CN102676451B - Method for separating mesenchymal stem cells from placenta - Google Patents
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Abstract
The invention relates to a method for separating mesenchymal stem cells from placenta. The method comprises the following steps: (a) taking placental cotyledon, and fully washing by using a phosphate buffer solution (PBS) to remove residual blood from the placenta; (b) cutting the placental cotyledon into blocks, adding a PBS containing tissue digestive enzyme, and incubating and digesting at 37DEG C; (c) filtering the tissue blocks by using a copper net, and grinding if necessary to promote filtration; (d) centrifuging the collected filtrate, separating mononuclear cells, suspending the obtained cells by using a mesenchymal stem cell (MSC) culture medium, and culturing in a 5 percent CO2 incubator at 37DEG C; and (e) after the dispersed cells form clones, selecting the clone cells, respectively culturing by using an MSC culture medium, and after the cells are fused, performing digestion and passage by using pancreatin to obtain the mesenchymal stem cells of the placenta. By the method, high purity mesenchymal stem cells of the placenta can be obtained.
Description
Technical field
The present invention relates to the method for separate stem cells from placenta, particularly the method for separating mesenchymal stem cell from placenta.
Background technology
Mescenchymal stem cell (mesenchymal stem cell, MSC) for example the mankind's mescenchymal stem cell is separated the earliest from marrow, 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 external specified conditions, chondrocyte, adipocyte, endotheliocyte, neurocyte, 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).Up-to-date research shows that mescenchymal stem cell has immunomodulatory and Hematopoiesis Support affect, and is easy to foreign gene importing expression.Therefore mescenchymal stem cell tissue-engineered bone, cartilage and the cardiac muscle seed cell in building still not, important carrier cell in 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, and people success separation and Culture from the Various Tissues such as liver, kidney, pancreas, muscle, cartilage, skin, peripheral blood go out mescenchymal stem cell.
The mescenchymal stem cell of reporting is at present mainly derived from marrow, adopts density gradient centrifugation to obtain.Although separation method is easy, donor is got marrow need to experience a more painful operation, and in the process of drawing materials and after drawing materials, has very high infection chance; Because the content of MSC in human bone marrow is extremely rare, every 10
5～10
6in individual mononuclearcell, approximately only have 1, and along with the increase at age, in marrow, the quantity of mescenchymal stem cell, propagation and differentiation capability all significantly decline, it is restricted in research with in applying especially clinical application.The placenta that originates from embryonic development period extraembryonic mesoderm is comprised of interstitial, blood vessel and nurse cell, contains a large amount of mesenchyme compositions.Up-to-date research shows to contain abundant stem cell in placenta, and from 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.
Thereby the existing method that separate stem cells is set up placenta stem-cell storehouse from placenta still has shortcomings, for example purity is not enough and/or quantity is not high, and then demonstrates these methods and still can not meet people's expectation.The for example invention of CN 101270349A (Chinese Patent Application No. 200810061267.6, open day on September 24th, 2008) disclosed being entitled as " placenta mesenchyma stem cell separation and amplification in vitro cultural method "; The invention of CN 101693884A (Chinese Patent Application No. 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 "; The invention that CN 102146359A (Chinese Patent Application No. 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.These methods are remaining to be further improved aspect the purity of extract and/or the rate of recovery.
This area still need to have new from placenta the method, the particularly method of separating mesenchymal stem cell from placenta of separate stem cells.
Summary of the invention
The object of the invention is to solve the existing defect of obtaining placenta mesenchyma stem cell method, simply a large amount of separating mesenchymal stem cells optionally set up the method in placenta stem-cell storehouse from placenta of a kind of practicality are provided.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 the method for separating mesenchymal stem cell from placenta, and the method comprises the following steps:
(a) get placental lobules, with PBS damping fluid, fully rinse, to remove blood residual in placenta;
(b) placental lobules is cut into bulk, adds the PBS damping fluid that contains tissue digestion enzyme, then hatch digestion at 37 ℃;
(c) tissue block is filtered with copper mesh, grind if desired to impel filtration;
(d) filtered liquid of collecting is centrifugal, separated mononuclearcell, then the cell that suspends and obtain with MSC substratum, then at 37 ℃, 5%CO
2in incubator, cultivate;
(e) after disseminated cell forms clone, each clone cell of picking, cultivates respectively with MSC substratum, after cytogamy, with trysinization, goes down to posterity, and obtains 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 associated 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 rinses placental lobules remove residual blood in placenta with PBS damping fluid.
According to the method for first aspect present invention, wherein said step (a) is within four hours postpartum, under aseptic condition, placental lobules is cut, with the PBS damping fluid containing 10% volume foetal calf serum (FBS), fully rinsed placental lobules and remove residual blood in placental lobules.
According to the method for first aspect present invention, the tissue digestion enzyme in 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 described step (b) comprises: dispase, pancreatin, deoxyribonuclease I (DNase I), collagenase IV, Unidasa.In one embodiment, in step (b), in described PBS damping fluid, comprise about 0.1mg/mLdispase, about 0.25mg/mL pancreatin, about 0.25mg/mL DNase I, about 1mg/mL collagenase IV and about 1mg/mL Unidasa.
According to the method for first aspect present invention, in step (b), at 37 ℃, hatch 10～30 minutes, preferably 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 about 1cm
3the tissue block of size.
According to the method for first aspect present invention, in step (c), tissue block is filtered and with syringe, ground simultaneously with copper mesh.In one embodiment, in step (c), tissue block and cell suspension one are reinstated to 160～240 orders (preferably 200 orders) copper mesh and filter and use syringe piston tissue abrasion piece simultaneously.
According to the method for first aspect present invention, in step (d), described MSC substratum is the PBS damping fluid containing 10%FBS.In an embodiment of either side of the present invention, described MSC substratum is the PBS damping fluid containing 10%FBS.In one embodiment, in step (d), by the separated mononuclearcell of density gradient centrifugation for the filtered liquid of collecting, washing, then the cell that suspends and obtain with MSC substratum, then at 37 ℃, 5%CO
2in incubator, cultivate.In one embodiment, in step (d), the cell suspension of collecting after filtering is joined in centrifuge tube, centrifugal 10 minutes of 1000rpm, outwells supernatant, with the PBS damping fluid re-suspended cell containing 10%FBS.
According to the method for first aspect present invention, in step (e), after disseminated cell forms clone, each clone cell of picking, cultivates respectively with MSC substratum, after cell 60～90% merges, with trysinization, goes down to posterity, and obtains placenta mesenchyma stem cell.In one embodiment, in step (e), when being dispersed in attached cell, form after 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 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 programmed cooling process.
According to the method for first aspect present invention, in step (g), described placenta mesenchyma stem cell is present in 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).
According to the method for first aspect present invention, in step (h), described database comprises and all relevant data of preserved cell, includes 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 comprises the following steps: under aseptic condition, placental lobules is cut, fully rinsed placental lobules remove residual blood in placenta with PBS damping fluid.Again placental lobules is cut into 1cm
3the tissue block of size, add contain Various Tissues digestive ferment PBS damping fluid at 37 ℃, hatch 15 minutes, tissue block is filtered and with syringe, to be ground simultaneously with copper mesh.Collect the separated mononuclearcell of density gradient centrifugation for liquid after filtration, after washing, with the cell that the suspension of MSC substratum obtains, be put into 37 ℃ of 5%CO
2in incubator, cultivate.After disseminated cell forms clone, each clone cell is chosen with MSC substratum and cultivated respectively, after cell 80～90% merges, with trysinization, go down to posterity, obtain placenta mesenchyma stem cell.By 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 to molecular genetics diagnosis, preserve all related datas of cell, set up the database of placenta stem-cell and carry out associated with freeze-stored cell.
According to the method for first aspect present invention, the method comprises the following steps: within four hours postpartum, under aseptic condition, placental lobules is cut, fully rinsed placental lobules remove residual blood in placental lobules with the PBS damping fluid containing 10% volume FBS.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 appropriate FBS to stop digestion.Again tissue block and cell suspension one are reinstated to 200 order copper mesh and filtered and use syringe piston tissue abrasion piece simultaneously, collect cell suspension after filtering and join in centrifuge tube 1000rpm centrifugal 10 minutes, outwell supernatant, with the PBS damping fluid re-suspended cell containing 10%FBS.Then use density gradient centrifugation separated and collected mononuclearcell, the cell that suspends and obtain with MSC substratum after 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.Finally culturing bottle is put into 37 ℃ of 5%CO
2in incubator, cultivate.After 72 hours, full dose is changed liquid and is removed non-adherent cell, has the shuttle type attached cell being dispersed in culturing bottle, adds fresh MSC nutrient solution to continue to cultivate.About 10 days, be dispersed in attached cell and form after clone, with 0.05% trypsinase/2mM EDTA digestion, after cell counting, according to 3000 cells/cm
2the cultivation of going down to posterity; Afterwards, when cell reaches 70% left and right and merges, had digestive transfer culture is cultivated, and obtains placenta mesenchyma stem cell.
In addition,, in first aspect present invention method, provide a kind of placenta mesenchyma stem cell.Therefore second aspect present invention provides a kind of placenta mesenchyma stem cell.
According to the placenta mesenchyma stem cell of second aspect present invention, it obtains according to method described in the arbitrary embodiment of first aspect present invention.
According to the placenta mesenchyma stem cell of second aspect present invention, its cell purity is greater than 95%.In one embodiment, described placenta mesenchyma stem cell is after in 3 generations, went down to posterity above, 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 to herein by reference.
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 being mutually useful in if desired can be done suitable modification.
In the present invention, term " placenta mesenchyma stem cell " refers to the mescenchymal stem cell that derives from placenta.Therefore in the present invention, particularly relate in linguistic context of the present invention, term " placenta mesenchyma stem cell " can exchange and use with " placenta stem-cell ", " stem cell ", " mescenchymal stem cell ", unless separately had clearly and indicated.
In the present invention, term " PBS damping fluid " or " PBS " refer to phosphate buffered saline buffer.Those skilled in the art know the generality formula of the PBS using under situation of the present invention and compound method and their general aspects for example pH value or pH scope.
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 special culture media that mescenchymal stem cell is cultivated.
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 still have a large amount of stem cells after perfusion, be trapped in placenta tissue, can not be effectively 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 profit is preserved in this way placenta mesenchyma stem cell and is set up placenta stem-cell storehouse.The present inventor, summing up on the basis of separation and Culture mescenchymal stem cell in the past, utilizes Various Tissues digestive ferment mixture slaking placental lobules tissue block, and in conjunction with stationary culture, success separation in placenta obtains a large amount of mescenchymal stem cells.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 in placenta is inmature compared with adult stem cell, 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 placenta stem-cell storehouse, for further investigation and the clinical treatment of stem cell lay the foundation later.
Owing to containing abundant hemopoietic stem cell in 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 to be chilled in the medium-term and long-term preservation of profound hypothermia liquid nitrogen of-196 degrees Celsius, set up placenta stem-cell storehouse, for stem-cell therapy is in the future preserved seed.
The object of this invention is to provide simply a large amount of separating mesenchymal stem cells 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, with PBS damping fluid, fully rinse placental lobules and remove residual blood in placenta.Again placental lobules is cut into 1cm
3the tissue block of size, add contain Various Tissues digestive ferment PBS damping fluid at 37 ℃, hatch 15 minutes, tissue block is filtered and with syringe, to be ground simultaneously with copper mesh.Collect the separated mononuclearcell of density gradient centrifugation for liquid after filtration, after washing, with the cell that the suspension of MSC substratum obtains, be put into 37 ℃ of 5%CO
2in incubator, cultivate.After disseminated cell forms clone, each clone cell is chosen with MSC substratum and cultivated respectively, after cell 80～90% merges, with trysinization, go down to posterity, by 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 to molecular genetics diagnosis, preserve all related datas of cell, set up the database of placenta stem-cell and carry out associated with freeze-stored cell.
The method according to this invention, for example, according to the method for the embodiment of the present invention 1,2, weight is that the placenta of 750 grams can get 8 * 10
9mononuclearcell, through adherent culture average 1 * 10
6in mononuclearcell, can obtain 12 clones, after evaluation, on average have 3 clones to there is the characteristic of mescenchymal stem cell, in 750g placenta, can get 24000 mescenchymal stem cells.These stem cells can reach Cell Biology Experiment and clinical treatment desired number very soon through external Short-term Culture.Yet the inventor finds, according to the method for embodiment in CN1548529A mono-, to be that the placenta of 750 grams is approximately separable obtain 3000 mescenchymal stem cells for weight, and after 3 generations went down to posterity, cell purity is greater than and is about 95%.In one embodiment of the invention, in PBS damping fluid described in step (b), comprise about 0.1mg/mL dispase, about 0.25mg/mL pancreatin, about 0.25mg/mL DNase I, about 1mg/mL collagenase IV and about 1mg/mL Unidasa; The inventor finds 5 kinds of enzymes in this PBS damping fluid, and while 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 above-mentioned 750g placenta far away; In addition, the concentration of 5 kinds of enzymes is done suitably to change, particularly lower than above-mentioned concentration separately 50% or higher than above-mentioned concentration separately 200% time, mescenchymal stem cell yield and cell purity are all obviously poor than the result that can get 24000 mescenchymal stem cells in above-mentioned 750g placenta; For example the dispase concentration in PBS damping fluid be 0.04mg/mL (lower than the above-mentioned concentration of the present invention 50%) or for 0.25mg/mL (higher than the above-mentioned concentration of the present invention 200%) time, in 750g placenta, approximately can get 8000 mescenchymal stem cells and cell minuent 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 described PBS damping fluid.
The present invention is simple to operate, convenient and practical, can obtain a large amount of mescenchymal stem cells, and 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 the separated placenta of perfusion method, adherent culture acquisition.It is few that this method is got cell quantity, and donor is being got the possibility that all has infection after marrow is got in marrow neutralization.The present invention's success is separated in placenta obtains the higher mescenchymal stem cell of a large amount of purity, and uses this method to set up 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 inmatureer, and wide material sources are conveniently easy to get, and therefore method of the present invention will have prospect widely in the clinical application of stem cell.
Accompanying drawing explanation
Fig. 1 is the morphological observation of cell growth under microscope.Wherein: A is for cultivating the attached cell being dispersed in as seen afterwards for 3 days; B forms clone for 7～10 days; 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 in each figure " counts " represents 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 for cultivating the DNA content of 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 in each figure " counts " represents count number, and X-coordinate mark " DNA Content " represents 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
By the following examples, can conduct further description the present invention, yet scope of the present invention is not limited to following embodiment.One of skill in the art can understand, and is not deviating under the prerequisite of 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 test.Although be well known in the art for realizing many materials and the working method that the object of the invention used, 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, with the PBS damping fluid containing 10% volume FBS, fully rinse placental lobules and remove residual blood in 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 appropriate FBS to stop digestion.Again tissue block and cell suspension one are reinstated to 200 order copper mesh and filtered and use syringe piston tissue abrasion piece simultaneously, collect cell suspension after filtering and join in centrifuge tube 1000rpm centrifugal 10 minutes, outwell supernatant, with the PBS damping fluid re-suspended cell containing 10%FBS.Then use density gradient centrifugation separated and collected mononuclearcell, the cell that suspends and obtain with MSC substratum after 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.Finally culturing bottle is put into 37 ℃ of 5%CO
2in incubator, cultivate.After 72 hours, full dose is changed liquid, removes non-adherent cell, has the shuttle type attached cell being dispersed in culturing bottle, then adds fresh MSC nutrient solution to continue to cultivate.
the cultivation and frozen of going down to posterity of embodiment 2, placenta MSC
After approximately 10 days, wait being dispersed in attached cell, form after clone, with 0.05% trypsinase/2mMEDTA digestion, after cell counting, according to 3000 cells/cm
2the cultivation of going down to posterity.Afterwards, when cell reaches 70% left and right and merges, had digestive transfer culture is cultivated.After digestion, get 3 * 10
6cell joins in 1ml cells frozen storing liquid (containing 50% low sugar DMEM nutrient solution, 40%FBS, 10% dimethyl sulfoxide (DMSO)), finally enters into liquid nitrogen pipe frozen through programmed cooling.
the Identification of Biological Characteristics of embodiment 3, placenta MSC
1, Growth of Cells and Morphological Characteristics thereof
By the separation and Culture of embodiment 1 and embodiment 2, placenta mononuclearcell was cultivated after 72 hours can obviously see fusiformis attached cell (Figure 1A) under the microscope, about 10 days, turbine-like cell clone (Figure 1B, Fig. 1 D) can be formed, after had digestive transfer culture, the adherent layer (Fig. 1 C, Fig. 1 E, Fig. 1 F) that merge 80% left and right can be formed.In culturing process, find the relative homogeneous of this cellular form, rate of propagation is fast, and adherent speed is fast, and easily by trysinization, more than being passaged to for 15 generations, 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, Flow cytometry cell surface marker, dynamically observes the variation of cell surface marker in culturing process.Digestion collecting cell, gets 8 * 10 after counting
6individual cell, packing 16 pipes; PBS washes once, the centrifugal 10min of 1500rpm; Abandon supernatant, residual 100～200 μ l, piping and druming mixes 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 to establish a pipe be blank; At 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 to mix cell, and 1% paraformaldehyde of 200 μ l is fixed, put 4 ℃ to be measured, 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 hematopoietic cell surface marker is CD14, CD31, CD34 (HSPC and endotheliocyte are positive), CD45 (white corpuscle is positive), CD54 (ICAM-1), CD80 (B7-1), the lasting feminine gender of CD86 (B7-2), HLA-DR (MHC-II quasi-molecule), CD29 and CD44 (acceptor of scleroproein and transparency grease hydrochlorate, stroma cell is expressed), CD73 (being 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 in 3 generations, went down to posterity above, cellular constituent homogeneous, purity is more than 95%.(Fig. 2)
3, the cell cycle of Flow cytometry placenta MSC
When cell grows to 80% left and right and merges, digestion collecting cell approximately 1 * 10
6individual, PBS washes once, adds 70% ethanol to fix, and 4 ℃ to be measured.During detection, the first centrifugal ethanol that goes, then wash once with PBS, add RNase I 500u, 37 ℃ of reaction 30min, 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%.Result shows that the cell of vitro culture has typical stem cells hyperplasia feature, only has a few cell 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, digestion counting, makes cell suspension (2 * 104/ml) with the LG-DMEM substratum of 10%FBS, every hole inoculation 0.5ml in 24 orifice plates, 37 ℃, 5%CO
2, under saturated humidity, cultivate.Get 3 multiple holes every day, living cell counting number after Trypan Blue, calculating mean value, Continuous Observation 7 days.Take incubation time as transverse axis, and cell count is the longitudinal axis, draws cell growth curve.With Patterson formula, calculate cell in the doubling time of logarithmic phase, i.e. Td=Tlg2/Lg (Nt/No), Td: doubling time (h), T: cell increases to Nt time used (h), N: cell count by No.
By Cytometric result drafting every day cell growth curve, calculate the doubling time.By cell growth curve, can be found out, cell at 2-4 days in exponential phase of growth.According to formula calculate the 5th generation cell in the doubling time of exponential phase of growth, be respectively 22.6h.(Fig. 4)
5, the evaluation of placenta MSC multi-lineage potential
(1) osteogenic induction
3 above MSC of generation, by 1 * 10
5six orifice plates are inoculated in/hole, are put in 37 ℃, 5%CO
2, under saturated humidity, in MSC substratum, cultivate after 24h, use instead containing 10% DMEM-HG through 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, within every 3 days, half amount is changed liquid, coinduction 2-4 week.Alkaline phosphatase staining identifies that scleroblast forms, and Von Kossa dyeing identifies that bone tubercle forms.
Containing 10% DMEM-HG through screening FBS, add dexamethasone 0.1 μ M, ascorbyl phosphate 50 μ M, β-phospho-glycerol 10mM to cultivate 1 week, cellular form occurs significantly to change, from fusiform inoblast sample, become polygon, be similar to neuronal cell sample, cell periphery occurs that long filament shape is outstanding, and can extend towards periphery.Continue to cultivate 2 weeks above after, in cell matrix, there is calcified plaque, mineralizer engenders, and starts to form the little junction structure of multilayer, to cultivating after 4 weeks, visible obvious calcification tubercle.In the time of 2 weeks, alkaline phosphatase staining is strong positive reaction, reaches more than 95%, and the control group of not induced is most of negative, only less than 5%, is shown as the weak positive, shows that cell transforms to scleroblast.Von Kossa dyeing can be dyed black by the calcium depositing in bone tubercle, and the visible a large amount of black bone tubercle of induction group, have obvious three-dimensional arrangement, and control group does not all have positive reaction at any time.(Fig. 5)
(2) Adipogenic induction
3 above MSC of generation, by 1 * 10
5/ hole is inoculated in six orifice plates, is put in 37 ℃, 5%CO
2, under saturated humidity, in MSC substratum, cultivate after 24h, use instead containing 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, under saturated humidity, cultivate, within every 3 days, half amount is changed liquid, coinduction 2 weeks, oil red dyeing identifies that fat drips formation.
Containing 10% DMEM-HG through screening FBS, add dexamethasone 1 μ M, INDOMETHACIN 200 μ M, IBMX 0.5mM, Regular Insulin 10 μ g/ml to cultivate 3 days, there is form and change in cell, by fusiform inoblast sample, is shunk and shorten gradually, and 90% above cell becomes cube or polygon; Cultured continuously 7 days, has small fat to ooze existing in visible cell under mirror, along with the prolongation of incubation time, fat drips and increases gradually and merge, and when cultivating 2 weeks, merges as seen agglomerating fat and drips and be full of whole cell.The fat producing in oil red O stain visible cell is dyed redness by specificity.(Fig. 6)
(3) become chondrocyte induction
3 generation above cell, according to every pipe 2 * 10
5cell divides and installs to 15ml polypropylene centrifuge tube, low-speed centrifugal makes cell in test tube, form micelle, 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 saturated humidity, within every 3 days, half amount is changed liquid, cultured continuously 2 weeks.
Induce after 2 weeks cell micelle is broken up to smear, dye visible II Collagen Type VI of alcian blue (Alcian blue) forms extracellular matrix and is blue, and control group dyes without indigo plant.(Fig. 7)
6, RT-PCR detects placenta MSC multi-lineage potential
Collect the cell after induction, application Trizol reagent extracts cell total rna, and what take carries out RT-PCR as template, and reverse transcription and PCR operation are carried out according to RT-PCR test kit specification sheets, and primer sequence is as shown in table 1.
Table 1.RT-PCR primer sequence and specificity thereof
In Fig. 8, be respectively from left to right: placenta MSC cell, induction become adipocyte, induction is that scleroblast, induction are chondrocyte's RT-PCR electrophoresis photo, wherein from left to right, Normal swimming lane is the front cellular gene expression situation of induction, and Adipogenic swimming lane, Osteogenic swimming lane, Chondrogenic swimming lane are cellular gene expression situation after inducing.Result shows, external evoked rear cell can be expressed serial specific mrna: cell expressing PPAR-γ after Adipogenic induction, cell expressing osteopontin (Osteopontin) after osteogenic induction, cell expressing collagen I I (Collagen II) after one-tenth chondrocyte induction, illustrate that resulting MSC cell has skeletonization, becomes fat, becomes cartilage differentiation ability, meets generally acknowledged MSC standard.
By the detection of above a series of data targets, demonstrate the MSC that the separation of application the inventive method obtains, have to the ability of scleroblast, adipocyte, Chondrocyte Differentiation, the MSC that proved inventive method obtains has 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 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 associated.
Claims (1)
1. the method for separating mesenchymal stem cell from placenta, the method comprises the following steps:
(a) get placental lobules, with PBS damping fluid, fully rinse, to remove blood residual in placenta;
(b) placental lobules is cut into bulk, adds the PBS damping fluid that contains tissue digestion enzyme, then hatch digestion in 10 ~ 20 minutes at 37 ℃;
(c) tissue block is filtered with copper mesh, grind to impel filtration;
(d) filtered liquid of collecting is centrifugal, separated mononuclearcell, then the cell that suspends and obtain with MSC substratum, then at 37 ℃, 5%CO
2in incubator, cultivate;
(e) after disseminated cell forms clone, each clone cell of picking, cultivates respectively with MSC substratum, after cytogamy, with trysinization, goes down to posterity, and obtains 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 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 it is associated that this database and the freeze-stored cell of step (g) are carried out,
Wherein, in step (b), in described PBS damping fluid, comprise 0.1 mg/mL dispase, 0.25 mg/mL pancreatin, 0.25 mg/mL DNase I, 1 mg/mL collagenase IV and 1 mg/mL Unidasa.
2. according to the method for claim 1, wherein said step (a) is within four hours postpartum, under aseptic condition, placental lobules is cut, with the PBS damping fluid containing 10% volume foetal calf serum, fully rinsed placental lobules and remove residual blood in placental lobules.
3. according to the process of claim 1 wherein in step (b), at 37 ℃, hatch 10 minutes.
4. according to the method for claim 1, in step (b), at 37 ℃, hatch 20 minutes.
5. according to the method for claim 1, in step (d), described MSC substratum is the PBS damping fluid containing 10%FBS.
6. according to the method for claim 1, in step (e), after disseminated cell forms clone, each clone cell of picking, cultivates respectively with MSC substratum, after cell 60～90% merges, with trysinization, goes down to posterity, and obtains placenta mesenchyma stem cell.
7. according to the method for claim 1, in step (h), described database comprises and all relevant data of preserved cell, comprising: the biological characteristics detected result of cell, multi-lineage potential qualification result, fetus and father and mother's thereof detail file.
8. according to the method for claim 1 to 7 any one, the cell purity of the placenta mesenchyma stem cell that wherein obtained is greater than 95%.
9. according to the method for claim 1, the method comprises the following steps:
Within four hours postpartum, under aseptic condition, placental lobules is cut, with the PBS damping fluid containing 10% volume FBS, fully rinsed placental lobules and remove residual blood in placental lobules;
First placental lobules is cut into 1cm
3the tissue block of size, join in the PBS damping fluid that contains 0.1 mg/mL dispase, 0.25 mg/mL pancreatin, 0.25 mg/mL DNase I, 1 mg/mL collagenase IV, 1 mg/mL Unidasa 37 ℃ of digestion 15 minutes, add appropriate FBS to stop digestion;
Again tissue block and cell suspension one are reinstated to 200 order copper mesh and filtered and use syringe piston tissue abrasion piece simultaneously, collect cell suspension after filtering and join in centrifuge tube 1000rpm centrifugal 10 minutes, outwell supernatant, with the PBS damping fluid re-suspended cell containing 10%FBS;
Then use density gradient centrifugation separated and collected mononuclearcell, the cell that suspends and obtain with MSC substratum after PBS damping fluid washing mononuclearcell, the density of inoculation mononuclearcell is the every 75cm of Tissue Culture Flask internal surface area
21 * 10
7mononuclearcell is 10ml MSC substratum altogether;
Finally culturing bottle is put into 37 ℃ of 5%CO
2in incubator, cultivate, after 72 hours, full dose is changed liquid and is removed non-adherent cell, has the shuttle type attached cell being dispersed in culturing bottle, adds fresh MSC nutrient solution to continue to cultivate;
In the time of 10 days, be dispersed in attached cell and form after clone, with 0.05% trypsinase/2mM EDTA digestion, after cell counting, according to 3000 cells/cm
2the cultivation of going down to posterity;
Afterwards, when cell reaches 70% fusion, had digestive transfer culture is cultivated, and obtains placenta mesenchyma stem cell;
For gained placenta mesenchyma stem cell, detect at least one item of following items: cytoactive, HLA-ABC/DR join type;
Placenta mesenchyma stem cell after gained is gone down to posterity is frozen in liquid nitrogen;
The database of the placenta stem-cell that foundation comprises above information, and make this database carry out associated with freeze-stored cell.
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| CN102586184B (en) * | 2011-09-05 | 2013-10-23 | 博雅干细胞科技有限公司 | Method for establishing placental mesenchyme stem cell library |
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| CN101161249B (en) * | 2006-10-12 | 2011-07-20 | 周胜利 | Method for extracting original mesenchyma and hematopoiesis trunks/ancestral cell from caesarean birth placenta |
| CN102586184B (en) * | 2011-09-05 | 2013-10-23 | 博雅干细胞科技有限公司 | Method for establishing placental mesenchyme stem cell library |
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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 |