CN102676451A - 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 human mescenchymal stem cell is separated from marrow the earliest; Derive from mesoblastic one type of tissue stem cell with multidirectional differentiation potential and self ability; Has ability (Caplan AI.Mesenchymal stem cells.J Orthop Res.1991 in vivo with under the external specified conditions to multiple adult cytodifferentiation such as scleroblast, chondrocyte, adipocyte, endotheliocyte, neurocyte, myocyte, liver cells; 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 effect, and is easy to foreign gene importing expression.Therefore the seed cell of mescenchymal stem cell during still tissue-engineered bone, cartilage and cardiac muscle make up; Important carrier cell in the gene therapy; And, in HSCT and organ transplantation, be with a wide range of applications because mescenchymal stem cell promotes hematopoietic reconstitution and the anti-host response function of inhibition of transplant.Mescenchymal stem cell has the characteristic of external adherent growth, utilizes this specific character, people successfully from multiple tissues such as liver, kidney, pancreas, muscle, cartilage, skin, peripheral blood separation and Culture go out mescenchymal stem cell.
The mescenchymal stem cell of being reported at present is mainly derived from marrow, adopts density gradient centrifugation to obtain.Though separation method is easy, donor is got marrow need experience a relatively more painful operation, and has very high infection chance in the process of drawing materials and after drawing materials; Because the content of MSC is extremely rare among the human bone marrow, per 10
5～10
6Approximately have only 1 in the individual mononuclearcell, and along with the increase at age, the quantity of mescenchymal stem cell, propagation and differentiation capability descend significantly all in the marrow, make it in research with use in the especially clinical application and be restricted.Originate from the placenta of embryonic development period extraembryonic mesoderm and form, contain a large amount of mesenchyme compositions by a matter, blood vessel and nurse cell.Up-to-date research shows and contains abundant stem cell in the placenta, and separation and Culture goes out these multipotential stem cells and will open up a brand-new and abundant source for experimental study and clinical application from placenta.
The existing method that thereby separate stem cells is set up the placenta stem-cell storehouse from placenta still has many shortcomings, and for example purity is not enough and/or quantity is not high, and then demonstrates the expectation that these methods still can not satisfy people.The for example invention of CN 101270349A (one Chinese patent application numbers 200810061267.6, open day on September 24th, 2008) disclosed being entitled as " placenta mesenchyma stem cell separates and the amplification in vitro cultural method "; The invention of CN 101693884A (one Chinese patent application numbers 200910117522.9, open day on April 14th, 2010) disclosed being entitled as method of separating and extracting stem cells " a kind of from placenta, umbilical cord or fatty tissue "; CN 102146359A (one Chinese patent application numbers 201110005964.1, open day on August 10th, 2011) disclosed being entitled as " extracted the method for primary mesenchymal stem cells and serum-free amplification " from placenta invention.These methods are further improved remaining aspect the purity of extract and/or the recovery.
This area still need new from placenta the method, the particularly method of separating mesenchymal stem cell from placenta of separate stem cells.
Summary of the invention
The objective of the invention is to solve the existing defective of obtaining the placenta mesenchyma stem cell method, simply a large amount of separating mesenchymal stem cells and randomly 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 height that is obtained and/or the cell recovery are 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 this method may further comprise the steps:
(a) get placental lobules, fully wash, to remove residual blood in the placenta with the PBS damping fluid;
(b) placental lobules is cut into bulk, adds the PBS damping fluid that contains the tissue digestion enzyme, hatch digestion at 37 ℃ again;
(c) tissue block is filtered with copper mesh, grind in case of necessity to impel filtration;
(d) filtered liq of collecting is centrifugal, separate mononuclearcell, the cell that suspends and obtained with the MSC substratum again is then at 37 ℃, 5%CO
2Cultivate in the incubator;
(e) treat that disseminated cell forms the clone after, each clone cell of picking is cultivated respectively with the MSC substratum, treat cytogamy after, go down to posterity with trysinization, promptly get placenta mesenchyma stem cell;
And optional following one or more steps:
(f) to step (e) gained placenta mesenchyma stem cell, detect at least one item of following project: 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 DB of the placenta stem-cell that comprises above information, and make this DB carry out related with the freeze-stored cell of step (g).
According to the method for first aspect present invention, wherein said step (a) is under aseptic condition, placental lobules to be cut, and fully washes placental lobules with the PBS damping fluid and removes residual blood in the placenta.
Method according to first aspect present invention; Wherein said step (a) is within four hours postpartum; Under aseptic condition, placental lobules is cut, fully washed placental lobules with the PBS damping fluid that contains 10% volume foetal calf serum (FBS) and remove residual blood in the placental lobules.
According to the method for first aspect present invention, the tissue digestion enzyme in the wherein said step (b) is to be selected from following one or more: dispase, pancreatin, deoxyribonuclease I (DNase I), collagenase IV, Unidasa.In one embodiment, the tissue digestion enzyme in the said step (b) comprises: dispase, pancreatin, deoxyribonuclease I (DNase I), collagenase IV, Unidasa.In one embodiment, in step (b), 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 in the said PBS damping fluid.
According to the method for first aspect present invention, in step (b), hatched 10～30 minutes at 37 ℃, preferred 10～20 minutes, for example 10 minutes, 15 minutes or 20 minutes.
According to the method for first aspect present invention, in step (b), placental lobules is cut into about 1cm
3The tissue block of size.
According to the method for first aspect present invention, in step (c), tissue block is ground with syringe with the copper mesh filtration simultaneously.In one embodiment, in step (c), tissue block and cell suspension one are reinstated 160～240 orders (preferred 200 orders) copper mesh filter and use syringe piston tissue abrasion piece simultaneously.
According to the method for first aspect present invention, in step (d), said MSC substratum is the PBS damping fluid that contains 10%FBS.In an embodiment aspect arbitrary of the present invention, said MSC substratum is the PBS damping fluid that contains 10%FBS.In one embodiment, in step (d), the filtered liq of collecting is separated mononuclearcell with density gradient centrifugation, washing, the cell that suspends and obtained with the MSC substratum again is then at 37 ℃, 5%CO
2Cultivate in the incubator.In one embodiment, in step (d), the cell suspension of collecting after filtering is joined in the centrifuge tube, centrifugal 10 minutes of 1000rpm outwells supernatant, with the PBS damping fluid re-suspended cell that contains 10%FBS.
According to the method for first aspect present invention, in step (e), treat that disseminated cell forms the clone after, each clone cell of picking is cultivated respectively with the MSC substratum, treat cell 60～90% fusions after, go down to posterity with trysinization, promptly get placenta mesenchyma stem cell.In one embodiment, in step (e), after being dispersed in attached cell formation clone, with 0.05% trypsinase/2mM EDTA digestion, the cultivation of going down to posterity.
According to the method for first aspect present invention, in step (f), it is to utilize the trypan blue staining to count the number of frozen front and back viable cell that said cytoactive detects.
According to the method for first aspect present invention, in step (f), said cell contamination detects and utilizes small amounts of cells to cultivate, and detects cell and whether receives the pollution of fungi and bacterium.In one embodiment; Whether it is to utilize the etiology method that said cell contamination detects, detect cell and receive and be selected from following one or multinomial infection: hepatitis B two double, 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 said 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 said HLA-ABC/DR joins type.
According to the method for first aspect present invention, in step (g), said placenta mesenchyma stem cell is frozen in liquid nitrogen through the programmed cooling process.
According to the method for first aspect present invention, in step (g), said placenta mesenchyma stem cell is present in the cells frozen storing liquid.In one embodiment, this cells frozen storing liquid comprises 50% low sugar DMEM nutrient solution, 40%FBS, 10% DMSO 99.8MIN..
Method according to first aspect present invention; In step (h); Comprise in the said DB and all relevant data of the cell of being preserved, include but not limited to: the biological characteristics detected result of cell, multidirectional differentiation 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, this method may further comprise the steps: under aseptic condition, placental lobules is cut, fully washed placental lobules with the PBS damping fluid and remove residual blood in the placenta.Again placental lobules is cut into 1cm
3The tissue block of size, add contain multiple tissue digestion enzyme the PBS damping fluid hatched 15 minutes at 37 ℃, tissue block is filtered with copper mesh grinds with syringe simultaneously.The liquid of collecting after filtering separates mononuclearcell with density gradient centrifugation, and the washing back is put into 37 ℃ of 5%CO with the cell that the suspension of MSC substratum is obtained
2Cultivate in the incubator.After treating that disseminated cell forms the clone, each clone cell chosen with the MSC substratum cultivate respectively, after cell 80～90% fusions, go down to posterity, promptly get placenta mesenchyma stem cell with trysinization.With the frozen and relevant fetus information of record in liquid nitrogen of the cell after going down to posterity; And the biological characteristics and the multidirectional differentiation potential that carry out cell are identified; And pair cell carries out the molecular genetics diagnosis; Preserve all related datas of cell, set up the DB of placenta stem-cell and carry out related with freeze-stored cell.
According to the method for first aspect present invention, this method may further comprise the steps: under aseptic condition, placental lobules is cut within four hours postpartum, fully washed placental lobules with the PBS damping fluid that contains 10% volume FBS and remove residual blood in the placental lobules.Earlier placental lobules is cut into 1cm
3The tissue block of size joins in the PBS damping fluid that contains 0.1mg/mL dispase, 0.25mg/mL pancreatin, 0.25mg/mL DNase I, 1mg/mL collagenase IV, 1mg/mL Unidasa 37 ℃ of digestion 15 minutes, adds an amount of FBS and stops digestion.Again tissue block and cell suspension one are reinstated 200 order copper mesh and filter and use syringe piston tissue abrasion piece simultaneously, collect cell suspension after filtering and joined in the centrifuge tube 1000rpm centrifugal 10 minutes, outwell supernatant, with the PBS damping fluid re-suspended cell that contains 10%FBS.Use density gradient centrifugation separated and collected mononuclearcell then, the cell that suspends and obtained with the MSC substratum behind the PBS damping fluid washing mononuclearcell, the density of inoculation mononuclearcell is every 75cm
2(Tissue Culture Flask internal surface area) 1 * 10
7Mononuclearcell is 10ml MSC substratum altogether.At last culturing bottle is put into 37 ℃ of 5%CO
2Cultivate in the incubator.Full dose is changed liquid and is removed non-adherent cell after 72 hours, has the shuttle type attached cell that is dispersed in the culturing bottle, adds fresh MSC nutrient solution and continues to cultivate.About 10 days, after being dispersed in attached cell and forming the clone, digest with 0.05% trypsinase/2mM EDTA, after the cell counting, according to 3000 cells/cm
2The cultivation of going down to posterity; Afterwards, had digestive transfer culture is cultivated when about cell reaches 70%, merging, and promptly gets placenta mesenchyma stem cell.
In addition, in the first aspect present invention method, a kind of placenta mesenchyma stem cell is provided.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 the said method of 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, said placenta mesenchyma stem cell is after going down to posterity more than 3 generations, and cell purity is greater than 95%.
Be further described in the face of the present invention down.The document that the present invention quoted, and the document of being quoted in the document, their full content is incorporated this paper by reference into.
In the present invention; In arbitrary technical scheme of the arbitrary aspect of the present invention; Its arbitrary technical characterictic is equally applicable to arbitrary embodiment of arbitrary aspect of the present invention, as long as they can not cause contradiction, and this being useful in each other in case of necessity can be done suitable modification.
In the present invention, term " placenta mesenchyma stem cell " is meant the mescenchymal stem cell that derives from placenta.Therefore in the present invention, particularly relate in the linguistic context of the present invention, term " placenta mesenchyma stem cell " can exchange with " placenta stem-cell ", " stem cell ", " mescenchymal stem cell " and use, and indicates only if having clearly in addition.
In the present invention, term " PBS damping fluid " perhaps " PBS " be meant phosphate buffered saline buffer.Generality prescription and compound method and their general aspects that those skilled in the art know the PBS that under situation of the present invention, uses be pH value or pH scope for example.
In the present invention, term " placenta " is meant newborn infant's placenta, is meant the placenta within 4 hours postpartum especially.
In the present invention, term " MSC substratum " is meant the special culture media that mescenchymal stem cell is cultivated.
Contriver of the present invention once utilized perfusion method separation and Culture mescenchymal stem cell from placenta, had obtained the very high mescenchymal stem cell of purity.But be trapped in the placenta tissue through still having a large amount of stem cells behind the perfusion, can not be separated effectively.Therefore can think, adopt perfusion method can not obtain mescenchymal stem cell to greatest extent.
The invention discloses a kind of from placenta the methods of a large amount of separating mesenchymal stem cells, and utilize this method to preserve placenta mesenchyma stem cell and set up the placenta stem-cell storehouse.Contriver of the present invention utilizes multiple tissue digestion enzyme mixture slaking placental lobules tissue block summing up on the basis of separation and Culture mescenchymal stem cell in the past, and in conjunction with the adherent culture method, success separates in placenta and obtaining a large amount of mescenchymal stem cells.The mescenchymal stem cell purity that the inventive method obtains is high, quantity is many, has the biological characteristics identical with mesenchymal stem cells MSCs, can be to differentiation such as scleroblast, chondrocyte, adipocyte, endotheliocyte, neurocyte.Because stem cell is inmature than adult stem cell in the placenta; Content is abundant; Be with a wide range of applications clinically; We use conventional cell cryopreservation method that mescenchymal stem cell is frozen as bleeding of the umbilicus, set up the placenta stem-cell storehouse, for further investigation and the clinical treatment of stem cell lay the foundation later on.
Because contain abundant hemopoietic stem cell in the bleeding of the umbilicus, people set up unbilical blood bank and store these important Biological resources of umbilical hemopoietic stem cell, for multiple disease in the blood system and disease of immune system provide a kind of treatment means.Same placenta mesenchyma stem cell is as a kind of more importantly stem cell resource; We use conventional cell cryopreservation method it to be chilled in-196 degrees centigrade medium-term and long-term preservation of profound hypothermia liquid nitrogen; Set up the placenta stem-cell storehouse, for stem-cell therapy is in the future preserved seed.
The purpose of this invention is to provide simply a large amount of separating mesenchymal stem cells and set up the method in placenta stem-cell storehouse from placenta of a kind of practicality; Comprise the steps: under aseptic condition, placental lobules to be cut, fully wash placental lobules with the PBS damping fluid and remove residual blood in the placenta.Again placental lobules is cut into 1cm
3The tissue block of size, add contain multiple tissue digestion enzyme the PBS damping fluid hatched 15 minutes at 37 ℃, tissue block is filtered with copper mesh grinds with syringe simultaneously.The liquid of collecting after filtering separates mononuclearcell with density gradient centrifugation, and the washing back is put into 37 ℃ of 5%CO with the cell that the suspension of MSC substratum is obtained
2Cultivate in the incubator.After treating that disseminated cell forms the clone, each clone cell chosen with the MSC substratum cultivate respectively, after cell 80～90% fusions; Go down to posterity with trysinization; With the frozen and relevant fetus information of record in liquid nitrogen of the cell after going down to posterity, and carry out the biological characteristics and the evaluation of multidirectional differentiation potential of cell, and pair cell carries out the molecular genetics diagnosis; Preserve all related datas of cell, set up the DB of placenta stem-cell and carry out related with freeze-stored cell.
According to the method for the invention, for example according to the method for the embodiment of the invention 1,2, the placenta that weight is 750 grams can get 8 * 10
9Mononuclearcell is through adherent culture average 1 * 10
6Can obtain 12 clones in the mononuclearcell, on average have 3 clones to have the characteristic of mescenchymal stem cell after the evaluation, promptly can get 24000 mescenchymal stem cells in the 750g placenta.These stem cells can reach Cell Biology Experiment and clinical treatment desired number very soon through external Short-term Culture.Yet, inventor's discovery, according to the method for embodiment among the CN1548529A one, about separable 3000 mescenchymal stem cells that obtain of placenta that weight is 750 grams, after 3 generations went down to posterity, cell purity was greater than being about 95%.In one embodiment of the invention, in PBS damping fluid described in the step (b), comprise 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 when lacking any one or more, mescenchymal stem cell yield and cell purity all do not reach the result that can get 24000 mescenchymal stem cells in the above-mentioned 750g placenta far away; In addition; The concentration of 5 kinds of enzymes is done suitably to change; Particularly be lower than above-mentioned concentration separately 50% or be higher than 200% o'clock of above-mentioned concentration separately, mescenchymal stem cell yield and cell purity are all obvious than getting the poor as a result of 24000 mescenchymal stem cells in the above-mentioned 750g placenta; For example when the dispase concentration in the PBS damping fluid be 0.04mg/mL (be lower than the above-mentioned concentration of the present invention 50%) or during for 0.25mg/mL (be higher than the above-mentioned concentration of the present invention 200%), can get 8000 mescenchymal stem cells approximately in the 750g placenta and the cell minuent is lower than 90%.Therefore; One embodiment of the invention are in step (b), comprise 0.05～0.2mg/mL dispase, 0.125～0.5mg/mL pancreatin, 0.125～0.5mg/mL DNase I, 0.5～2mg/mL collagenase IV and 0.5～2mg/mL Unidasa in the said PBS damping fluid.
The present invention is simple to operate, and is convenient and practical, can obtain a large amount of mescenchymal stem cells, and the differentiation performance is good, has the ability to cytodifferentiation such as scleroblast, adipocyte, chondrocyte, endotheliocyte, neurocyte.Comparison with existing method: MSC mainly adopts modus operandi extraction donor marrow or perfusion method to separate placenta at present, and adherent culture obtains.It is few that this method is got cell quantity, and donor is being got the possibility that infection is all arranged after marrow is got in the marrow neutralization.The present invention's success separates the higher mescenchymal stem cell of a large amount of purity of acquisition in placenta, and uses this method to set up the placenta stem-cell storehouse and lay in this stem cell that has application prospect.This method is simple and easy to do, and because placenta is the same with bleeding of the umbilicus, the cell composition is inmature, and wide material sources conveniently are easy to get, and therefore method of the present invention will have broad application prospect in the clinical application of stem cell.
Description of drawings
Fig. 1 is the morphological observation of microscopically cell growth.Wherein: A cultivates the visible attached cell that is dispersed in after 3 days; B is 7～10 days formation clones; C is for to form fine and close attached cell through screening and cloning; D, E, F are the dyeing of Rui Shi Ji's nurse Sa.
Fig. 2 is flow cytometry identification of M SC surface marker result.Ordinate zou " counts " expression count number among each figure.
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 of cell is in (G stationary phase
0/ G
1Phase, 96.66%), few cell is in the propagation phase (S phase, 3.25%).P6 is for cultivating the dna content of hexabasic cell, and in the analysis of cells cycle, visible most of cell is in (G stationary phase
0/ G
1Phase, 96.35%), few cell is in the propagation phase (S phase, 2.54%).Ordinate zou " counts " expression count number among each figure, X-coordinate mark " DNA Content " expression dna content.
Fig. 4 is placenta MSC cell growth curve figure.
Fig. 5 is the osteogenic induction cytological map that the multidirectional differentiation potential of placenta MSC is identified.
Fig. 6 is the one-tenth fat inducing cell figure that the multidirectional differentiation potential of placenta MSC is identified.
Fig. 7 is the one-tenth chondrocyte induction cytological map that the multidirectional differentiation potential of placenta MSC is identified.
Fig. 8 detects the electrophoresis photo of the multidirectional differentiation potential of placenta MSC for RT-PCR.
Embodiment
Can further describe the present invention through following embodiment, yet scope of the present invention is not limited to following embodiment.One of skill in the art can understand, and under the prerequisite that does not deviate from the spirit and scope of the present invention, can carry out various variations and modification to the present invention.The present invention carries out generality and/or concrete description to the material and the TP that are used in the test.Though for realizing that employed many materials of the object of the invention and working method are well known in the art, the present invention still does detailed as far as possible description at this.
The separation of embodiment 1, placenta MSC
Within four hours postpartum, under aseptic condition, placental lobules is cut, fully wash placental lobules with the PBS damping fluid that contains 10% volume FBS and remove residual blood in the placental lobules.Earlier placental lobules is cut into 1cm
3The tissue block of size joins in the PBS damping fluid that contains 0.1mg/mL dispase, 0.25mg/mL pancreatin, 0.25mg/mL DNase I, 1mg/mL collagenase IV, 1mg/mL Unidasa 37 ℃ of digestion 15 minutes, adds an amount of FBS and stops digestion.Again tissue block and cell suspension one are reinstated 200 order copper mesh and filter and use syringe piston tissue abrasion piece simultaneously, collect cell suspension after filtering and joined in the centrifuge tube 1000rpm centrifugal 10 minutes, outwell supernatant, with the PBS damping fluid re-suspended cell that contains 10%FBS.Use density gradient centrifugation separated and collected mononuclearcell then, the cell that suspends and obtained with the MSC substratum behind the PBS damping fluid washing mononuclearcell, the density of inoculation mononuclearcell is every 75cm
2(Tissue Culture Flask internal surface area) 1 * 10
7Mononuclearcell is 10ml MSC substratum altogether.At last culturing bottle is put into 37 ℃ of 5%CO
2Cultivate in the incubator.Full dose is changed liquid after 72 hours, removes non-adherent cell, has the shuttle type attached cell that is dispersed in the culturing bottle, adds fresh MSC nutrient solution again and continues to cultivate.
The cultivation and frozen of going down to posterity of embodiment 2, placenta MSC
After about 10 days, after waiting to be dispersed in attached cell and forming the clone, digest with 0.05% trypsinase/2mMEDTA, after the cell counting, according to 3000 cells/cm
2The cultivation of going down to posterity.Had digestive transfer culture is cultivated when about cell reaches 70%, merging afterwards.Get 3 * 10 after the digestion
6Cell joins in the 1ml cells frozen storing liquid (containing 50% low sugar DMEM nutrient solution, 40%FBS, 10% DMSO 99.8MIN.), and it is frozen to enter into liquid nitrogen pipe at last through programmed cooling.
The biological characteristics of embodiment 3, placenta MSC is identified
1, cell growth and morphology characteristics thereof
Separation and Culture through embodiment 1 and embodiment 2; The placenta mononuclearcell was cultivated after 72 hours can obviously see fusiformis attached cell (Figure 1A) at microscopically; Turbine-like cell clone (Figure 1B, Fig. 1 D) can be formed about 10 days, the adherent layer (Fig. 1 C, Fig. 1 E, Fig. 1 F) of about 80% fusions can be formed after the had digestive transfer culture.In the culturing process, find the relative homogeneous of this cellular form, rate of propagation is fast, and adherent speed is fast, is prone to be passaged to more than 15 generations by trysinization, and its form and growth characteristic also do not have obvious change.
2, flow cytometry identification of M SC surface marker
Get respectively the 3rd, 6,9,12,15 generation cell, the Flow cytometry cell surface marker dynamic observes the variation of cell surface marker in the culturing process.The digestion collecting cell gets 8 * 10 behind the counting
6Individual cell, packing 16 pipes; PBS washes once, the centrifugal 10min of 1500rpm; Abandon supernatant, residual 100～200 μ l, piping and druming mixing cell; Add CD45, CD105, HLA-ABC, HLA-DR, each 10 μ l of UEA-1 antibody of CD14, CD29, CD31, CD34, CD44, CD54, CD73, CD80, CD86, CD166 antibody and the FITC mark of PE mark, and establish a pipe and be blank; Under 4 ℃, lucifuge reaction 30min; PBS washes once, the centrifugal 10min of 1500rpm; Directly the cell of mark is abandoned supernatant, adds 200 μ l PBS piping and druming mixing cell, and 1% Paraformaldehyde 96 of 200 μ l is fixed, put 4 ℃ to be measured, the upflowing cell instrument detects in 3 days.
Flow cytometer detects the surface marker of cell, dynamic observes the cell in the 3rd, 6,9,12,15 generations, does not have obviously to change.Not expressing the hematopoietic cell surface marker is CD14, CD31, CD34 (HSPC and endotheliocyte are positive), CD45 (white corpuscle is positive), CD54 (ICAM-1), CD80 (B7-1), CD86 (B7-2), the lasting feminine gender of 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 going down to posterity more than 3 generations, the cellular constituent homogeneous, purity is more than 95%.(Fig. 2)
3, the cell cycle of Flow cytometry placenta MSC
Cell grows to about 80% when merging, digestion collecting cell about 1 * 10
6Individual, PBS washes once, and the ethanol of adding 70% is fixed, and 4 ℃ to be measured.During detection, the centrifugal ethanol that goes is washed once with PBS more earlier, adds RNase I 500u, 37 ℃ of reaction 30min, and PBS washes once, adds propidium iodide (PI, final concentration 50 μ g/ml) 1ml, room temperature lucifuge reaction 20min, last machine testing cell DNA content.
Through measure the 3rd generation and the 6th generation cell dna content, cell cycle analysis, G
0/ G
1Phase, S phase and G
2M phase proportion is respectively 96.35%, 96.66%, 1.11%, 0.09% and 2.54%, 3.25%.The result shows that the cell of vitro culture has typical stem cells hyperplasia characteristics, promptly has only few cell to be in the active propagation phase (1.11%, 0.09%), and most cell is in quiescent stage (96.35%, 96.66%).(Fig. 3)
4, the drafting of placenta MSC growth curve and the mensuration of logarithmic phase doubling time
The cell in vegetative period of taking the logarithm, the digestion counting is processed cell suspension (every hole inoculation 0.5ml in 2 * 104/ml), 24 orifice plates, 37 ℃, 5%CO with the LG-DMEM substratum of 10%FBS
2, cultivate under the saturated humidity.Get 3 multiple holes every day, trypan blue dyeing back living cell counting number, calculating mean value was observed 7 days continuously.With the incubation time is transverse axis, and cell count is the longitudinal axis, draws cell growth curve.Calculate cell in the doubling time of logarithmic phase with the Patterson formula, i.e. Td=Tlg2/Lg (Nt/No), Td: the doubling time (h), T: cell increases to Nt used time (h), N: cell count by No.
Cytometric result draws cell growth curve through every day, calculates the doubling time.Can find out that by cell growth curve cell was in exponential phase of growth at 2-4 days.According to formula calculate the 5th generation cell be respectively 22.6h in the doubling time of exponential phase of growth.(Fig. 4)
5, the evaluation of the multidirectional differentiation potential of placenta MSC
(1) osteogenic induction
Above MSC of 3 generations is by 1 * 10
5Six orifice plates are inoculated in/hole, are put in 37 ℃, 5%CO
2, under the saturated humidity, cultivate 24h in the MSC substratum after, use instead and contain 10% through the DMEM-HG of screening FBS and add DEXAMETHASONE BP98 0.1 μ M, ascorbyl phosphate 50 μ M, β-phospho-glycerol 10mM, be put in 37 ℃, 5%CO
2, under saturated humidity, cultivate, amount was changed liquid in per 3 days half, coinduction 2-4 week.Alkaline phosphatase staining identifies that scleroblast forms, and Von Kossa dyeing identifies that the bone tubercle forms.
Containing 10% DMEM-HG through screening FBS; Adding DEXAMETHASONE BP98 0.1 μ M, ascorbyl phosphate 50 μ M, β-phospho-glycerol 10mM cultivated for 1 week; Cellular form takes place significantly to change, and becomes polygon by fusiform inoblast appearance, is similar to neuronal cell appearance; It is outstanding that the long filament shape appears in the cell periphery, and can extend towards periphery.Continue to cultivate 2 weeks above after, calcified plaque appears in the cell matrix, mineralizer engenders, and begins to form the little junction structure of multilayer, after cultivating for 4 weeks, visible obviously calcification tubercle.The alkaline phosphatase staining of 2 whens week is strong positive reaction, reaches more than 95%, and in addition the inductive control group is then most of not negative, only is shown as the weak positive less than 5%, shows that cell transforms to scleroblast.Von Kossa dyeing can be dyed black with sedimentary calcium in the bone tubercle, induces the visible a large amount of black bone tubercle of group, tangible three-dimensional arrangement is arranged, and control group does not all have positive reaction at any time.(Fig. 5)
(2) become fat to induce
Above MSC of 3 generations is by 1 * 10
5/ hole is inoculated in six orifice plates, is put in 37 ℃, 5%CO
2, under the saturated humidity, in the MSC substratum, cultivate 24h after, use instead and contain 10% high sugared DMEM, and add DEXAMETHASONE BP98 1 μ M, INDOMETHACIN BP99 60 μ M, IBMX 0.5mM, Regular Insulin 5 μ g/ml through screening FBS, be put in 37 ℃, 5%CO
2, cultivate under the saturated humidity, amount was changed liquid in per 3 days half, and in 2 weeks of coinduction, oil red dyeing identifies that fat drips formation.
Containing 10% DMEM-HG through screening FBS; Adding DEXAMETHASONE BP98 1 μ M, INDOMETHACIN BP99 200 μ M, IBMX 0.5mM, Regular Insulin 10 μ g/ml cultivated 3 days; Form promptly takes place and changes in cell, is shunk gradually by fusiform inoblast appearance to shorten, and 90% above cell becomes cube or polygon; Cultured continuously 7 days, there have small fat to ooze under the mirror in the visible cell to be existing, and along with the prolongation of incubation time, fat drips and increases gradually and merges, and when cultivating for 2 weeks, the agglomerating fat of visible fusion drips and is full of whole cell.The fat that produces in the oil red O stain visible cell is dyed redness by specificity.(Fig. 6)
(3) become chondrocyte induction
3 generations above cell, according to every pipe 2 * 10
5The cell branch installs to the 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 the saturated humidity, amount was changed liquid, 2 weeks of cultured continuously in per 3 days half.
After inducing for 2 weeks the cell micelle is broken up smear, the visible II Collagen Type VI of alcian blue (Alcian blue) dyeing forms extracellular matrix and is blue, and control group does not have indigo plant and dyes.(Fig. 7)
6, RT-PCR detects the multidirectional differentiation potential of placenta MSC
Cell after collection is induced is used Trizol reagent and is extracted cell total rna, be that template is carried out RT-PCR, reverse transcription and PCR operation are carried out according to RT-PCR test kit specification sheets, primer sequence is as shown in table 1.
Table 1.RT-PCR primer sequence and specificity thereof
Among Fig. 8; Be respectively from left to right: placenta MSC cell, induce and become adipocyte, induce, induce RT-PCR electrophoresis photo into the chondrocyte to scleroblast; Wherein from left to right; The Normal swimming lane is cellular gene expression situation before inducing, and Adipogenic swimming lane, Osteogenic swimming lane, Chondrogenic swimming lane are for inducing back cellular gene expression situation.The result shows; External evoked back cell can be expressed serial specific mrna: become fat to induce back cell expressing PPAR-γ; Cell expressing SPP1 (Osteopontin) behind the osteogenic induction; Cell expressing collagen I I (Collagen II) behind the one-tenth chondrocyte induction explains that resulting MSC cell has skeletonization, becomes fat, becomes the cartilage differentiation capability, meets generally acknowledged MSC standard.
Through the detection of above a series of data targets, demonstrate and use the MSC that the inventive method separation obtains, have ability to scleroblast, adipocyte, chondrocyte's differentiation, confirm that the MSC that the inventive method obtains has the stem cell characteristic.
The foundation in embodiment 4, placenta stem-cell storehouse
1, the detection of cytoactive
Utilize the trypan blue staining to count the number of frozen front and back viable cell.
2, the detection of cell contamination
Utilize small amounts of cells to cultivate, detect cell and whether receive the pollution of fungi and bacterium.Utilize the etiology method, detect cell whether receive hepatitis B two double, third liver, AIDS, cytomegalovirus, Epstein-Barr virus and syphilis, HbsAg, HbsAb, HBcAb, HbeAg, HbeAb, HCVAb, HIV-1/2Ab, CMV-IgM and EBV-IgA, TRUST and infect.
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 in cell source
Record fetus and father and mother's thereof detail file, and place on record.
6, the foundation of placenta stem-cell DB
After preserving normal placenta stem-cell, set up the DB of placenta stem-cell, comprising the first six data, and foundation and freeze-stored cell is related.
Claims (10)
1. the method for separating mesenchymal stem cell from placenta, this method may further comprise the steps:
(a) get placental lobules, fully wash, to remove residual blood in the placenta with the PBS damping fluid;
(b) placental lobules is cut into bulk, adds the PBS damping fluid that contains the tissue digestion enzyme, hatch digestion at 37 ℃ again;
(c) tissue block is filtered with copper mesh, grind in case of necessity to impel filtration;
(d) filtered liq of collecting is centrifugal, separate mononuclearcell, the cell that suspends and obtained with the MSC substratum again is then at 37 ℃, 5%CO
2Cultivate in the incubator;
(e) treat that disseminated cell forms the clone after, each clone cell of picking is cultivated respectively with the MSC substratum, treat cytogamy after, go down to posterity with trysinization, promptly get placenta mesenchyma stem cell;
And optional following one or more steps:
(f) to step (e) gained placenta mesenchyma stem cell, detect at least one item of following project: 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 DB of the placenta stem-cell that comprises above information, and make this DB carry out related with the freeze-stored cell of step (g).
2. according to the method for claim 1; Wherein said step (a) is within four hours postpartum; Under aseptic condition, placental lobules is cut, fully washed placental lobules with the PBS damping fluid that contains 10% volume foetal calf serum (FBS) and remove residual blood in the placental lobules.
3. according to the process of claim 1 wherein that tissue digestion enzyme in the said step (b) is to be selected from following one or more: dispase, pancreatin, deoxyribonuclease I (DNase I), collagenase IV, Unidasa.
4. according to the method for claim 1, in step (b), hatched 10～30 minutes at 37 ℃, preferred 10～20 minutes, for example 10 minutes, 15 minutes or 20 minutes.
5. according to the method for claim 1, in step (d), said MSC substratum is the PBS damping fluid that contains 10%FBS.
6. according to the method for claim 1, in step (e), treat that disseminated cell forms the clone after, each clone cell of picking is cultivated respectively with the MSC substratum, treat cell 60～90% fusions after, go down to posterity with trysinization, promptly get placenta mesenchyma stem cell.
7. according to the method for claim 1; In step (f); Whether it is to utilize the etiology method that said cell contamination detects, detect cell and receive and be selected from following one or multinomial infection: hepatitis B two double, 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.
8. according to the method for claim 1; In step (h); Comprise in the said DB and all relevant data of the cell of being preserved, comprising: the biological characteristics detected result of cell, multidirectional differentiation potential qualification result, cellular elements genetic diagnosis result, fetus and father and mother's thereof detail file.
9. according to each method of claim 1 to 9, the cell purity of the placenta mesenchyma stem cell that is wherein obtained is greater than 95%.
10. placenta mesenchyma stem cell, it is to obtain according to each method of claim 1-9.
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