.05% trypsinase/2mM EDTA digestion, the cultivation of going down to posterity.
According to the method for first aspect present invention, in step (f), it is to utilize trypan blue staining to count the number of frozen front and back viable cell that described cytoactive detects.
According to the method for first aspect present invention, in step (f), described cell contamination detects and utilizes a small amount of cell cultures, detects the pollution whether cell is subject to fungus and bacterium.In one embodiment, it is to utilize the etiology method that described cell contamination detects, and detects cell and whether is subject to being selected from following one or more infection: Hepatitis B virus, the third liver, virus of AIDS, cytomegalovirus, Epstein-Barr virus and syphilis, HbsAg, HbsAb, HBcAb, HbeAg, HbeAb, HCVAb, HIV-1/2Ab, CMV-IgM and EBV-IgA, TRUST.
According to the method for first aspect present invention, in step (f), it is the method for utilizing molecular genetics that described inherited disease detects, and detects freeze-stored cell and whether has inherited disease.
According to the method for first aspect present invention, in step (f), it is to detect cell HLA-ABC/DR phenotype that described HLA-ABC/DR joins type.
According to the method for first aspect present invention, in step (g), described placenta mesenchyma stem cell is frozen in liquid nitrogen through the programmed cooling process.
According to the method for first aspect present invention, in step (g), described placenta mesenchyma stem cell is present in the cells frozen storing liquid.In one embodiment, this cells frozen storing liquid comprises 50% low sugar DMEM nutrient solution, 40%FBS, 10% dimethyl sulfoxide (DMSO).
Method according to first aspect present invention, in step (h), comprise in the described database and all relevant data of the cell of preserving, include but not limited to: the biological characteristics detected result of cell, multi-lineage potential qualification result, cellular elements genetic diagnosis result, fetus and father and mother's thereof detail file.
According to the method for first aspect present invention, the method may further comprise the steps: under aseptic condition placental lobules is cut, fully washed placental lobules with the PBS damping fluid and remove residual blood in the placenta.Again placental lobules is cut into 1cm
3The tissue block of size, add contain the Various Tissues digestive ferment the PBS damping fluid hatched 15 minutes at 37 ℃, tissue block is filtered with copper mesh grinds with syringe simultaneously.The liquid of collecting after filtering separates mononuclearcell with density gradient centrifugation, and the cell that obtains with the suspension of MSC substratum after the washing is put into 37 ℃ of 5%CO
2Cultivate in the incubator.After disseminated cell forms the clone, each clone cell chosen with the MSC substratum cultivate respectively, after cell 80～90% fusions, go down to posterity with trysinization, namely get placenta mesenchyma stem cell.With the frozen and relevant fetus information of record in liquid nitrogen of the cell after going down to posterity, and the biological characteristics and the multi-lineage potential that carry out cell are identified, and cell is carried out molecular genetics diagnose, preserve all related datas of cell, set up the database of placenta stem-cell and carry out related with freeze-stored cell.
According to the method for first aspect present invention, the method may further comprise the steps: under aseptic condition placental lobules is cut within four hours postpartum, fully washed placental lobules with the PBS damping fluid that contains 10% volume FBS and remove residual blood in the placental lobules.First placental lobules is cut into 1cm
3The tissue block of size joins in the PBS damping fluid that contains 0.1mg/mL dispase, 0.25mg/mL pancreatin, 0.25mg/mL DNase I, 1mg/mL collagenase IV, 1mg/mL Unidasa 37 ℃ of digestion 15 minutes, adds an amount of FBS and stops digestion.Again tissue block and cell suspension one are reinstated 200 order copper mesh and filtered and use simultaneously syringe piston tissue abrasion piece, collect cell suspension after filtering and joined in the centrifuge tube 1000rpm centrifugal 10 minutes, outwell supernatant, with the PBS damping fluid re-suspended cell that contains 10%FBS.Then use density gradient centrifugation separated and collected mononuclearcell, the cell that suspends and obtain with the MSC substratum behind the PBS damping fluid washing mononuclearcell, the density of inoculation mononuclearcell is every 75cm
2(Tissue Culture Flask internal surface area) 1 * 10
7Mononuclearcell is 10ml MSC substratum altogether.At last culturing bottle is put into 37 ℃ of 5%CO
2Cultivate in the incubator.Full dose is changed liquid and is removed non-adherent cell after 72 hours, has the shuttle type attached cell that is dispersed in the culturing bottle, adds fresh MSC nutrient solution and continues to cultivate.About 10 days, after being dispersed in attached cell and forming the clone, digest with 0.05% trypsinase/2mM EDTA, after the cell counting, according to 3000 cells/cm
2The cultivation of going down to posterity; Afterwards, had digestive transfer culture is cultivated when merging about cell reaches 70%, namely gets placenta mesenchyma stem cell.
In the described method of setting up placental mesenchyme stem cell library of first aspect present invention, forgiven the method for separating mesenchymal stem cell from placenta.Therefore, second aspect present invention provides the method for separating mesenchymal stem cell from placenta, and the method may further comprise the steps:
(a) get placental lobules, fully wash with the PBS damping fluid, to remove residual blood in the placenta;
(b) placental lobules is cut into bulk, adds the PBS damping fluid that contains the tissue digestion enzyme, hatch digestion at 37 ℃ again;
(c) tissue block is filtered with copper mesh, grind in case of necessity to impel filtration;
(d) filtered liquid of collecting is centrifugal, separate mononuclearcell, the cell that suspends and obtain with the MSC substratum again is then at 37 ℃, 5%CO
2Cultivate in the incubator;
(e) after disseminated cell formed the clone, each clone cell of picking was cultivated respectively with the MSC substratum, after cytogamy, goes down to posterity with trysinization, namely gets placenta mesenchyma stem cell;
And optional following one or more steps:
(f) for step (e) gained placenta mesenchyma stem cell, detect at least one item of following items: cytoactive, cell contamination, inherited disease, HLA-ABC/DR join type;
(g) placenta mesenchyma stem cell after step (e) gained is gone down to posterity is frozen in liquid nitrogen;
(h) set up the database of the placenta stem-cell that comprises above information, and make this database carry out related with the freeze-stored cell of step (g).
According to the method for second aspect present invention, wherein said step (a) is under aseptic condition placental lobules to be cut, and fully washes placental lobules with the PBS damping fluid and removes residual blood in the placenta.
Method according to second aspect present invention, wherein said step (a) is within four hours postpartum, under aseptic condition, placental lobules is cut, fully washed placental lobules with the PBS damping fluid that contains 10% volume foetal calf serum (FBS) and remove residual blood in the placental lobules.
According to the method for second aspect present invention, the tissue digestion enzyme in the wherein said step (b) is to be selected from following one or more: dispase, pancreatin, deoxyribonuclease I (DNase I), collagenase IV, Unidasa.In one embodiment, the tissue digestion enzyme in the described step (b) comprises: dispase, pancreatin, deoxyribonuclease I (DNase I), collagenase IV, Unidasa.In one embodiment, in step (b), comprise approximately 0.1mg/mLdispase, approximately 0.25mg/mL pancreatin, approximately 0.25mg/mL DNase I, approximately 1mg/mL collagenase IV and about 1mg/mL Unidasa in the described PBS damping fluid.
According to the method for second aspect present invention, in step (b), hatched 10～30 minutes at 37 ℃, preferred 10～20 minutes, for example 10 minutes, 15 minutes or 20 minutes.
According to the method for second aspect present invention, in step (b), placental lobules is cut into approximately 1cm
3The tissue block of size.
According to the method for second aspect present invention, in step (c), tissue block is ground with syringe simultaneously with the copper mesh filtration.In one embodiment, in step (c), tissue block and cell suspension one are reinstated 160～240 orders (preferred 200 orders) copper mesh filter and use simultaneously syringe piston tissue abrasion piece.
According to the method for second aspect present invention, in step (d), described MSC substratum is the PBS damping fluid that contains 10%FBS.In an embodiment of either side of the present invention, described MSC substratum is the PBS damping fluid that contains 10%FBS.In one embodiment, in step (d), the filtered liquid of collecting is separated mononuclearcell with density gradient centrifugation, washing, the cell that suspends and obtain with the MSC substratum again is then at 37 ℃, 5%CO
2Cultivate in the incubator.In one embodiment, in step (d), the cell suspension of collecting after filtering is joined in the centrifuge tube, centrifugal 10 minutes of 1000rpm outwells supernatant, with the PBS damping fluid re-suspended cell that contains 10%FBS.
According to the method for second aspect present invention, in step (e), after disseminated cell formed the clone, each clone cell of picking was cultivated respectively with the MSC substratum, after cell 60～90% merges, went down to posterity with trysinization, namely got placenta mesenchyma stem cell.In one embodiment, in step (e), after being dispersed in attached cell formation clone, with 0.05% trypsinase/2mM EDTA digestion, the cultivation of going down to posterity.
According to the method for second aspect present invention, in step (f), it is to utilize trypan blue staining to count the number of frozen front and back viable cell that described cytoactive detects.
According to the method for second aspect present invention, in step (f), described cell contamination detects and utilizes a small amount of cell cultures, detects the pollution whether cell is subject to fungus and bacterium.In one embodiment, it is to utilize the etiology method that described cell contamination detects, and detects cell and whether is subject to being selected from following one or more infection: Hepatitis B virus, the third liver, virus of AIDS, cytomegalovirus, Epstein-Barr virus and syphilis, HbsAg, HbsAb, HBcAb, HbeAg, HbeAb, HCVAb, HIV-1/2Ab, CMV-IgM and EBV-IgA, TRUST.
According to the method for second aspect present invention, in step (f), it is the method for utilizing molecular genetics that described inherited disease detects, and detects freeze-stored cell and whether has inherited disease.
According to the method for second aspect present invention, in step (f), it is to detect cell HLA-ABC/DR phenotype that described HLA-ABC/DR joins type.
According to the method for second aspect present invention, in step (g), described placenta mesenchyma stem cell is frozen in liquid nitrogen through the programmed cooling process.
According to the method for second aspect present invention, in step (g), described placenta mesenchyma stem cell is present in the cells frozen storing liquid.In one embodiment, this cells frozen storing liquid comprises 50% low sugar DMEM nutrient solution, 40%FBS, 10% dimethyl sulfoxide (DMSO).
Method according to second aspect present invention, in step (h), comprise in the described database and all relevant data of the cell of preserving, include but not limited to: the biological characteristics detected result of cell, multi-lineage potential qualification result, cellular elements genetic diagnosis result, fetus and father and mother's thereof detail file.
According to the method for second aspect present invention, the method may further comprise the steps: under aseptic condition placental lobules is cut, fully washed placental lobules with the PBS damping fluid and remove residual blood in the placenta.Again placental lobules is cut into 1cm
3The tissue block of size, add contain the Various Tissues digestive ferment the PBS damping fluid hatched 15 minutes at 37 ℃, tissue block is filtered with copper mesh grinds with syringe simultaneously.The liquid of collecting after filtering separates mononuclearcell with density gradient centrifugation, and the cell that obtains with the suspension of MSC substratum after the washing is put into 37 ℃ of 5%CO
2Cultivate in the incubator.After disseminated cell forms the clone, each clone cell chosen with the MSC substratum cultivate respectively, after cell 80～90% fusions, go down to posterity with trysinization, namely get placenta mesenchyma stem cell.With the frozen and relevant fetus information of record in liquid nitrogen of the cell after going down to posterity, and the biological characteristics and the multi-lineage potential that carry out cell are identified, and cell is carried out molecular genetics diagnose, preserve all related datas of cell, set up the database of placenta stem-cell and carry out related with freeze-stored cell.
According to the method for second aspect present invention, the method may further comprise the steps: under aseptic condition placental lobules is cut within four hours postpartum, fully washed placental lobules with the PBS damping fluid that contains 10% volume FBS and remove residual blood in the placental lobules.First placental lobules is cut into 1cm
3The tissue block of size joins in the PBS damping fluid that contains 0.1mg/mL dispase, 0.25mg/mL pancreatin, 0.25mg/mL DNase I, 1mg/mL collagenase IV, 1mg/mL Unidasa 37 ℃ of digestion 15 minutes, adds an amount of FBS and stops digestion.Again tissue block and cell suspension one are reinstated 200 order copper mesh and filtered and use simultaneously syringe piston tissue abrasion piece, collect cell suspension after filtering and joined in the centrifuge tube 1000rpm centrifugal 10 minutes, outwell supernatant, with the PBS damping fluid re-suspended cell that contains 10%FBS.Then use density gradient centrifugation separated and collected mononuclearcell, the cell that suspends and obtain with the MSC substratum behind the PBS damping fluid washing mononuclearcell, the density of inoculation mononuclearcell is every 75cm
2(Tissue Culture Flask internal surface area) 1 * 10
7Mononuclearcell is 10ml MSC substratum altogether.At last culturing bottle is put into 37 ℃ of 5%CO
2Cultivate in the incubator.Full dose is changed liquid and is removed non-adherent cell after 72 hours, has the shuttle type attached cell that is dispersed in the culturing bottle, adds fresh MSC nutrient solution and continues to cultivate.About 10 days, after being dispersed in attached cell and forming the clone, digest with 0.05% trypsinase/2mM EDTA, after the cell counting, according to 3000 cells/cm
2The cultivation of going down to posterity; Afterwards, had digestive transfer culture is cultivated when merging about cell reaches 70%, namely gets placenta mesenchyma stem cell.
In addition, in first aspect present invention method or second aspect method, provide a kind of placenta mesenchyma stem cell.Therefore third aspect present invention provides a kind of placenta mesenchyma stem cell.
According to the placenta mesenchyma stem cell of third aspect present invention, it obtains according to the described method of the arbitrary embodiment of first aspect present invention or according to the described method of the arbitrary embodiment of first aspect present invention.
According to the placenta mesenchyma stem cell of third aspect present invention, its cell purity is greater than 95%.In one embodiment, described placenta mesenchyma stem cell is after going down to posterity more than 3 generations, and cell purity is greater than 95%.
Below the present invention is further illustrated.The document that the present invention quotes, and the document of quoting in the document, their full content is incorporated this paper by reference into.
In the present invention, in arbitrary technical scheme of either side of the present invention, its arbitrary technical characterictic is equally applicable to arbitrary embodiment of either side of the present invention, as long as they can not cause contradiction, and this mutually being useful in case of necessity can be done suitable modification.
In the present invention, term " placenta mesenchyma stem cell " refers to derive from the mescenchymal stem cell of placenta.Therefore in the present invention, particularly relate in the linguistic context of the present invention, term " placenta mesenchyma stem cell " can with " placenta stem-cell ", " stem cell ", " mescenchymal stem cell " Alternate, indicate unless have clearly in addition.
In the present invention, term " PBS damping fluid " or " PBS " refer to phosphate buffered saline buffer.Those skilled in the art know the generality prescription of the PBS that uses and compound method and their general aspects for example pH value or pH scope under situation of the present invention.
In the present invention, term " placenta " refers to newborn infant's placenta, refers to especially the placenta within 4 hours postpartum.
In the present invention, term " MSC substratum " refers to the mescenchymal stem cell special culture media.
The present inventor once utilized perfusion method separation and Culture mescenchymal stem cell from placenta, had obtained the very high mescenchymal stem cell of purity.But be trapped in the placenta tissue through still having a large amount of stem cells behind the perfusion, can not effectively be separated.Therefore can think, adopt perfusion method can not obtain to greatest extent mescenchymal stem cell.
The invention discloses a kind of from placenta the methods of a large amount of separating mesenchymal stem cells, and utilize this method to preserve placenta mesenchyma stem cell and set up the placenta stem-cell storehouse.The present inventor utilizes Various Tissues digestive ferment mixture slaking placental lobules tissue block summing up on the basis of separation and Culture mescenchymal stem cell in the past, and in conjunction with stationary culture, success separates in placenta and obtaining a large amount of mescenchymal stem cells.The mescenchymal stem cell purity that the inventive method obtains is high, quantity is many, has the biological characteristics identical with mesenchymal stem cells MSCs, can be to differentiation such as scleroblast, chondrocyte, adipocyte, endotheliocyte, neurocyte.Because stem cell is inmature than adult stem cell in the placenta, rich content, be with a wide range of applications clinically, we use conventional cell freezing method that mescenchymal stem cell is frozen as bleeding of the umbilicus, set up the placenta stem-cell storehouse, for further investigation and the clinical treatment of stem cell lay the foundation later on.
Because contain abundant hemopoietic stem cell in the bleeding of the umbilicus, people set up unbilical blood bank these important Biological resources of umbilical hemopoietic stem cell are stored, for multiple disease in the blood system and disease of immune system provide a kind for the treatment of means.Same placenta mesenchyma stem cell is as a kind of more importantly stem cell resource, we use conventional cell freezing method it to be chilled in-196 degrees centigrade the medium-term and long-term preservation of profound hypothermia liquid nitrogen, set up the placenta stem-cell storehouse, for stem-cell therapy is in the future preserved seed.
The purpose of this invention is to provide simply a large amount of separating mesenchymal stem cells and set up the method in placenta stem-cell storehouse from placenta of a kind of practicality, comprise the steps: under aseptic condition, placental lobules to be cut, fully wash placental lobules with the PBS damping fluid and remove residual blood in the placenta.Again placental lobules is cut into 1cm
3The tissue block of size, add contain the Various Tissues digestive ferment the PBS damping fluid hatched 15 minutes at 37 ℃, tissue block is filtered with copper mesh grinds with syringe simultaneously.The liquid of collecting after filtering separates mononuclearcell with density gradient centrifugation, and the cell that obtains with the suspension of MSC substratum after the washing is put into 37 ℃ of 5%CO
2Cultivate in the incubator.After disseminated cell forms the clone, each clone cell chosen with the MSC substratum cultivate respectively, after cell 80～90% merges, go down to posterity with trysinization, with the frozen and relevant fetus information of record in liquid nitrogen of the cell after going down to posterity, and carry out biological characteristics and the multi-lineage potential evaluation of cell, and cell is carried out the molecular genetics diagnosis, preserve all related datas of cell, set up the database of placenta stem-cell and carry out related with freeze-stored cell.
The method according to this invention, for example according to the embodiment of the invention 1,2 method, the placentas that weight is 750 grams can get 8 * 10
9Mononuclearcell is through adherent culture average 1 * 10
6Can obtain 12 clones in the mononuclearcell, on average have 3 clones to have the characteristic of mescenchymal stem cell after the evaluation, namely can get 24000 mescenchymal stem cells in the 750g placenta.These stem cells can reach Cell Biology Experiment and clinical treatment desired number very soon through external Short-term Culture.Yet, inventor's discovery, according to the method for embodiment among the CN1548529A one, about separable 3000 mescenchymal stem cells that obtain of placentas that weight is 750 grams, after 3 generations went down to posterity, cell purity was greater than being about 95%.In one embodiment of the invention, in PBS damping fluid described in the step (b), comprise approximately 0.1mg/mL dispase, approximately 0.25mg/mL pancreatin, approximately 0.25mg/mL DNase I, approximately 1mg/mL collagenase IV and about 1mg/mL Unidasa; The inventor finds 5 kinds of enzymes in this PBS damping fluid, and when lacking any one or more, mescenchymal stem cell yield and cell purity all do not reach the result that can get 24000 mescenchymal stem cells in the above-mentioned 750g placenta far away; In addition, the concentration of 5 kinds of enzymes is done suitably to change, particularly be lower than above-mentioned separately concentration 50% or be higher than above-mentioned separately concentration 200% the time, mescenchymal stem cell yield and cell purity are all obviously poor than the result that can get 24000 mescenchymal stem cells in the above-mentioned 750g placenta; For example when the dispase concentration in the PBS damping fluid be 0.04mg/mL (be lower than the above-mentioned concentration of the present invention 50%) or during for 0.25mg/mL (be higher than the above-mentioned concentration of the present invention 200%), approximately can get 8000 mescenchymal stem cells in the 750g placenta and the cell minuent is lower than 90%.Therefore, one embodiment of the invention are in step (b), comprise 0.05～0.2mg/mL dispase, 0.125～0.5mg/mL pancreatin, 0.125～0.5mg/mL DNase I, 0.5～2mg/mL collagenase IV and 0.5～2mg/mL Unidasa in the described PBS damping fluid.
The present invention is simple to operate, and is convenient and practical, can obtain a large amount of mescenchymal stem cells, and the differentiation performance is good, has to the ability of the cytodifferentiation such as scleroblast, adipocyte, chondrocyte, endotheliocyte, neurocyte.With now methodical comparison: at present MSC mainly adopts modus operandi to extract donor marrow or perfusion method separates placenta, the adherent culture acquisition.It is few that this method is got cell quantity, and donor is being got the possibility that infection is all arranged after marrow is got in the marrow neutralization.The present invention's success separates the higher mescenchymal stem cell of a large amount of purity of acquisition in placenta, and uses this method to set up the placenta stem-cell storehouse and lay in this stem cell that has application prospect.This method is simple and easy to do, and because placenta is the same with bleeding of the umbilicus, Cell Component is inmature, and wide material sources conveniently are easy to get, and therefore method of the present invention will have widely prospect in the clinical application of stem cell.
Description of drawings
Fig. 1 is the morphological observation of microscopically cell growth.Wherein: A is for cultivating the attached cell that as seen was dispersed in afterwards in 3 days; B is 7～10 days formation clones; C is for to form fine and close attached cell through screening and cloning; D, E, F are the dyeing of Rui Shi Ji's nurse Sa.
Fig. 2 is flow cytometry identification of M SC surface marker result.Ordinate zou among each figure " counts " expression count number.
Fig. 3 is the analytical results of placenta MSC cell cycle.Wherein, P3 is for cultivating the dna content of third generation cell, and in the analysis of cells cycle, visible most cells is in (G stationary phase
0/ G
1Phase, 96.66%), few cell is in proliferation period (S phase, 3.25%).P6 is the dna content of cultivating the cell in the 6th generation, and in the analysis of cells cycle, visible most cells is in (G stationary phase
0/ G
1Phase, 96.35%), few cell is in proliferation period (S phase, 2.54%).Ordinate zou among each figure " counts " expression count number, X-coordinate mark " DNA Content " expression dna content.
Fig. 4 is placenta MSC cell growth curve figure.
Fig. 5 is the osteogenic induction cytological map that placenta MSC multi-lineage potential is identified.
Fig. 6 is the Adipogenic induction cytological map that placenta MSC multi-lineage potential is identified.
Fig. 7 is the one-tenth chondrocyte induction cytological map that placenta MSC multi-lineage potential is identified.
Fig. 8 is the electrophoresis photo that RT-PCR detects placenta MSC multi-lineage potential.
Embodiment
Can conduct further description the present invention by the following examples, yet scope of the present invention is not limited to following embodiment.One of skill in the art can understand, and under the prerequisite that does not deviate from the spirit and scope of the present invention, can carry out various variations and modification to the present invention.The present invention carries out generality and/or concrete description to the material and the test method that use in the test.Although for realizing that the employed many materials of the object of the invention and working method are well known in the art, the present invention still does detailed as far as possible description at this.
The separation of embodiment 1, placenta MSC
Within four hours postpartum, under aseptic condition, placental lobules is cut, fully wash placental lobules with the PBS damping fluid that contains 10% volume FBS and remove residual blood in the placental lobules.First placental lobules is cut into 1cm
3The tissue block of size joins in the PBS damping fluid that contains 0.1mg/mL dispase, 0.25mg/mL pancreatin, 0.25mg/mL DNase I, 1mg/mL collagenase IV, 1mg/mL Unidasa 37 ℃ of digestion 15 minutes, adds an amount of FBS and stops digestion.Again tissue block and cell suspension one are reinstated 200 order copper mesh and filtered and use simultaneously syringe piston tissue abrasion piece, collect cell suspension after filtering and joined in the centrifuge tube 1000rpm centrifugal 10 minutes, outwell supernatant, with the PBS damping fluid re-suspended cell that contains 10%FBS.Then use density gradient centrifugation separated and collected mononuclearcell, the cell that suspends and obtain with the MSC substratum behind the PBS damping fluid washing mononuclearcell, the density of inoculation mononuclearcell is every 75cm
2(Tissue Culture Flask internal surface area) 1 * 10
7Mononuclearcell is 10ml MSC substratum altogether.At last culturing bottle is put into 37 ℃ of 5%CO
2Cultivate in the incubator.Full dose is changed liquid after 72 hours, removes non-adherent cell, has the shuttle type attached cell that is dispersed in the culturing bottle, adds fresh MSC nutrient solution again and continues to cultivate.
The cultivation and frozen of going down to posterity of embodiment 2, placenta MSC
Approximately after 10 days, after being dispersed in attached cell and forming the clone, digest with 0.05% trypsinase/2mMEDTA, after the cell counting, according to 3000 cells/cm
2The cultivation of going down to posterity.Had digestive transfer culture is cultivated when merging about cell reaches 70% afterwards.Get 3 * 10 after the digestion
6Cell joins in the 1ml cells frozen storing liquid (containing 50% low sugar DMEM nutrient solution, 40%FBS, 10% dimethyl sulfoxide (DMSO)), enters at last liquid nitrogen pipe through programmed cooling frozen.
The Identification of Biological Characteristics of embodiment 3, placenta MSC
1, Growth of Cells and Morphological Characteristics thereof
Separation and Culture by embodiment 1 and embodiment 2, the placenta mononuclearcell was cultivated after 72 hours can obviously see fusiformis attached cell (Figure 1A) at microscopically, turbine-like cell clone (Figure 1B, Fig. 1 D) can be formed about 10 days, about the 80% adherent layers (Fig. 1 C, Fig. 1 E, Fig. 1 F) that merge can be formed after the had digestive transfer culture.In the culturing process, find the relative homogeneous of this cellular form, rate of propagation is fast, and adherent speed is fast, easily by trysinization, is passaged to more than 15 generations, and its form and growth characteristic are also without obviously changing.
2, flow cytometry identification of M SC surface marker
Get respectively the 3rd, 6,9,12,15 generation cell, the Flow cytometry cell surface marker is dynamically observed the variation of cell surface marker in the culturing process.The digestion collecting cell gets 8 * 10 behind the counting
6Individual cell, packing 16 pipes; PBS washes once, the centrifugal 10min of 1500rpm; Abandon supernatant, residual 100～200 μ l, piping and druming mixing cell; Add CD45, CD105, HLA-ABC, HLA-DR, each 10 μ l of UEA-1 antibody of CD14, CD29, CD31, CD34, CD44, CD54, CD73, CD80, CD86, CD166 antibody and the FITC mark of PE mark, and establish a pipe and be blank; Under 4 ℃, lucifuge reaction 30min; PBS washes once, the centrifugal 10min of 1500rpm; Directly the cell of mark is abandoned supernatant, adds 200 μ l PBS piping and druming mixing cell, and 1% Paraformaldehyde 96 of 200 μ l is fixed, put 4 ℃ to be measured, the upflowing cell instrument detects in 3 days.
Flow cytometer detects the surface marker of cell, dynamically observes the cell in the 3rd, 6,9,12,15 generations, without obviously changing.Not expressing the hematopoietic cell surface marker is CD14, CD31, CD34 (HSPC and endotheliocyte are positive), CD45 (white corpuscle is positive), CD54 (ICAM-1), CD80 (B7-1), CD86 (B7-2), HLA-DR (MHC-II quasi-molecule) continues negative, CD29 and CD44 (acceptor of scleroproein and transparency grease hydrochlorate, stroma cell is expressed), CD73 (is SH-3,4), CD105 (being SH-2), CD166 (mesenchymal cell expression), HLA-ABC (MHC-I quasi-molecule) and UEA-1 (surface marker of endotheliocyte) are continuously the positive.After going down to posterity more than 3 generations, the cellular constituent homogeneous, purity is more than 95%.(Fig. 2)
3, the cell cycle of Flow cytometry placenta MSC
Cell grows to about 80% when merging, digestion collecting cell approximately 1 * 10
6Individual, PBS washes once, and the ethanol of adding 70% is fixed, and 4 ℃ to be measured.During detection, the centrifugal ethanol that goes is washed once with PBS more first, adds RNase I 500u, 37 ℃ of reaction 30min, and PBS washes once, adds propidium iodide (PI, final concentration 50 μ g/ml) 1ml, room temperature lucifuge reaction 20min, upper machine testing cell DNA content.
After measured the 3rd generation and the 6th generation cell dna content, cell cycle analysis, G
0/ G
1Phase, S phase and G
2M phase proportion is respectively 96.35%, 96.66%, 1.11%, 0.09%, and 2.54%, 3.25%.The result shows that the cell of vitro culture has typical stem cells hyperplasia characteristics, namely only has a few cell to be in active proliferation period (1.11%, 0.09%), and most cell is in quiescent stage (96.35%, 96.66%).(Fig. 3)
4, the drafting of placenta MSC growth curve and the mensuration of logarithmic phase doubling time
The cell in vegetative period of taking the logarithm, the digestion counting is made cell suspension (every hole inoculation 0.5ml in 2 * 104/ml), 24 orifice plates, 37 ℃, 5%CO with the LG-DMEM substratum of 10%FBS
2, cultivate under the saturated humidity.Get 3 multiple holes every day, living cell counting number behind the Trypan Blue, calculating mean value, Continuous Observation 7 days.Take incubation time as transverse axis, cell count is the longitudinal axis, draws cell growth curve.Calculate cell in the doubling time of logarithmic phase with the Patterson formula, i.e. Td=Tlg2/Lg (Nt/No), Td: the doubling time (h), T: cell increases to Nt used time (h), N: cell count by No.
Cytometric result draws cell growth curve by every day, calculates the doubling time.Can be found out by cell growth curve, cell was in exponential phase of growth at 2-4 days.According to formula calculate the 5th generation cell be respectively 22.6h in the doubling time of exponential phase of growth.(Fig. 4)
5, the evaluation of placenta MSC multi-lineage potential
(1) osteogenic induction
Above MSC of 3 generations is by 1 * 10
5Six orifice plates are inoculated in/hole, are put in 37 ℃, 5%CO
2, under the saturated humidity, cultivate 24h in the MSC substratum after, use instead and contain 10% through the DMEM-HG of screening FBS and add dexamethasone 0.1 μ M, ascorbyl phosphate 50 μ M, β-phospho-glycerol 10mM, be put in 37 ℃, 5%CO
2, under saturated humidity, cultivate, amount was changed liquid in per 3 days half, coinduction 2-4 week.Alkaline phosphatase staining identifies that scleroblast forms, and Von Kossa dyeing identifies that the bone tubercle forms.
Containing 10% DMEM-HG through screening FBS, adding dexamethasone 0.1 μ M, ascorbyl phosphate 50 μ M, β-phospho-glycerol 10mM cultivated for 1 week, cellular form occurs significantly to change, become polygon by fusiform inoblast sample, be similar to the neuronal cell sample, it is outstanding that the long filament shape appears in the cell periphery, and can extend towards periphery.Continue to cultivate 2 weeks above after, calcified plaque appears in the cell matrix, mineralizer engenders, and begins to form the little junction structure of multilayer, after cultivating for 4 weeks, visible obviously calcification tubercle.Alkaline phosphatase staining is strong positive reaction during 2 week, reaches more than 95%, and the control group of not induced is then most of negative, only is shown as the weak positive less than 5%, shows that cell transforms to scleroblast.Von Kossa dyeing can be dyed black with the calcium that deposits in the bone tubercle, induces the visible a large amount of black bone tubercle of group, obvious three-dimensional arrangement is arranged, and control group does not all have positive reaction at any time.(Fig. 5)
(2) Adipogenic induction
Above MSC of 3 generations is by 1 * 10
5/ hole is inoculated in six orifice plates, is put in 37 ℃, 5%CO
2, under the saturated humidity, in the MSC substratum, cultivate 24h after, use instead and contain 10% DMEM in high glucose through screening FBS, and add dexamethasone 1 μ M, INDOMETHACIN 60 μ M, IBMX 0.5mM, Regular Insulin 5 μ g/ml, be put in 37 ℃, 5%CO
2, cultivate under the saturated humidity, amount was changed liquid in per 3 days half, and in 2 weeks of coinduction, oil red dyeing identifies that fat drips formation.
Containing 10% DMEM-HG through screening FBS, adding dexamethasone 1 μ M, INDOMETHACIN 200 μ M, IBMX 0.5mM, Regular Insulin 10 μ g/ml cultivated 3 days, form namely occurs and changes in cell, is shunk gradually by fusiform inoblast sample to shorten, and 90% above cell becomes cube or polygon; Cultured continuously 7 days has small fat to ooze existing in the visible cell under the mirror, along with the prolongation of incubation time, fat drips and increases gradually and merges, to cultivating 2 when all, as seen merges agglomerating fat and drips and be full of whole cell.The fat that produces in the oil red O stain visible cell is dyed redness by specificity.(Fig. 6)
(3) become chondrocyte induction
3 generations above cell, according to every pipe 2 * 10
5Cell divides and installs to the 15ml polypropylene centrifuge tube, low-speed centrifugal makes cell form micelle in test tube, in containing the DMEM-HG of 2.5%FBS, add Regular Insulin, Transferrins,iron complexes, each 6.25 μ g/ml of Sodium Selenite, BSA 1.25 μ g/ml, Sodium.alpha.-ketopropionate 1mM/L, xitix phosphoric acid 37.5 μ g/ml, TGF-β
150ng/ml is put in 37 ℃, 5%CO
2, cultivate under the saturated humidity, amount was changed liquid, 2 weeks of cultured continuously in per 3 days half.
After inducing for 2 weeks the cell micelle is broken up smear, the visible II Collagen Type VI of alcian blue (Alcian blue) dyeing forms extracellular matrix and is blue, and control group dyes without indigo plant.(Fig. 7)
6, RT-PCR detects placenta MSC multi-lineage potential
Cell after collection is induced is used Trizol reagent and is extracted cell total rna, take carry out RT-PCR for template, reverse transcription and PCR operate and carry out according to RT-PCR test kit specification sheets, primer sequence is as shown in table 1.
Table 1.RT-PCR primer sequence and specificity thereof
Among Fig. 8, be respectively from left to right: placenta MSC cell, induce and become adipocyte, induce as scleroblast, induce the RT-PCR electrophoresis photo into the chondrocyte, wherein from left to right, the Normal swimming lane is for inducing front cellular gene expression situation, and Adipogenic swimming lane, Osteogenic swimming lane, Chondrogenic swimming lane are for inducing rear cellular gene expression situation.The result shows, external evoked rear cell can be expressed serial specific mrna: cell expressing PPAR-γ behind the Adipogenic induction, cell expressing osteopontin (Osteopontin) behind the osteogenic induction, cell expressing collagen I I (Collagen II) behind the one-tenth chondrocyte induction, illustrate that resulting MSC cell has skeletonization, becomes fat, becomes the cartilage differentiation ability, meets generally acknowledged MSC standard.
By the detection of above a series of data targets, demonstrate and use the MSC that the inventive method separation obtains, have to the ability of scleroblast, adipocyte, Chondrocyte Differentiation, the MSC that the proved inventive method obtains has the stem cell characteristic.
The foundation in embodiment 4, placenta stem-cell storehouse
1, the detection of cytoactive
Utilize trypan blue staining to count the number of frozen front and back viable cell.
2, the detection of cell contamination
Utilize a small amount of cell cultures, detect the pollution whether cell is subject to fungus and bacterium.Utilize the etiology method, detect whether cell is subject to Hepatitis B virus, the third liver, AIDS, cytomegalovirus, Epstein-Barr virus and syphilis, HbsAg, HbsAb, HBcAb, HbeAg, HbeAb, HCVAb, HIV-1/2Ab, CMV-IgM and EBV-IgA, TRUST infects.
3, the detection of inherited disease
Utilize the method for molecular genetics, detect freeze-stored cell and whether have inherited disease.
4, HLA-ABC/DR joins type
Detect cell HLA-ABC/DR phenotype, and place on record.
5, the investigation of cell derived
Record fetus and father and mother's thereof detail file, and place on record.
6, the foundation of placenta stem-cell database
After preserving normal placenta stem-cell, set up the database of placenta stem-cell, comprising the first six data, and foundation and freeze-stored cell is related.
Claims (5)
1. set up the method for placental mesenchyme stem cell library, the method may further comprise the steps:
(a) fully wash placental lobules with the PBS damping fluid, to remove residual blood in the placenta;
(b) placental lobules is cut into bulk, adds the PBS damping fluid that contains the tissue digestion enzyme, hatch digestion at 37 ℃ again;
(c) tissue block is filtered with copper mesh, grind in case of necessity to impel filtration;
(d) filtered liquid of collecting is centrifugal, separate mononuclearcell, the cell that suspends and obtain with the MSC substratum again is then at 37 ℃, 5%CO
2Cultivate in the incubator;
(e) after disseminated cell formed the clone, each clone cell of picking was cultivated respectively with the MSC substratum, after cytogamy, goes down to posterity with trysinization, namely gets placenta mesenchyma stem cell;
(f) for step (e) gained placenta mesenchyma stem cell, detect at least one item of following items: cytoactive HLA-ABC/DR joins type;
(g) placenta mesenchyma stem cell after step (e) gained is gone down to posterity is frozen in liquid nitrogen;
(h) set up the database of the placenta mesenchyma stem cell that comprises above information, and make this database carry out related with the freeze-stored cell of step (g); And
In step (b), comprise 0.1mg/mL dispase, 0.25mg/mL pancreatin, 0.25mg/mL DNase I, 1mg/mL collagenase IV and 1mg/mL Unidasa in the described PBS damping fluid.
2. according to claim 1 method, wherein said step (a) is within four hours postpartum, under aseptic condition, placental lobules is cut, fully washed placental lobules with the PBS damping fluid that contains 10% volume foetal calf serum and remove residual blood in the placental lobules.
3. according to claim 1 method in step (b), was hatched 10～30 minutes at 37 ℃.
4. according to claim 1 method, in step (e), after disseminated cell formed the clone, each clone cell of picking was cultivated respectively with the MSC substratum, after cell 60～90% fusions, went down to posterity with trysinization, namely got placenta mesenchyma stem cell.
5. according to claim 1 method, in step (h), comprise in the described database and all relevant data of the cell of preserving, comprising: the biological characteristics detected result of cell, multi-lineage potential qualification result, fetus and father and mother's thereof detail file.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201210044638 CN102586184B (en) | 2011-09-05 | 2012-02-27 | Method for establishing placental mesenchyme stem cell library |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110258696.4 | 2011-09-05 | ||
| CN201110258696 | 2011-09-05 | ||
| CN 201210044638 CN102586184B (en) | 2011-09-05 | 2012-02-27 | Method for establishing placental mesenchyme stem cell library |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102586184A CN102586184A (en) | 2012-07-18 |
| CN102586184B true CN102586184B (en) | 2013-10-23 |
Family
ID=46475420
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201210044638 Active CN102586184B (en) | 2011-09-05 | 2012-02-27 | Method for establishing placental mesenchyme stem cell library |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102586184B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102676451A (en) * | 2011-09-05 | 2012-09-19 | 博雅干细胞科技有限公司 | Method for separating mesenchymal stem cells from placenta |
Families Citing this family (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103320382A (en) * | 2013-02-08 | 2013-09-25 | 周胜利 | Method for extracting and purifying multi-source stem cells from placenta and umbilical cord |
| CN103173405B (en) * | 2013-03-21 | 2017-11-07 | 北京京蒙高科干细胞技术有限公司 | A kind of simple separation of Human plactnta myofibroblast mother cell and authentication method |
| CN104818264A (en) * | 2015-05-29 | 2015-08-05 | 广州赛莱拉干细胞科技股份有限公司 | Digestive enzyme composition, and preparation and application thereof |
| CN105238748A (en) * | 2015-10-27 | 2016-01-13 | 上海百众源生物科技有限公司 | Preparation method of placenta-source decidua parietalis mesenchymal stem cells by separation and refrigeration |
| CN106702499A (en) * | 2017-01-13 | 2017-05-24 | 天津百恩生物科技有限公司 | Canine mesenchymal stem cell bank and construction method thereof |
| CN106834257B (en) * | 2017-03-23 | 2020-06-30 | 天晴干细胞股份有限公司 | Mixed enzyme for separating placenta mesenchymal stem cells and separation method thereof |
| CN107299082B (en) * | 2017-08-02 | 2020-09-15 | 广州中科博雅干细胞科技有限公司 | Method for separating placenta mesenchymal cells from tissues and culturing into mesenchymal stem cells |
| CN115777691A (en) * | 2018-04-19 | 2023-03-14 | 重庆斯德姆生物技术有限公司 | Method for cryopreserving and recovering placenta tissues and preparing mesenchymal stem cells |
| CN108795853B (en) * | 2018-05-28 | 2021-08-24 | 天津博雅秀岩生物技术有限公司 | Method for preparing canine fetal membrane mesenchymal stem cells and canine fetal membrane mesenchymal stem cells |
| CN108728408B (en) * | 2018-05-28 | 2021-08-24 | 天津博雅秀岩生物技术有限公司 | Canine fetal membrane mesenchymal stem cell, preparation method and culture medium used by same |
| CN109554345A (en) * | 2018-11-21 | 2019-04-02 | 新格元(南京)生物科技有限公司 | A kind of digestive juice and its method that Tissues of Human Adenocarcinoma of Pancreas is separated into single living cell |
| CN110840914B (en) * | 2019-11-23 | 2021-07-06 | 博雅干细胞科技有限公司 | Method for alleviating or improving vascular disorders using cell therapeutic agent |
| CN110974847B (en) * | 2019-12-25 | 2021-07-06 | 博雅干细胞科技有限公司 | Method for treating lower limb ischemic diseases by using stem cells |
| CN113403260A (en) * | 2020-12-09 | 2021-09-17 | 海南优尼科尔生物科技有限公司 | Preparation and culture method of placenta stem cells |
| CN113100225A (en) * | 2021-02-24 | 2021-07-13 | 河南省银丰生物工程技术有限公司 | Cell preparation protective solution for treatment and preparation method thereof |
| CN113100224A (en) * | 2021-02-24 | 2021-07-13 | 河南省银丰生物工程技术有限公司 | Mesenchymal stem cell cryopreservation solution and activity research method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1786154A (en) * | 2005-10-18 | 2006-06-14 | 天津昂赛细胞基因工程有限公司 | Human placenta, umbilical cord mesenchyma stemcell stock and its construction method |
| CN102676451A (en) * | 2011-09-05 | 2012-09-19 | 博雅干细胞科技有限公司 | Method for separating mesenchymal stem cells from placenta |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2009288781B2 (en) * | 2008-09-02 | 2015-01-29 | Pluri Biotech Ltd | Adherent cells from placenta tissue and use thereof in therapy |
| US9175262B2 (en) * | 2009-11-30 | 2015-11-03 | Pluristerm Ltd. | Adherent cells from placenta and use of same in disease treatment |
-
2012
- 2012-02-27 CN CN 201210044638 patent/CN102586184B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1786154A (en) * | 2005-10-18 | 2006-06-14 | 天津昂赛细胞基因工程有限公司 | Human placenta, umbilical cord mesenchyma stemcell stock and its construction method |
| CN102676451A (en) * | 2011-09-05 | 2012-09-19 | 博雅干细胞科技有限公司 | Method for separating mesenchymal stem cells from placenta |
Non-Patent Citations (3)
| Title |
|---|
| 人胎盘源贴壁细胞支持脐血CD34+细胞体外扩增;张毅 等;《中国实验血液学杂志》;20031220;第6卷(第11期);正文"材料与方法" * |
| 张毅 等.人胎盘源贴壁细胞支持脐血CD34+细胞体外扩增.《中国实验血液学杂志》.2003,第6卷(第11期),第560-564页. |
| 霍思维.人胎盘来源间充质干细胞支持脐血造血干细胞体外扩增.《中国优秀博硕士学位论文全文数据库（硕士） 基础科学辑》.2006,(第12期),A006-189. * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102676451A (en) * | 2011-09-05 | 2012-09-19 | 博雅干细胞科技有限公司 | Method for separating mesenchymal stem cells from placenta |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102586184A (en) | 2012-07-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102586184B (en) | Method for establishing placental mesenchyme stem cell library | |
| CN102676451B (en) | Method for separating mesenchymal stem cells from placenta | |
| CN102660497B (en) | Method for freezing and reviving umbilical cord tissues and for separating and increasing stem cells | |
| CN107236704B (en) | From the method for placenta separating mesenchymal stem cell and the digestive enzyme compositions used | |
| CN102807966B (en) | Method for freezing and thawing placental whole cells and separating and expanding stem cells | |
| CN102660502B (en) | Methods for freezing and thawing whole cell of umbilical cord and separating and augmenting stem cell | |
| CN102002475B (en) | Method for obtaining fat adult stem cells of human and method for establishing stem cell library | |
| CN107299082B (en) | Method for separating placenta mesenchymal cells from tissues and culturing into mesenchymal stem cells | |
| CN102660501A (en) | Method for separating and amplifying mesenchymal stem cell from fresh tissue of umbilical cord | |
| CN102106342B (en) | Method for storing mesenchymal stem cells and method for culturing mesenchymal stem cells | |
| CN102639694B (en) | Methods of preparing mesenchymal stem cells, compositions and kit thereof | |
| CN102550542B (en) | Establishment for serum-free freezing medium and adipose-derived stem cell library of adipose-derived stem cells | |
| CN103805562B (en) | Cultivate the serum free medium of placenta mesenchyma stem cell | |
| CN109234229B (en) | Method for separating mesenchymal stem cells from placental blood vessels and digestive enzyme composition used in same | |
| CN101914490B (en) | Human amnion mesenchymal stem cell serum-free culture medium and culture method thereof | |
| CN102660503A (en) | Method for separating and amplifying mesenchymal stem cells from umbilical cord | |
| CN107022521A (en) | Decidua vera tissue freezing, the method recovered and be separately cultured mescenchymal stem cell | |
| CN1548529A (en) | Separation method of buffering stem cell in human placenta | |
| CN102965335A (en) | Kit for mesenchymal stem cell culture and application thereof | |
| CN104560871A (en) | Culturing method of mesenchymal stem cells of menstrual blood | |
| CN108486050B (en) | Method for preparing mesenchymal stem cells from umbilical cord of dog | |
| CN108456657A (en) | Dog umbilical cord mesenchymal stem cells and preparation method thereof and cryopreservation methods | |
| CN104694464A (en) | Clinical dental pulp stem cell and preparation method thereof | |
| CN109628388B (en) | Isolation of mesenchymal stem cells from placental blood vessels with digestive enzyme composition | |
| CN110317781B (en) | Method for freezing and resuscitating mesenchymal stem cells |
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 | ||
| CB03 | Change of inventor or designer information | ||
| CB03 | Change of inventor or designer information |
Inventor after: Huo Siwei Inventor after: Chen Junfeng Inventor after: Xu Xiaochun Inventor after: Li Yishu Inventor before: Huo Siwei Inventor before: Chen Junfeng Inventor before: Zhang Yi Inventor before: Xu Xiaochun Inventor before: Li Yishu |