CN107236704B

CN107236704B - From the method for placenta separating mesenchymal stem cell and the digestive enzyme compositions used

Info

Publication number: CN107236704B
Application number: CN201710454588.1A
Authority: CN
Inventors: 许晓椿; 陆晗燕; 朱业峰
Original assignee: BOYA STEM CELL TECHNOLOGY Co Ltd
Current assignee: BOYA STEM CELL TECHNOLOGY Co Ltd
Priority date: 2017-06-15
Filing date: 2017-06-15
Publication date: 2019-09-20
Anticipated expiration: 2037-06-15
Also published as: CN107236704A

Abstract

The present invention relates to the methods from placenta separating mesenchymal stem cell and the digestive enzyme compositions used.Specifically, the method includes (a) to take placental lobules, is sufficiently rinsed with PBS buffer solution, to remove remaining blood in placenta；(b) placental lobules is cut into bulk, the PBS buffer solution containing tissue digestion enzyme is added, then digest in 37 DEG C of incubations；(c) tissue block is filtered with copper mesh, is ground when necessary to promote to filter；(d) filtered fluid of collection is centrifuged, separates mononuclearcell, then with MSC culture medium suspension cell obtained, then in 37 DEG C, 5%CO₂It is cultivated in incubator；(e) after disseminated cell forms clone, each clone cell of picking is cultivated respectively with MSC culture medium, after cell fusion, with pancreatin had digestive transfer culture to get placenta mesenchyma stem cell.The placenta mesenchyma stem cell of high-purity can be obtained by the method for the invention, and low in cost, cell high income.

Description

From the method for placenta separating mesenchymal stem cell and the digestive enzyme compositions used

Technical field

The present invention relates to the method for separating stem cell from placenta, in particular to the separating mesenchymal stem cell from placenta Method particularly relates to a kind of digestive enzyme compositions using unique formula of the present invention to fill from placenta compartment The method of matter stem cell.The efficiency from placenta separating mesenchymal stem cell can be effectively improved using the method for the present invention.

Background technique

The mescenchymal stem cell of mescenchymal stem cell (mesenchymal stem cell, MSC) such as mankind be earliest from It is separated in marrow, it is dry thin from mesoblastic a kind of tissue with multi-lineage potential and self-renewal capacity Born of the same parents, in vivo under external specified conditions have to osteoblast, cartilage cell, fat cell, endothelial cell, nerve cell, Ability (the Caplan AI.Mesenchymal stem cells.J of a variety of adult cell differentiation such as myocyte, liver cell 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).Most It is new research shows that mescenchymal stem cell has immunological regulation and Hematopoiesis Support affect, and be easy to foreign gene and import expression. Therefore the mescenchymal stem cell not still seed cell in tissue-engineered bone, cartilage and cardiac muscle building, it is important in gene therapy Carrier cell, and since mescenchymal stem cell promotes hematopoietic reconstitution and the anti-host response function of inhibition of transplant, in hematopoiesis It is with a wide range of applications in stem cell transplantation and organ transplant.Mescenchymal stem cell has the characteristic of growth-arrested in vitro, Using this characteristic, people have succeeded to be divided from the Various Tissues such as liver, kidney, pancreas, muscle, cartilage, skin, peripheral blood From turning out mescenchymal stem cell.

The mescenchymal stem cell reported at present is mainly derived from marrow, is obtained using density-gradient centrifugation method.Although point It is easy from method, but the operation that donor takes marrow to need to undergo a comparison painful, and had very during materials and after materials High infection chance；Since the content of MSC in human bone marrow is extremely rare, every 10⁵~10⁶Only about 1 in a mononuclearcell It is a, and with the increase at age, quantity, proliferation and the differentiation capability of mescenchymal stem cell are remarkably decreased in marrow, make it It is restricted in research and application especially clinical application.Placenta originating from embryonic development period extraembryonic mesoderm be by Matter, blood vessel and trophocyte composition, contain a large amount of mesenchyma ingredient.It is newest research shows that rich in dry thin in placenta Born of the same parents, be separately cultured out from placenta these multipotential stem cells will be opened up for experimental study and clinical application one it is brand-new and abundant Source.

Existing method of the stem cell to establish placenta stem-cell library that separate from placenta still has shortcomings, such as pure Degree is insufficient, and/or quantity is not high, and then shows that these methods are not able to satisfy the expectation of people still.Such as CN101270349A Entitled " placenta mesenchyma stem cell disclosed in (Chinese Patent Application No. 200810061267.6, publication date September in 2008 24 days) The invention of separation and amplification in vitro cultural method "；CN101693884A (Chinese Patent Application No. 200910117522.9, it is open Day on April 14th, 2010) disclosed in entitled " a method of the separating and extracting stem cells from placenta, umbilical cord or adipose tissue " Invention；It is entitled disclosed in CN102146359A (Chinese Patent Application No. 201110005964.1, publication date August in 2011 10 days) The invention of " method of primary mesenchymal stem cells and serum-free amplification is extracted from placenta ".In addition, Chinese Patent Application No. 201210044648X discloses a kind of method of separating mesenchymal stem cell from placenta.Purity of these methods in extract And/or it is remained to be further improved in terms of the rate of recovery.

This field remains a need for the new method that stem cell is separated from placenta, especially efficiently separates from placenta The method of mescenchymal stem cell.In addition, this field still need it is new for the separating mesenchymal stem cell methods from placenta Used in digestive enzyme compositions, to improve the method efficiency of the separating mesenchymal stem cell from placenta.

Summary of the invention

Present invention aim to address the existing deficiencies for obtaining placenta mesenchyma stem cell method, provide a kind of practical, simple List, efficiently a large amount of separating mesenchymal stem cells and the method for optionally establishing placenta stem-cell library from placenta.Meanwhile this hair Bright another object is that the method for a large amount of separating mesenchymal stem cells from placenta to be above-mentioned provides a kind of digestive enzyme compositions. The inventors discovered that cell purity obtained is high using special operating method and the digestive enzyme compositions of special prescription And/or cell recoveries are high.It is accomplished the present invention is based on this discovery.

Therefore, first aspect present invention provides the method for the separating mesenchymal stem cell from placenta, this method include with Lower step:

(a) placental lobules is taken, is sufficiently rinsed with PBS buffer solution, to remove remaining blood in placenta；

(b) placental lobules is cut into bulk, the PBS buffer solution containing tissue digestion enzyme is added, then digest in 37 DEG C of incubations；

(c) tissue block is filtered with copper mesh, is ground when necessary to promote to filter；

(d) filtered fluid of collection is centrifuged, separates mononuclearcell, then with MSC culture medium suspension cell obtained, so Afterwards in 37 DEG C, 5%CO₂It is cultivated in incubator；

(e) after disseminated cell forms clone, each clone cell of picking is cultivated with MSC culture medium, respectively to cell fusion Afterwards, with pancreatin had digestive transfer culture to get placenta mesenchyma stem cell；

And optional following one or more steps:

(f) for placenta mesenchyma stem cell obtained by step (e), detect following items at least one of: it is cell activity, thin Born of the same parents' pollution, hereditary disease, HLA-ABC/DR distribution type；

(g) placenta mesenchyma stem cell after passage obtained by step (e) is frozen in liquid nitrogen；

(h) database of the placenta stem-cell comprising information above is established, and keeps the database and freezing for step (g) thin Born of the same parents are associated.

According to method of the first aspect of the present invention, wherein the step (a) is aseptically to cut placental lobules, Remaining blood in placental lobules removal placenta is sufficiently rinsed with PBS buffer solution.

According to method of the first aspect of the present invention, wherein the step (a) is within four hours postpartum, in aseptic condition It is lower to cut placental lobules, placental lobules removal placenta is sufficiently rinsed with the PBS buffer solution containing 10% volume fetal calf serum (FBS) Remaining blood in leaflet.

According to method of the first aspect of the present invention, wherein the tissue digestion enzyme in the step (b) is selected from following one Kind is a variety of: dispase, pancreatin, deoxyribonuclease I (DNase I), clostridiopetidase A IV, hyaluronidase.Implement at one In scheme, the tissue digestion enzyme in the step (b) includes: dispase, pancreatin, deoxyribonuclease I (DNase I), glue Protoenzyme IV, hyaluronidase.It in one embodiment, include about 0.1mg/mL in the PBS buffer solution in step (b) Dispase (it is also known as neutral proteinase or dispase), about 0.25mg/mL pancreatin, about 0.25mg/mL DNase I (its also Referred to as deoxyribonuclease I), about 1mg/mL clostridiopetidase A IV and about 1mg/mL hyaluronidase.

According to method of the first aspect of the present invention, the PBS buffer solution wherein containing tissue digestion enzyme described in step (b) is Include in PBS buffer solution: 0.02mg/mL dispase, 0.05mg/mL pancreatin, 0.02mg/mL DNase I, 0.05mg/mL Clostridiopetidase A IV and 0.2mg/mL hyaluronidase.

According to method of the first aspect of the present invention, the PBS buffer solution wherein containing tissue digestion enzyme described in step (b) is Include in PBS buffer solution: 0.02mg/mL dispase, 0.05mg/mL pancreatin, 0.02mg/mL DNase I, 0.05mg/mL Clostridiopetidase A IV and 0.2mg/mL hyaluronidase and 0.2~0.5mg/mL sodium glutamate and 2~5mg/mL mannose, such as Contain about 0.2mg/mL, about 0.3mg/mL, the sodium glutamate of about 0.4mg/mL or about 0.5mg/mL and about in the buffer The mannose of 2mg/mL, about 3mg/mL, about 4mg/mL or about 5mg/mL.It certainly, should the PBS buffer solution containing tissue digestion enzyme Used is the PBS buffer solution with liquid medium, that is, is also buffered comprising PBS in the PBS buffer solution containing tissue digestion enzyme Liquid；Although not referring in the above-mentioned PBS buffer solution composition containing tissue digestion enzyme, this field does not have to generally also in such situation It refers to.It has been had now surprisingly been found that, add a small amount of sodium glutamate and sweet in the PBS buffer solution of Xiang Hanyou tissue digestion enzyme simultaneously Dew alcohol and the concentration for greatly reducing other five kinds of tissue digestion enzymes, still are able to obtain excellent effect, this will be had extremely Significance, reason is that the price of five kinds of digestive ferments is very expensive and sodium glutamate and mannitol are extremely cheap, can be with It substantially reduces methodology cost and satisfied technical effect can be obtained, for example, the above-mentioned group containing sodium glutamate and mannose Object is closed, cost includes about 0.1mg/mL dispase, 0.25mg/mL pancreatin, 0.25mg/mL DNase I, 1mg/mL The 5% of clostridiopetidase A IV and 1mg/mL hyaluronidase and the composition cost without sodium glutamate and mannose, and can make Methodology totle drilling cost reduces about 90%.

According to method of the first aspect of the present invention, in step (b), 37 DEG C be incubated for 10~30 minutes, preferably 10~20 Minute, such as 10 minutes, 15 minutes or 20 minutes.

According to method of the first aspect of the present invention, in step (b), placental lobules is cut into about 1cm³The tissue of size Block.

According to method of the first aspect of the present invention, in step (c), tissue block is filtered with copper mesh and is ground simultaneously with syringe Mill.In one embodiment, in step (c), tissue block and cell suspension one are reinstated into 160~240 mesh (preferably 200 mesh) Copper mesh filtering while with syringe piston tissue abrasion block.

According to method of the first aspect of the present invention, in step (d), by the filtered fluid of collection density-gradient centrifugation method point From mononuclearcell, washing, then with MSC culture medium suspension cell obtained, then in 37 DEG C, 5%CO₂It is trained in incubator It supports.In one embodiment, in step (d), filtered cell suspension will be collected and be added in centrifuge tube, 1000rpm Centrifugation 10 minutes, outwells supernatant, and cell is resuspended with the PBS buffer solution containing 10%FBS.

According to method of the first aspect of the present invention, in step (e), after disseminated cell forms clone, picking is respectively cloned Cell is cultivated respectively with MSC culture medium, dry to get placenta mesenchyma with pancreatin had digestive transfer culture after the fusion of cell 60~90% Cell.In one embodiment, in step (e), when be dispersed in attached cell formed clone after, with 0.05% trypsase/ 2mM EDTA digestion, secondary culture.

According to method of the first aspect of the present invention, in step (f), the cytoactive detection is to utilize Trypan Blue Method counts the number for freezing front and back living cells.

According to method of the first aspect of the present invention, in step (f), the cell contamination detection is trained using a small amount of cell Support, detection cell whether the pollution by fungi and bacterium.In one embodiment, the cell contamination detection is to utilize disease Former method, detection cell whether by be selected from following one or more infection: Hepatitis B virus, hepatitis, AIDS virus, Cytomegalovirus, Epstein-Barr virus and syphilis, HbsAg, HbsAb, HBcAb, HbeAg, HbeAb, HCVAb, HIV-1/2Ab, CMV-IgM With EBV-IgA, TRUST.

According to method of the first aspect of the present invention, in step (f), the hereditary disease detection is to utilize molecular genetics Method, detection freeze-stored cell whether there is hereditary disease.

According to method of the first aspect of the present invention, in step (f), the HLA-ABC/DR distribution type is detection cell HLA- ABC/DR phenotype.

According to method of the first aspect of the present invention, in step (g), the placenta mesenchyma stem cell is cooled down through program Process freezes in liquid nitrogen.

According to method of the first aspect of the present invention, in step (g), the placenta mesenchyma stem cell is present in cell jelly In liquid storage.In one embodiment, which includes 50% low sugar DMEM culture solution, 40%FBS, 10% dimethyl Sulfoxide.

According to method of the first aspect of the present invention, the cell institute in step (h), in the database including and being saved There are relevant data, including but not limited to: the biological characteristics testing result of cell, multi-lineage potential qualification result, cell Molecular genetics diagnostic result, fetus and its particulars of parent.

According to method of the first aspect of the present invention, method includes the following steps: aseptically cutting placental lobules Under, remaining blood in placental lobules removal placenta is sufficiently rinsed with PBS buffer solution.Placental lobules is cut into 1cm again³Size Tissue block is added the PBS buffer solution containing Various Tissues digestive ferment and is incubated for 15 minutes at 37 DEG C, and tissue block copper mesh is filtered together When ground with syringe.Filtered liquid density-gradient centrifugation method separation mononuclearcell is collected, is cultivated after washing with MSC Base suspension cell obtained is put into 37 DEG C of 5%CO₂It is cultivated in incubator.It is after disseminated cell forms clone, each clone is thin Born of the same parents choose to be cultivated respectively with MSC culture medium, dry to get placenta mesenchyma with pancreatin had digestive transfer culture after cell 80~90% merges Cell.Cell after passage is frozen in liquid nitrogen and records related fetus information, and carries out the biological characteristics of cell and more It is identified to differentiation potential, and molecular genetics diagnosis is carried out to cell, save all related datas of cell, it is dry to establish placenta The database of cell is simultaneously associated with freeze-stored cell.

According to method of the first aspect of the present invention, method includes the following steps: postpartum four hours inherence aseptic conditions It is lower to cut placental lobules, it is sufficiently rinsed in placental lobules removal placental lobules and is remained with the PBS buffer solution containing 10% volume FBS Blood.Placental lobules is first cut into 1cm³The tissue block of size is added to containing 0.1mg/mL dispase, 0.25mg/mL Pancreatin, 0.25mg/mL DNase I, 1mg/mL clostridiopetidase A IV, 1mg/mL hyaluronidase PBS buffer solution in 37 DEG C digestion 15 Minute, appropriate FBS is added and terminates digestion.Tissue block and cell suspension one are reinstated into the filtering of 200 mesh copper mesh again while using syringe Piston tissue abrasion block collects filtered cell suspension and is added to 1000rpm centrifugation 10 minutes in centrifuge tube, outwells supernatant, Cell is resuspended with the PBS buffer solution containing 10%FBS.Then mononuclearcell, PBS buffering are separated and collected with density-gradient centrifugation method Liquid uses MSC culture medium suspension cell obtained after washing mononuclearcell, and the density for being inoculated with mononuclearcell is every 75cm² (Tissue Culture Flask internal surface area) 1 × 10⁷The total 10ml MSC culture medium of mononuclearcell.Culture bottle is finally put into 37 DEG C 5% CO₂It is cultivated in incubator.Full dose changes liquid removal non-adherent cell after 72 hours, and it is adherent thin to have the shuttle-type being dispersed in culture bottle Born of the same parents are added fresh MSC culture solution and continue to cultivate.10 days or so, after being dispersed in attached cell formation clone, with 0.05% tryptose Enzyme/2mM EDTA digestion, after cell count, according to 3000 cells/cm²Carry out secondary culture；Later, when cell reaches 70% left side Had digestive transfer culture culture is when right fusion to get placenta mesenchyma stem cell.

According to method of the first aspect of the present invention, method includes the following steps: postpartum four hours inherence aseptic conditions It is lower to cut placental lobules, it is sufficiently rinsed in placental lobules removal placental lobules and is remained with the PBS buffer solution containing 10% volume FBS Blood.Placental lobules is first cut into 1cm³The tissue block of size is added to containing 0.1mg/mL dispase, 0.25mg/mL Pancreatin, 0.25mg/mL DNase I, 1mg/mL clostridiopetidase A IV, 1mg/mL hyaluronidase, 0.2~0.5mg/mL sodium glutamate It is digested 15 minutes with 37 DEG C in the PBS buffer solution of 2~5mg/mL mannose, appropriate FBS is added and terminates digestion.Again by tissue block and Cell suspension one reinstates the filtering of 200 mesh copper mesh while with syringe piston tissue abrasion block, collects filtered cell suspension and adds Enter into centrifuge tube 1000rpm to be centrifuged 10 minutes, outwell supernatant, cell is resuspended with the PBS buffer solution containing 10%FBS.Then it uses Density-gradient centrifugation method separates and collects mononuclearcell, is obtained after PBS buffer solution washing mononuclearcell with the suspension of MSC culture medium The cell obtained, the density for being inoculated with mononuclearcell is every 75cm²(Tissue Culture Flask internal surface area) 1 × 10⁷Mononuclearcell is total 10ml MSC culture medium.Culture bottle is finally put into 37 DEG C of 5%CO₂It is cultivated in incubator.After 72 hours full dose change liquid go unless Attached cell has the shuttle-type attached cell being dispersed in culture bottle, fresh MSC culture solution is added and continues to cultivate.It 10 days or so, dissipates After attached cell forms clone, digested with 0.05% trypsase/2mM EDTA, after cell count, according to 3000 cells/ cm²Carry out secondary culture；Later, when cell reaches 70% or so fusion, had digestive transfer culture culture is dry thin to get placenta mesenchyma Born of the same parents.

In addition, providing a kind of placenta mesenchyma stem cell in first aspect present invention method.Therefore the present invention second Aspect provides a kind of placenta mesenchyma stem cell.

Placenta mesenchyma stem cell according to a second aspect of the present invention is any embodiment party according to a first aspect of the present invention What case the method obtained.

Placenta mesenchyma stem cell according to a second aspect of the present invention, cell purity are greater than 95%.In an embodiment party In case, for the placenta mesenchyma stem cell after 3 more than generation pass on, cell purity is greater than 95%.

Further, third aspect present invention provides used in a kind of method from placenta separating mesenchymal stem cell Digestive enzyme compositions, the digestive enzyme compositions are the PBS buffer solution containing tissue digestion enzyme, should the PBS containing tissue digestion enzyme Buffer is in PBS buffer solution comprising selected from following one or more digestive ferments: dispase, pancreatin, DNA Enzyme I (DNase I), clostridiopetidase A IV, hyaluronidase.In one embodiment, comprising following in the digestive enzyme compositions Digestive ferment: dispase, pancreatin, deoxyribonuclease I (DNase I), clostridiopetidase A IV, hyaluronidase.

Digestive enzyme compositions according to a third aspect of the present invention, the digestive enzyme compositions are the PBS containing the digestive ferment Buffer.In one embodiment, include in the PBS buffer solution containing the digestive ferment: about 0.1mg/mL dispase, about 0.25mg/mL pancreatin, about 0.25mg/mL DNase I, about 1mg/mL clostridiopetidase A IV and about 1mg/mL hyaluronidase.

Digestive enzyme compositions according to a third aspect of the present invention, the digestive enzyme compositions are the PBS containing the digestive ferment Buffer.In one embodiment, include in the PBS buffer solution containing the digestive ferment: 0.02mg/mL dispase, 0.05mg/mL pancreatin, 0.02mg/mL DNase I, 0.05mg/mL clostridiopetidase A IV and 0.2mg/mL hyaluronidase.

Digestive enzyme compositions according to a third aspect of the present invention, which are in PBS buffer solution, includes: 0.02mg/mL Dispase, 0.05mg/mL pancreatin, 0.02mg/mL DNase I, 0.05mg/mL clostridiopetidase A IV and 0.2mg/mL hyaluronic acid Contain about 0.2mg/mL, about in enzyme and 0.2~0.5mg/mL sodium glutamate and 2~5mg/mL mannose, such as the buffer The sodium glutamate of 0.3mg/mL, about 0.4mg/mL or about 0.5mg/mL and about 2mg/mL, about 3mg/mL, about 4mg/mL or about The mannose of 5mg/mL.

Digestive enzyme compositions according to a third aspect of the present invention, wherein described from the side of placenta separating mesenchymal stem cell Method comprising following steps:

And optional following one or more steps:

Digestive enzyme compositions according to a third aspect of the present invention, wherein the step (a) is aseptically by placenta Leaflet is cut, and remaining blood in placental lobules removal placenta is sufficiently rinsed with PBS buffer solution.

Digestive enzyme compositions according to a third aspect of the present invention, wherein the step (a) be within four hours postpartum, Placental lobules is cut under aseptic condition, sufficiently rinses placental lobules with the PBS buffer solution containing 10% volume fetal calf serum (FBS) Remove remaining blood in placental lobules.

Digestive enzyme compositions according to a third aspect of the present invention, wherein the tissue digestion enzyme in the step (b) is to be selected from Following is one or more: dispase, pancreatin, deoxyribonuclease I (DNase I), clostridiopetidase A IV, hyaluronidase.? In one embodiment, the tissue digestion enzyme in the step (b) includes: dispase, pancreatin, deoxyribonuclease I (DNase I), clostridiopetidase A IV, hyaluronidase.In one embodiment, it in step (b), is wrapped in the PBS buffer solution Containing about 0.1mg/mL dispase (it is also known as neutral proteinase or dispase), about 0.25mg/mL pancreatin, about 0.25mg/mL DNase I (it is also known as deoxyribonuclease I), about 1mg/mL clostridiopetidase A IV and about 1mg/mL hyaluronidase.

Digestive enzyme compositions according to a third aspect of the present invention, the wherein PBS containing tissue digestion enzyme described in step (b) Buffer is in PBS buffer solution: 0.02mg/mL dispase, 0.05mg/mL pancreatin, 0.02mg/mL DNase I, 0.05mg/mL clostridiopetidase A IV and 0.2mg/mL hyaluronidase.

Digestive enzyme compositions according to a third aspect of the present invention, the wherein PBS containing tissue digestion enzyme described in step (b) Buffer is in PBS buffer solution: 0.02mg/mL dispase, 0.05mg/mL pancreatin, 0.02mg/mL DNase I, 0.05mg/mL clostridiopetidase A IV and 0.2mg/mL hyaluronidase and 0.2~0.5mg/mL sodium glutamate and 2~5mg/mL are sweet Dew sugar, such as the glutamic acid in the buffer containing about 0.2mg/mL, about 0.3mg/mL, about 0.4mg/mL or about 0.5mg/mL The mannose of sodium and about 2mg/mL, about 3mg/mL, about 4mg/mL or about 5mg/mL.Certainly, tissue digestion enzyme should be contained Matching liquid medium used in PBS buffer solution is the PBS buffer solution, that is, is also wrapped in the PBS buffer solution containing tissue digestion enzyme Containing PBS buffer solution；Although not referred in the above-mentioned PBS buffer solution composition containing tissue digestion enzyme, this field in such situation Do not have to generally also refer to.It has been had now surprisingly been found that, add a small amount of paddy in the PBS buffer solution of Xiang Hanyou tissue digestion enzyme simultaneously Propylhomoserin sodium and mannitol and the concentration for greatly reducing other five kinds of tissue digestion enzymes, still are able to obtain excellent effect, this will There is meaning of crucial importance, reason be that the price of five kinds of digestive ferments is very expensive and sodium glutamate and mannitol extremely Cheaply, methodology cost can be substantially reduced and satisfied technical effect can be obtained, for example, above-mentioned containing sodium glutamate and sweet Reveal the composition of sugar, cost includes about 0.1mg/mL dispase, 0.25mg/mL pancreatin, 0.25mg/mL DNase I, the 5% of 1mg/mL clostridiopetidase A IV and 1mg/mL hyaluronidase and the composition cost without sodium glutamate and mannose, and And methodology totle drilling cost can be made to reduce about 90%.

Digestive enzyme compositions according to a third aspect of the present invention are incubated for 10~30 minutes in step (b) at 37 DEG C, excellent It selects 10~20 minutes, such as 10 minutes, 15 minutes or 20 minutes.

Placental lobules is cut into about 1cm in step (b) by digestive enzyme compositions according to a third aspect of the present invention³Size Tissue block.

Tissue block copper mesh is filtered while being used in step (c) by digestive enzyme compositions according to a third aspect of the present invention Syringe grinding.In one embodiment, in step (c), it is (excellent that tissue block and cell suspension one are reinstated into 160~240 mesh Select 200 mesh) copper mesh filtering while with syringe piston tissue abrasion block.

Digestive enzyme compositions according to a third aspect of the present invention, in step (d), by the filtered fluid density gradient of collection Centrifugal process separates mononuclearcell, washing, then with MSC culture medium suspension cell obtained, then in 37 DEG C, 5%CO₂Culture It is cultivated in case.In one embodiment, in step (d), filtered cell suspension will be collected and be added in centrifuge tube, 1000rpm is centrifuged 10 minutes, outwells supernatant, and cell is resuspended with the PBS buffer solution containing 10%FBS.

Digestive enzyme compositions according to a third aspect of the present invention after disseminated cell forms clone, are chosen in step (e) Each clone cell is taken, is cultivated respectively with MSC culture medium, after the fusion of cell 60~90%, with pancreatin had digestive transfer culture to get placenta Mescenchymal stem cell.In one embodiment, in step (e), after being dispersed in attached cell formation clone, with 0.05% pancreas Protease/2mM EDTA digestion, secondary culture.

Digestive enzyme compositions according to a third aspect of the present invention, in step (f), the cytoactive detection is to utilize platform Expect that blue decoration method counts the number for freezing front and back living cells.

Digestive enzyme compositions according to a third aspect of the present invention, in step (f), the cell contamination detection is using on a small quantity Cell culture, detection cell whether the pollution by fungi and bacterium.In one embodiment, the cell contamination, which detects, is Using aetology method, whether cell is detected by selected from following one or more infection: Hepatitis B virus, hepatitis, Chinese mugwort Grow virus, cytomegalovirus, Epstein-Barr virus and syphilis, HbsAg, HbsAb, HBcAb, HbeAg, HbeAb, HCVAb, HIV-1/2Ab, CMV-IgM and EBV-IgA, TRUST.

Digestive enzyme compositions according to a third aspect of the present invention, in step (f), the hereditary disease detection is to utilize molecule The method of science of heredity, detection freeze-stored cell whether there is hereditary disease.

Digestive enzyme compositions according to a third aspect of the present invention, in step (f), the HLA-ABC/DR distribution type is detection Cell HLA-ABC/DR phenotype.

Digestive enzyme compositions according to a third aspect of the present invention, in step (g), the placenta mesenchyma stem cell be through Program temperature-fall period freezes in liquid nitrogen.

Digestive enzyme compositions according to a third aspect of the present invention, in step (g), the placenta mesenchyma stem cell exists In cells frozen storing liquid.In one embodiment, the cells frozen storing liquid include 50% low sugar DMEM culture solution, 40%FBS, 10% dimethyl sulfoxide.

Digestive enzyme compositions according to a third aspect of the present invention include and are saved in step (h) in the database All relevant data of cell, including but not limited to: the biological characteristics testing result of cell, multi-lineage potential identification knot Fruit, cellular elements genetic diagnosis result, the particulars of fetus and its parent.

Digestive enzyme compositions according to a third aspect of the present invention, the method from placenta separating mesenchymal stem cell include Following steps: aseptically cutting placental lobules, is sufficiently rinsed in placental lobules removal placenta and is remained with PBS buffer solution Blood.Placental lobules is cut into 1cm again³The PBS buffer solution containing Various Tissues digestive ferment is added 37 in the tissue block of size It DEG C is incubated for 15 minutes, tissue block is filtered with copper mesh and is ground simultaneously with syringe.Collect filtered liquid density gradient from Heart method separates mononuclearcell, is put into 37 DEG C of 5%CO with MSC culture medium suspension cell obtained after washing₂It is trained in incubator It supports.After disseminated cell forms clone, each clone cell is chosen and is cultivated respectively with MSC culture medium, cell 80~90% merges Afterwards, with pancreatin had digestive transfer culture to get placenta mesenchyma stem cell.Cell after passage is frozen in liquid nitrogen and records related tire Youngster's information, and biological characteristics and the multi-lineage potential identification of cell are carried out, and molecular genetics diagnosis is carried out to cell, All related datas for saving cell, establish the database of placenta stem-cell and are associated with freeze-stored cell.

Digestive enzyme compositions according to a third aspect of the present invention, the method from placenta separating mesenchymal stem cell include Following steps: aseptically cutting placental lobules within four hours postpartum, with the PBS buffer solution for containing 10% volume FBS Sufficiently rinse remaining blood in placental lobules removal placental lobules.Placental lobules is first cut into 1cm³The tissue block of size, adds Enter to containing 0.1mg/mL dispase, 0.25mg/mL pancreatin, 0.25mg/mL DNase I, 1mg/mL clostridiopetidase A IV, 1mg/ It is digested 15 minutes for 37 DEG C in the PBS buffer solution of mL hyaluronidase, appropriate FBS is added and terminates digestion.Again by tissue block and cell Suspension one reinstates the filtering of 200 mesh copper mesh while with syringe piston tissue abrasion block, collects filtered cell suspension and is added to 1000rpm is centrifuged 10 minutes in centrifuge tube, outwells supernatant, and cell is resuspended with the PBS buffer solution containing 10%FBS.Then density is used Gradient centrifugation separates and collects mononuclearcell, and PBS buffer solution uses the suspension of MSC culture medium obtained after washing mononuclearcell Cell, the density for being inoculated with mononuclearcell is every 75cm²(Tissue Culture Flask internal surface area) 1 × 10⁷The total 10ml of mononuclearcell MSC culture medium.Culture bottle is finally put into 37 DEG C of 5%CO₂It is cultivated in incubator.It is non-adherent thin to change liquid removal for full dose after 72 hours Born of the same parents have the shuttle-type attached cell being dispersed in culture bottle, fresh MSC culture solution are added and continues to cultivate.It 10 days or so, is dispersed in adherent After cell forms clone, digested with 0.05% trypsase/2mM EDTA, after cell count, according to 3000 cells/cm²It carries out Secondary culture；Later, when cell reach 70% or so fusion when had digestive transfer culture culture to get placenta mesenchyma stem cell.

Digestive enzyme compositions according to a third aspect of the present invention, the method from placenta separating mesenchymal stem cell include Following steps: aseptically cutting placental lobules within four hours postpartum, with the PBS buffer solution for containing 10% volume FBS Sufficiently rinse remaining blood in placental lobules removal placental lobules.Placental lobules is first cut into 1cm³The tissue block of size, adds Enter to containing 0.1mg/mL dispase, 0.25mg/mL pancreatin, 0.25mg/mL DNase I, 1mg/mL clostridiopetidase A IV, 1mg/ 15 points are digested for 37 DEG C in the PBS buffer solution of mL hyaluronidase, 0.2~0.5mg/mL sodium glutamate and 2~5mg/mL mannose Clock is added appropriate FBS and terminates digestion.It is living with syringe simultaneously that tissue block and cell suspension one are reinstated into the filtering of 200 mesh copper mesh again Tissue abrasion's block is filled in, filtered cell suspension is collected and is added to 1000rpm centrifugation 10 minutes in centrifuge tube, outwell supernatant, use Cell is resuspended in PBS buffer solution containing 10%FBS.Then mononuclearcell, PBS buffer solution are separated and collected with density-gradient centrifugation method MSC culture medium suspension cell obtained is used after washing mononuclearcell, the density for being inoculated with mononuclearcell is every 75cm²It is (thin Born of the same parents' culture bottle internal surface area) 1 × 10⁷The total 10ml MSC culture medium of mononuclearcell.Culture bottle is finally put into 37 DEG C of 5%CO₂Training It supports and is cultivated in case.Full dose changes liquid removal non-adherent cell after 72 hours, has the shuttle-type attached cell being dispersed in culture bottle, is added Fresh MSC culture solution continues to cultivate.10 days or so, after being dispersed in attached cell formation clone, with 0.05% trypsase/2mM EDTA digestion, after cell count, according to 3000 cells/cm²Carry out secondary culture；Later, when cell reaches 70% or so fusion When had digestive transfer culture culture to get placenta mesenchyma stem cell.

The present invention is further illustrated below.Cited text in document cited in the present invention and the document It offers, their full content is incorporated herein by reference.

In the present invention, in any technical solution of either side of the present invention, any technical characteristic is equally applicable to this Any embodiment in either invention face, as long as they will not cause contradiction, and this be mutually applicable in if necessary may be used To be suitably modified.

In the present invention, term " placenta mesenchyma stem cell " refers to the mescenchymal stem cell from placenta.Therefore exist In the present invention, more particularly in context of the invention, term " placenta mesenchyma stem cell " can with " placenta stem-cell ", " stem cell ", " mescenchymal stem cell " are used interchangeably, unless otherwise clearly indicating.

In the present invention, term " PBS buffer solution " or " PBS " refer to phosphate buffer.Those skilled in the art are ripe Know the general formula and preparation method and their general aspects such as pH value or pH of the PBS used under situation of the present invention Range, and these PBS buffer solution are usually the pre-mixing liquor (or prewired powder) that can be obtained through commercial channels, such as this The PBS of invention field is usually the commercialization buffer of pH7.4 (± 0.1), such as the PBS buffer solution of HyClone brand；Ability It include 137mM sodium chloride, 2.7nM potassium chloride and 10mM phosphate radical in PBS buffer solution composition when the application of domain classics, in this hair In bright if not otherwise specified, PBS used using when composition be the composition.

In the present invention, term " placenta " refers to newborn fetal placenta, particularly relates to the placenta within 4 hours postpartum.

In the present invention, term " MSC culture medium " refers to the special culture media of mescenchymal stem cell culture, and the present invention is to contain The PBS buffer solution culture medium of the DMEM-F12 of 10%FBS.

The present inventor was once separately cultured mescenchymal stem cell using perfusion method from placenta, and it is very high to obtain purity Mescenchymal stem cell.But still suffer from a large amount of stem cells after perfusion and be trapped in placenta tissue, cannot effectively by It separates.It is understood that mescenchymal stem cell cannot be obtained to greatest extent using perfusion method.

The method of the invention discloses a kind of from placenta a large amount of separating mesenchymal stem cells, and save with this method Placenta mesenchyma stem cell simultaneously establishes placenta stem-cell library.The present inventor was separately cultured mesenchyma in summary in the past and did carefully It is successful from placenta in conjunction with stationary culture using Various Tissues digestive ferment mixture slaking placental lobules tissue block on the basis of born of the same parents In isolated a large amount of mescenchymal stem cells.Mescenchymal stem cell that the method for the present invention obtains purity is high, quantity are more, have and bone The identical biological characteristics of bone marrow-drived mesenchymal stem, can be thin to osteoblast, cartilage cell, fat cell, endothelial cell, nerve The differentiation such as born of the same parents.Due in placenta stem cell compared with adult stem cell naivety, rich content, before clinically having a wide range of applications Scape, we freeze mescenchymal stem cell with conventional cell freezing method as bleeding of the umbilicus, establish placenta stem-cell Library lays the foundation for the further investigation and clinical treatment of later stem cell.

Due to candidate stem cell rich in bleeding of the umbilicus, people establish unbilical blood bank, and umbilical hemopoietic stem cell, this is important Living resources store, provide a kind for the treatment of means for a variety of diseases in the blood system and disease of immune system.Same placenta As a kind of more importantly stem cell resource, we are freezed mescenchymal stem cell with conventional cell freezing method It is saved for a long time in -196 degrees Celsius of profound hypothermia liquid nitrogen, establishes placenta stem-cell library, save kind for stem-cell therapy in the future Son.

The object of the present invention is to provide a kind of practical separating mesenchymal stem cells a large amount of simply from placenta and establish tire The method of disk stem cell bank, includes the following steps: aseptically to cut placental lobules, is sufficiently rinsed with PBS buffer solution Placental lobules removes remaining blood in placenta.Placental lobules is cut into 1cm again³The tissue block of size is added containing there are many groups The PBS buffer solution for knitting digestive ferment is incubated for 15 minutes at 37 DEG C, and tissue block is filtered with copper mesh and is ground simultaneously with syringe.It collects Filtered liquid separates mononuclearcell with density-gradient centrifugation method, with MSC culture medium suspension cell obtained after washing It is put into 37 DEG C of 5%CO₂It is cultivated in incubator.After disseminated cell forms clone, each clone cell is chosen with MSC culture medium point It does not cultivate, after cell 80~90% merges, with pancreatin had digestive transfer culture, the cell after passage is frozen in liquid nitrogen and records correlation Fetus information, and biological characteristics and the multi-lineage potential identification of cell are carried out, and molecular genetics is carried out to cell and is examined It is disconnected, all related datas of cell are saved, the database of placenta stem-cell is established and is associated with freeze-stored cell.

According to the method for the present invention, such as 1,2 method according to embodiments of the present invention, the placenta that a weight is 750 grams 8 × 10 can be got⁹Mononuclearcell, by adhere-wall culture average 1 × 10⁶12 clones can be obtained in mononuclearcell, reflected Averagely there are 3 clones that there is the characteristic of mescenchymal stem cell after fixed, i.e., can get 24000 mescenchymal stem cells in 750g placenta Clone.These stem cells can quickly reach number needed for Cell Biology Experiment and clinical treatment by external Short-term Culture Amount.However, the inventors discovered that, according to the method for embodiment one in CN1548529A, the placenta that a weight is 750 grams is about Separable to obtain 3000 mescenchymal stem cell clones, after the passage of 3 generations, cell purity is greater than about 95%.In the present invention one It include about 0.1mg/mL dispase, about 0.25mg/mL pancreas in the PBS buffer solution described in step (b) in a embodiment Enzyme, about 0.25mg/mL DNase I, about 1mg/mL clostridiopetidase A IV and about 1mg/mL hyaluronidase；The inventors discovered that should 5 kinds of enzymes in PBS buffer solution, when lacking any one or more, mescenchymal stem cell yield and cell purity are much not achieved The result of 24000 mescenchymal stem cells clone can be got in above-mentioned 750g placenta；In addition, the concentration of 5 kinds of enzymes makees appropriate variation, Especially in 50% lower than above-mentioned respective concentration or 200% higher than above-mentioned respective concentration, mescenchymal stem cell yield It is obviously poorer than the result of 24000 mescenchymal stem cells clone can be got in above-mentioned 750g placenta with cell purity；Such as when Dispase concentration in PBS buffer solution is 0.04mg/mL (lower than the 50% of the above-mentioned concentration of the present invention) or is 0.25mg/mL When (higher than the 200% of the above-mentioned concentration of the present invention), 8000 mescenchymal stem cell clones and cell can be about got in 750g placenta Purity is lower than 90%.Therefore, one embodiment of the invention be in step (b), in the PBS buffer solution comprising 0.05~ 0.2mg/mL dispase, 0.125~0.5mg/mL pancreatin, 0.125~0.5mg/mL DNase I, 0.5~2mg/mL collagen Enzyme IV and 0.5~2mg/mL hyaluronidase.

In addition, the present inventor provides the test (i.e. complementary testing 1 to complementary testing 3) supplemented as follows here.Supplement examination It tests 1: carrying out the separation of placenta MSC, secondary culture referring to the embodiment of the present invention 1 to embodiment 2 and its freeze, referring next to implementation Example 3 and embodiment 4 carry out the detection of relevant item, and different is only that will be cut into 1cm in the operation of embodiment 1³The placenta of size Leaflet tissue block is added to containing 0.02mg/mL dispase, 0.05mg/mL pancreatin, 0.02mg/mL DNase I, 0.05mg/ The PBS buffer solution of mL clostridiopetidase A IV and 0.2mg/mL hyaluronidase and 0.3mg/mL sodium glutamate and 3mg/mL mannose In, carry out digestion and subsequent processing.Complementary testing 2: the separation of the reference embodiment of the present invention 1 to the progress of embodiment 2 placenta MSC, It secondary culture and its freezes, the detection of relevant item is carried out referring next to embodiment 3 and embodiment 4, different is only to implement 1cm will be cut into the operation of example 1³The placental lobules tissue block of size is added to containing 0.02mg/mL dispase, 0.05mg/mL Pancreatin, 0.02mg/mL DNase I, 0.05mg/mL clostridiopetidase A IV and 0.2mg/mL hyaluronidase and 0.2mg/mL paddy ammonia In the PBS buffer solution of sour sodium and 5mg/mL mannose, digestion and subsequent processing are carried out.Complementary testing 3: referring to the embodiment of the present invention 1 carries out the separation of placenta MSC, secondary culture to embodiment 2 and its freezes, and carries out referring next to embodiment 3 to embodiment 4 related The detection of project, different is only that will be cut into 1cm in the operation of embodiment 1³The placental lobules tissue block of size be added to containing 0.02mg/mL dispase, 0.05mg/mL pancreatin, 0.02mg/mL DNase I, 0.05mg/mL clostridiopetidase A IV and 0.2mg/ In the PBS buffer solution of mL hyaluronidase and 0.5mg/mL sodium glutamate and 2mg/mL mannose, digestion and subsequent place are carried out Reason.The results show that being tied obtained by complementary testing 1 to 3 three complementary testing acquired results of complementary testing and embodiment 1 to embodiment 4 Fruit is almost the same, in addition in terms of mescenchymal stem cell yield it is higher than embodiment 1 to embodiment 4, such as complementary testing 1 is to benefit It fills in test 3 to separate respectively from 750g placenta and obtains 30000~32000 mescenchymal stem cell clones, such as supplement examination It tests 1 and is separated from 750g placenta and obtain 31500 mescenchymal stem cells clone, complementary testing 1 is supplemented to complementary testing 3 three Test gained cell purity is in 96~98% ranges, such as complementary testing 1, after 3 more than generation pass on, cell component is uniform, It is similar with Fig. 2 result, purity 95% or more, in another example in complementary testing 1 Flow cytometry placenta MSC cell cycle When obtain similar with Fig. 3 as a result, the i.e. DNA content in the 3rd generation and the 6th generation cell after measured, cell cycle analysis, the G0/G1 phase, S phase and G2M phase proportion are respectively 96.32%, 96.75%, 1.13%, 0.09% and 2.52%, 3.30%, as a result table The cell of bright in vitro culture has the characteristics that typical stem cells hyperplasia, i.e., only a few cell is in active proliferation period (1.13%, 0.09%), most cell are in quiescent stage (96.32%, 96.75%).In further complementary testing, Above-mentioned complementary testing 1 is respectively referred to, different is only to be not added with sodium glutamate in digestive juice or be not added with mannitol, is as a result shown Show that mescenchymal stem cell clone yield display reduces in the case where being not added with alternative one, is down to 21000 clone/750g respectively With 18000 clone/750g, and gained cell purity is respectively 93.3% and 93.7%.

In addition, the present inventor provides the test (i.e. complementary testing 4 to complementary testing 5) supplemented as follows here.Supplement examination It tests 4: carrying out the separation of placenta MSC, secondary culture referring to the embodiment of the present invention 1 to embodiment 2 and its freeze, referring next to implementation Example 3 and embodiment 4 carry out the detection of relevant item, and different is only that will be cut into 1cm in the operation of embodiment 1³The placenta of size Leaflet tissue block is added to the #264E1 type digestion enzyme mixation (proportion of #264E1 type digestion enzyme mixation are as follows: collagenase type I 1 μ g/ml, 0.5 μ g/ml of neutral proteinase, 2 μ g/ml of deoxyribonuclease, PBS 100ml) in, carry out digestion and subsequent place Reason.Complementary testing 5: it to the separation of the progress of embodiment 2 placenta MSC, secondary culture and its freezes, connects referring to the embodiment of the present invention 1 The detection of relevant item is carried out referring to embodiment 3 and embodiment 4, different is only that will be cut into 1cm in the operation of embodiment 1³ The placental lobules tissue block of size is added to (the preparation of #500E1 type digestion enzyme mixation of #500E1 type digestion enzyme mixation Are as follows: II Collagenase Type 250U/ml, neutral proteinase 100U/ml, hyaluronidase 10U/ml, 37 DEG C are dissolved in DMEM/F12 training Support base, the filter degerming of 0.22 μm of filtering) in, carry out digestion and subsequent processing.The results show that complementary testing 4 is to complementary testing 5 Two complementary testing acquired results are significantly poorer than embodiment 1 to embodiment 4 acquired results, such as in mescenchymal stem cell yield Aspect can only separate respectively from 750g placenta obtains 15000~17000 mescenchymal stem cell clones and gained cell purity In 91~93% ranges.

Operation of the present invention is simple, convenient and practical, can obtain a large amount of mescenchymal stem cell, and differentiation performance is good, have at The ability of the cell differentiations such as osteocyte, fat cell, cartilage cell, endothelial cell, nerve cell.Compared with existing method: MSC mainly uses modus operandi to extract donor bone marrow or perfusion method separation placenta, adhere-wall culture acquisition at present.The method gets cell number Amount is few, and donor in taking marrow and takes the possibility for having infection after marrow.Present invention success separation from placenta obtains a large amount of pure Higher mescenchymal stem cell is spent, and establishes placenta stem-cell library with this method to lay in the dry thin of this great application prospect Born of the same parents.The method is simple and easy to do, and since placenta is as bleeding of the umbilicus, Cell Component is inmatureer, from a wealth of sources, is conveniently easy to get, therefore this The method of invention will have extensive prospect in the clinical application of stem cell.

Detailed description of the invention

Fig. 1 is under microscope to the morphological observation of cell growth.Wherein: A be after cultivating 3 days it is visible be dispersed in it is adherent thin Born of the same parents；B is the clone of formation in 7~10 days；C is 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 " counts " indicates count number in each figure.

Fig. 3 is the analysis result of placenta MSC cell cycle.Wherein, P3 is the DNA content for cultivating third generation cell, analysis Cell cycle, it is seen that most cells are in resting stage (G₀/G₁Phase, 96.66%), few cell be in proliferation period (the S phase, 3.25%).P6 is the DNA content for cultivating the cell in the 6th generation, analyzes the cell cycle, it is seen that most cells are in resting stage (G₀/G₁Phase, 96.35%), few cell is in proliferation period (S phase, 2.54%).Ordinate " counts " indicates to count in each figure Amount, abscissa mark " DNA Content " indicate DNA content.

Fig. 4 is placenta MSC cell growth curve figure.

Fig. 5 is the osteogenic induction cytological map of placenta MSC multi-lineage potential identification.

Fig. 6 is the Adipogenic induction cytological map of placenta MSC multi-lineage potential identification.

Fig. 7 is the identification of placenta MSC multi-lineage potential into chondrocyte induction cytological map.

Fig. 8 is the electrophoresis photographs that RT-PCR detects placenta MSC multi-lineage potential.

Specific embodiment

The present invention can be further described by the following examples, however, the scope of the present invention and unlimited In following embodiments.One of skill in the art, can be with it is understood that under the premise of without departing substantially from the spirit and scope of the present invention Various change and modification are carried out to the present invention.The present invention carries out the material and test method arrived used in test general And/or specific description.Although to realize the present invention many materials and operating method used in purpose be it is known in the art that But the present invention still makees description as detailed as possible herein.

The separation of embodiment 1, placenta MSC

Within four hours postpartum, aseptically placental lobules is cut, is buffered with the PBS containing 10% volume FBS Liquid sufficiently rinses remaining blood in placental lobules removal placental lobules.Placental lobules is first cut into 1cm³The tissue block of size, Be added to containing 0.1mg/mL dispase, 0.25mg/mL pancreatin, 0.25mg/mL DNase I, 1mg/mL clostridiopetidase A IV, It is digested 15 minutes for 37 DEG C in the PBS buffer solution of 1mg/mL hyaluronidase, appropriate FBS is added and terminates digestion.Again by tissue block and Cell suspension one reinstates the filtering of 200 mesh copper mesh while with syringe piston tissue abrasion block, collects filtered cell suspension and adds Enter into centrifuge tube 1000rpm to be centrifuged 10 minutes, outwell supernatant, cell is resuspended with the PBS buffer solution containing 10%FBS.Then it uses Density-gradient centrifugation method separates and collects mononuclearcell, is obtained after PBS buffer solution washing mononuclearcell with the suspension of MSC culture medium The cell obtained, the density for being inoculated with mononuclearcell is every 75cm²(Tissue Culture Flask internal surface area) 1 × 10⁷Mononuclearcell is total 10ml MSC culture medium.Culture bottle is finally put into 37 DEG C of 5%CO₂It is cultivated in incubator.Full dose changes liquid after 72 hours, go unless Attached cell has the shuttle-type attached cell being dispersed in culture bottle, adds fresh MSC culture solution and continue to cultivate.

Embodiment 2, placenta MSC secondary culture and its freeze

After about 10 days, after being dispersed in attached cell and forming clone, digested with 0.05% trypsase/2mM EDTA, carefully After born of the same parents count, according to 3000 cells/cm²Carry out secondary culture.Later, when cell reaches 70% or so fusion, had digestive transfer culture is trained It supports.3 × 10 are taken after digestion⁶Cell is added to 1ml cells frozen storing liquid (containing 50% low sugar DMEM culture solution, 40%FBS, 10% 2 Methyl sulfoxide) in, it enters finally into liquid nitrogen pipe and freezes by program cooling.

The Identification of Biological Characteristics of embodiment 3, placenta MSC

1, cell growth and its Morphological Characteristics

By being separately cultured for embodiment 1 and embodiment 2, the culture of placenta mononuclearcell under the microscope may be used after 72 hours It obviously sees shuttle shape attached cell (Figure 1A), will form within 10 days or so turbine-like cell clone (Figure 1B, Fig. 1 D), after had digestive transfer culture It will form the leapfrog structure (Fig. 1 C, Fig. 1 E, Fig. 1 F) of 80% or so fusion.In incubation, it is found that this cellular morphology is relatively equal One, growth rate is fast, and adherent speed is fast, is easily digested by pancreatin, and it is more than generation to be passaged to 15, and form and growth characteristic are also without obvious Change.

2, flow cytometry identification of M SC surface marker

The 3rd, 6,9,12,15 generation cells, Flow cytometry cell surface marker, dynamic observation incubation are taken respectively The variation of middle cell surface marker.Cell is collected in digestion, takes 8 × 10 after counting⁶A cell, 16 pipe of packing；PBS is washed once, 1500rpm is centrifuged 10min；Supernatant is abandoned, 100~200 μ l are remained, piping and druming mixes cell；CD14, CD29 of addition PE label, CD31, CD34, CD44, CD54, CD73, CD80, CD86, CD166 antibody and FITC label CD45, CD105, HLA-ABC, Each 10 μ l of HLA-DR, UEA-1 antibody, and a pipe is set as blank control；At 4 DEG C, it is protected from light 30min；PBS is washed once, 1500rpm is centrifuged 10min；The cell directly marked abandons supernatant, and 200 μ l PBS of addition, which are blown and beaten, mixes cell, and more than the 1% of 200 μ l Polyformaldehyde is fixed, and 4 DEG C of to be measured, flow cytometer detections in 3 days are set.

The surface marker of flow cytomery cell, dynamic observation the 3rd, 6,9,12, the cell in 15 generations, without obviously changing Become.Hematopoietic cell surface marker i.e. CD14, CD31, CD34 (HSPC and endothelial cell are positive), CD45 (leucocyte sun are not expressed Property), CD54 (ICAM-1), CD80 (B7-1), CD86 (B7-2), HLA-DR (MHC-II class molecule) continue feminine gender, CD29 and CD44 (receptor of fibrin and transparency grease hydrochlorate, stroma cell expression), CD73 (i.e. SH-3,4), CD105 (i.e. SH-2), CD166 (mesenchymal cell expression), HLA-ABC (MHC-I class molecule) and UEA-1 (surface marker of endothelial cell) are continuously sun Property.After 3 more than generation pass on, cell component is uniform, and purity is 95% or more.(Fig. 2)

3, the cell cycle of Flow cytometry placenta MSC

Cell is long to when 80% or so fusion, and cell about 1 × 10 is collected in digestion⁶It is a, PBS wash once be added 70% second Alcohol is fixed, and 4 DEG C to be measured.When detection, first ethyl alcohol is removed in centrifugation, then is washed once with PBS, and RNase I 500u, 37 DEG C of reactions are added 30min, PBS are washed once, and propidium iodide (PI, 50 μ g/ml of final concentration) 1ml is added, and room temperature is protected from light 20min, upper machine testing Cell DNA content.

The DNA content in the 3rd generation and the 6th generation cell after measured, cell cycle analysis, G₀/G₁Phase, S phase and G₂M phase institute accounting Example is respectively 96.35%, 96.66%, 1.11%, 0.09% and 2.54%, 3.25%.The result shows that the cell of in vitro culture Has the characteristics that typical stem cells hyperplasia, i.e., only a few cell is in active proliferation period (1.11%, 0.09%), most of Cell be in quiescent stage (96.35%, 96.66%).(Fig. 3)

4, the drafting of placenta MSC growth curve and the measurement of logarithmic growth phase doubling time

Logarithmic growth phase cell, digestion count, with the LG-DMEM culture medium of 10%FBS be made cell suspension (2 × 10⁴/ ml), every hole is inoculated with 0.5ml in 24 orifice plates, and 37 DEG C, 5%CO₂, cultivate under saturated humidity.3 multiple holes, trypan blue dye are taken daily Living cell counting number after color calculates average value, is observed continuously 7 days.Using incubation time as horizontal axis, cell number is the longitudinal axis, is drawn thin Intracellular growth curve.Cell is calculated in the doubling time of logarithmic growth phase, i.e. Td=Tlg2/Lg (Nt/ with Patterson formula No), Td: the doubling time (h), T: cell increases to the time (h) used in Nt as No, N: cell number.

Cell growth curve is drawn by the result of daily cell count, calculates the doubling time.It can by cell growth curve To find out, cell was in exponential phase of growth at the 2-4 days.The 5th generation cell is calculated from the formula in the multiplication of exponential phase of growth Time is respectively 22.6h.(Fig. 4)

5, the identification of placenta MSC multi-lineage potential

(1) osteogenic induction

The 3 generations above MSC, by 1 × 10⁵/ hole is inoculated with six orifice plates, is put in 37 DEG C, 5%CO₂, under saturated humidity, MSC culture medium After middle culture for 24 hours, uses instead and screened the DMEM-HG of FBS containing 10% and 0.1 μM of dexamethasone, 50 μ of ascorbyl phosphate is added M, β-phosphoglycerol 10mM, is put in 37 DEG C, 5%CO₂, cultivate under saturated humidity, every 3 days half amounts change liquid, coinduction 2-4 weeks. Alkaline phosphatase staining identification osteoblast is formed, and Von Kossa dyeing identification bone tubercle is formed.

, through screening the DMEM-HG of FBS, 0.1 μM of dexamethasone, 50 μM of ascorbyl phosphate, β-phosphorus is being added containing 10% Acid glycerol 10mM is cultivated 1 week, and apparent change occurs for cellular morphology, becomes polygonal, class from fusiform fibroblast sample It is similar to neuronal cell sample, it is prominent that long filiform occurs in cell periphery, and can extend to surrounding.After continuing culture 2 weeks or more, cell Occurs calcified plaque in matrix, mineralizer gradually appears, and initially forms the small junction structure of multilayer, until after culture 4 weeks, it is seen that obvious Calcium scoring.Alkaline phosphatase staining is in strong positive reaction at 2 weeks, reaches 95% or more, and the control group not induced is then Most of is feminine gender, is shown as weakly positive only less than 5%, shows that cell is converted to osteoblast.Von Kossa dyeing can The calcium deposited in bone tubercle is dyed into black, the visible a large amount of black bone tubercle of induction group has apparent stereochemical structure, and compares Group is at any time all without positive reaction.(Fig. 5)

(2) Adipogenic induction

The 3 generations above MSC, by 1 × 10⁵/ hole is inoculated in six orifice plates, is put in 37 DEG C, 5%CO₂, under saturated humidity, trained in MSC Support base in cultivate for 24 hours after, use instead containing 10% through screen FBS DMEM in high glucose, and be added 1 μM of dexamethasone, 60 μM of indocin, IBMX 0.5mM, 5 μ g/ml of insulin, are put in 37 DEG C, 5%CO₂, cultivate under saturated humidity, every 3 days half amounts change liquid, coinduction 2 Week, oil red dyeing identification fat drips are formed.

, through screening the DMEM-HG of FBS, 1 μM of dexamethasone, 200 μM of indocin, IBMX 0.5mM, pancreas is being added containing 10% Island 10 μ g/ml of element are cultivated 3 days, and morphologic change occurs for cell, are gradually tapered up and are shortened by fusiform fibroblast sample, and 90% The above cell becomes cube or polygonal；Continuous culture 7 days, has small fat drips to occur, with culture under mirror in visible cell The extension of time, fat drips are gradually increased and merge, until when cultivating 2 weeks, it is seen that merge pockets of fat drips full of entire cell.Oil red The fat generated in O dyeing visible cell dyes red by specificity.(Fig. 6)

(3) at chondrocyte induction

The 3 generations above cell, according to every pipe 2 × 10⁵Cell is dispensed into 15ml polypropylene centrifuge tube, and low-speed centrifugal makes cell exist Micelle is formed in test tube, and insulin, transferrins, each 6.25 μ g/ of sodium selenite are added in the DMEM-HG containing 2.5%FBS Ml, BSA 1.25 μ g/ml, Sodium Pyruvate 1mM/L, 37.5 μ g/ml of ascorbic acid phosphoric acid, TGF-β₁50ng/ml, be put in 37 DEG C, 5%CO₂, cultivate under saturated humidity, every 3 days half amounts change liquid, continuous culture 2 weeks.

Cell micelle is broken up into smear after inducing 2 weeks, alcian blue (Alcian blue) dyes visible II Collagen Type VI and formed carefully Extracellular matrix is blue, and control group is contaminated without indigo plant.(Fig. 7)

6, RT-PCR detects placenta MSC multi-lineage potential

Cell after collecting induction, extracts cell total rna using Trizol reagent, carries out RT-PCR using it as template, instead Transcription and PCR operation are carried out according to RT-PCR kit specification, Primer, primer sequence, PCR product size, special Property etc. is as shown in the table 1 of its [0086]~[0087] CN102676451A.

In Fig. 8, it is respectively as follows: placenta MSC cell from left to right, induction becomes fat cell, induction is osteoblast, induction For the RT-PCR electrophoresis photographs of cartilage cell, wherein from left to right, Normal swimming lane is cellular gene expression situation before inducing, Adipogenic swimming lane, Osteogenic swimming lane, Chondrogenic swimming lane are cellular gene expression situation after induction.As a result table Bright, external evoked rear cell can express serial specific mrna: cell expression PPAR- γ after Adipogenic induction, after osteogenic induction Cell expresses osteopontin (Osteopontin), at cell expression collagen I I (Collagen II) after chondrocyte induction, illustrates institute Obtained MSC cell have skeletonization, at fat, at cartilage differentiation ability, meet generally acknowledged MSC standard.

By the detection of above series of data target, show the MSC isolated using the method for the present invention, have to Osteoblast, fat cell, the ability of Chondrocyte Differentiation, it was demonstrated that the MSC that the method for the present invention obtains has stem cell properties.

The foundation of embodiment 4, placenta stem-cell library

1, the detection of cell activity

The number for freezing front and back living cells is counted using trypan blue staining.

2, the detection of cell contamination

Using a small amount of cell culture, detect cell whether the pollution by fungi and bacterium.Utilize aetology method, detection Cell whether by Hepatitis B virus, hepatitis, AIDS, cytomegalovirus, Epstein-Barr virus and syphilis, HbsAg, HbsAb, HBcAb, HbeAg, HbeAb, HCVAb, HIV-1/2Ab, CMV-IgM and EBV-IgA, TRUST infection.

3, the detection of hereditary disease

Using the method for molecular genetics, detecting freeze-stored cell whether there is hereditary disease.

4, HLA-ABC/DR distribution type

Cell HLA-ABC/DR phenotype is detected, and is placed on record.

5, the investigation of cell origin

Fetus and its particulars of parent are recorded, and are placed on record.

6, the foundation of placenta stem-cell database

After saving normal placenta stem-cell, the database of placenta stem-cell is established, including the first six data, and Foundation is associated with freeze-stored cell.

Claims

1. the method for separating mesenchymal stem cell from placenta, method includes the following steps:

(b) placental lobules is cut into bulk, the PBS buffer solution containing tissue digestion enzyme is added, then be incubated for 10 ~ 20 minutes at 37 DEG C Digestion；

(c) tissue block is filtered with copper mesh, is ground to promote to filter；

(d) filtered fluid of collection is centrifuged, separates mononuclearcell, then with MSC culture medium suspension cell obtained, then existed 37℃、5%CO₂It is cultivated in incubator；

(e) after disseminated cell forms clone, each clone cell of picking is cultivated respectively with MSC culture medium, after cell fusion, With pancreatin had digestive transfer culture to get placenta mesenchyma stem cell；

(f) for placenta mesenchyma stem cell obtained by step (e), following items at least one is detected: cell activity, HLA- ABC/DR distribution type；

(h) establish the database of the placenta stem-cell comprising information above, and make the freeze-stored cell of the database and step (g) into Row association,

Wherein, in step (b), in the PBS buffer solution comprising 0.02mg/mL dispase, 0.05mg/mL pancreatin, 0.02mg/mL DNase I, 0.05mg/mL clostridiopetidase A IV, 0.2mg/mL hyaluronidase, 0.2 ~ 0.5mg/mL sodium glutamate and 2 ~ 5mg/mL mannose.

2. the method according to claim 1, wherein the step (a) is within four hours postpartum, aseptically by tire Disk leaflet is cut, and is sufficiently rinsed with the PBS buffer solution containing 10% volume fetal calf serum remaining in placental lobules removal placental lobules Blood.

3. the method according to claim 1, wherein being incubated for 10 minutes in step (b) at 37 DEG C.

4. the method according to claim 1, wherein being incubated for 20 minutes in step (b) at 37 DEG C.

5. the method according to claim 1, wherein including 0.2mg/mL, 0.3mg/ in the PBS buffer solution in step (b) The sodium glutamate of mL, 0.4mg/mL or 0.5mg/mL and the mannose of 2mg/mL, 3mg/mL, 4mg/mL or 5mg/mL.

6. the method according to claim 1, in step (e), after disseminated cell forms clone, each clone cell of picking is used MSC culture medium is cultivated respectively, after the fusion of cell 60~90%, with pancreatin had digestive transfer culture to get placenta mesenchyma stem cell.

7. the method according to claim 1 includes all related to the cell saved in step (h), in the database Data, comprising: the biological characteristics testing result of cell, multi-lineage potential qualification result, fetus and its parent it is detailed Data.

8. method according to any one of claims 1 to 7, wherein the cell purity of placenta mesenchyma stem cell obtained is greater than 95%。

9. digestive enzyme compositions used in a kind of method from placenta separating mesenchymal stem cell, the digestive enzyme compositions are PBS buffer solution containing tissue digestion enzyme, being somebody's turn to do the PBS buffer solution containing tissue digestion enzyme is in PBS buffer solution comprising following Digestive ferment: dispase, pancreatin, deoxyribonuclease I, that is, DNase I, clostridiopetidase A IV, hyaluronidase；Disappear containing described Change enzyme PBS buffer solution in include: 0.02mg/mL dispase, 0.05mg/mL pancreatin, 0.02mg/mL DNase I, 0.05mg/mL clostridiopetidase A IV, 0.2mg/mL hyaluronidase, 0.2 ~ 0.5mg/mL sodium glutamate and 2 ~ 5mg/mL mannose.