CN1548529A - Separation method of buffering stem cell in human placenta - Google Patents
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Abstract
The present invention discloses the separation method of mesenchymal stem cell in human placenta. On the basis of past separation of tissue cell, the present inventor separates from placenta mesenchymal stem cell with high purity via perfusion process based on the special anatomical structure of placenta. Identification result shows that the mesenchymal stem cell separated from placenta has the biological characteristic and polydirectional differentiation capacity as the reported mesenchymal stem cell of marrow. Owing to the infantile cell component and wide source of placenta, like cord blood, the present invention will have wide clinical application foreground.
Description
Technical field
The present invention relates to a kind of separation method of cell, relate in particular to a kind of separation method of people's placenta mesenchyma stem cell.
Background technology
Human mesenchymal stem cell (mesenchymal stem cell, MSC) be the tissue stem cell that an isolated class has multidirectional differentiation potential and self ability in marrow the earliest, derive from the mesoderm of embryo's emergence period, in vivo with external specified conditions under can be to scleroblast, the chondrocyte, adipocyte, the myocyte, Tenocyte cell, liver cell even neurogliocyte differentiation (1.Caplan AI.Mesenchymalstem cells.J Orthop Res.1991,9:641-650.2.Pittenger MF, Mackay AM, Beck SC, et al.Multilineage potential of adult human mesenchymal stem cells.Science.1999; 284:143-147.).MSC is easy to separation and purification from marrow, carry out effective amplification in vitro, its biological action result of study is shown, MSC has the bone marrow microenvironment of improvement, promote hematopoiesis to transplant effect (the 1.Kadereit S of back hematopoietic reconstitution, Deeds LS, Haynesworth S, et al.Expansion ofLTC-ICs and Maintenance of p21 and BCL-2 Expression in Cord BloodCD34+/CD38-Early Progenitors Cultured over Human MSCs as a Feeder Layer.Stem cell.2002; 20:573-582.2.Koc ON, Gerson SL, Cooper BW, et al.Rapidhematopoietic recovery after coinfusion of autologous-blood stem cells andculture-expanded marrow mesenchymal stem cells in advanced breast cancerpatients receiving high-dose chemotherapy.J Clin Oncol.2000; 18:307-316.3.Klyushnenlova E, Mosca J, McIntosh K.Human mesenchymal stem cells suppressallogeneic T cell responses in vitro:implications for allogeneic transplantation.Blood.1998; 92:642a.).The immunological characteristic aspect, MSC does not express or only expresses the MHC II quasi-molecule that can ignore level, the MSC of uncorrelated donor does not cause the allosome lymphocyte reaction, can reduce allosome immune response (1.McIntosh K, Klyushnenkova E, Shustova V, et al.Suppression ofalloreactive T cell reaponse by human mesenchymal stem cells involves CD8+cells.Blood.1999; 94:133a.2.Klyushnenkova E, Shustova V, Mosca J, et al.Human mesenchymal stem cells induce unresponsiveness in preactivated but notnative alloantigen specific T cells.EXP Hematol.1999; 27:122.).Based on above-mentioned feature, make it after finding, become the desirable engineering cell that cell therapy, gene therapy, effectively plays a role rapidly, and organizational project important seed cell in bone or the cartilaginous tissue injury repairing especially.
The MSC that is 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 has only 1 in the individual mononuclearcell, and increase along with the age, the quantity of MSC, propagation and differentiation capability all significantly descend (Rao MS, Mattson MP.Stem cells and aging:expanding the possibilities.Mech Ageing Dev.2001 in the marrow; 122:713-734.), make it in research with use in the especially clinical application and be restricted.
Recently, existing mesenchymal cell with stem/progenitor cells characteristic is separated (1.Ralf H.Isolation of Primary and Immortalized CD34-Hematopoietic and Mesenchymal Stem Cells from Various Sources.StemCells.2000 from comprising in the tissues such as bone, cartilage, muscle, tendon; 18:1-9.2.Deans RJ, Moseley AB.Mesenchymal stem cells:biology andpotential clinical uses.Exp Hematol.2000; 28:875-884.), it is exactly the mesoderm that derives from embryonic development period that these tissues have a common feature.
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.In fetal development period, the multipotential stem cell of extraembryonic mesoderm has a spot of not being recruited to be broken up, and be present in the placenta tissue with static form, if can from placenta, separation and Culture go out MSC, will open up a brand-new and abundant source for experimental study and clinical application.U.S.'s hematology annual meeting in 2000, report such as Jaroscak adopts enzyme digestion, placenta tissue is shredded with surgical scissors, after adding histaminase digestion certain hour, filter and obtain cell suspension, be inoculated in the substratum, after cultivating after a while, in the visible culturing bottle attached cell is arranged, continue to cultivate, the placenta attached cell that obtains is adopted the Flow cytometry cell surface marker, the result shows placenta attached cell and marrow MSC not obviously difference on the cell surface marker dyeing property of acquisition, but the placenta attached cell is heterogeneous, cell surface marker SH2, SH3, SH4 etc. are positive; Do not express CD45, PECAM, pointing out it is non-hematopoietic cell and endotheliocyte, illustrate and have MSC composition (Jaroscak J in the placenta, Smith T, Haynesworth S, et al.Preliminary characterization of the surface stainingof placental derived adherent cells:A potential new souece of stroma for umbilicolcord blood (UCB) expansion.THE AMERICAN SOCIETY OFHEMATOLOGY, December 1-5,2000.).Employing enzyme digestions such as Japanese scholar Watanabe in 2002 or tissue culture method are isolated MSC in conjunction with the flow cytometry separating method from placenta, concrete grammar is mainly placenta tissue is shredded, histaminase digestion or directly tissue block cultivation, the required cell of selected by flow cytometry apoptosis, after continuing to cultivate for some time, carry out cytobiology and identify that also inducing cell is to becoming fat, skeletonization with become cartilage differentiation, further inducing cell neuralward cytodifferentiation (Watanabe N, Igura K, Nagamura-Inoue T, et al.Multilineage potential of human placenta-derivedmesenchymal cells.THE AMERICAN SOCIETY OF HEMATOLOGY, December 1-5,2002).But separation method recited above is all comparatively loaded down with trivial details.
Summary of the invention
The objective of the invention is to disclose a kind of method of easy separation of human placenta mesenchyma stem cell (MSC), this method may further comprise the steps:
According to the anatomical structure of placenta, under aseptic condition, insert conduit from umbilical blood vessels and form the circulating liquid perfusion system.With the DMEM that contains heparin or IMDM substratum as perfusate.Residual blood is washed out by the arteriovenous circulation with perfusate earlier, hatch with perfusate then.Adopt density gradient centrifugation to separate mononuclearcell from perfusate, cultivate with the MSC substratum suspension cell that obtains the washing back.After treating that disseminated cell forms the clone, each clone cell chosen with the MSC substratum cultivate respectively, after cell 80～90% merges, go down to posterity with trysinization, the cell that reaches more than 3 generations is frozen in liquid nitrogen, and carry out the biological characteristics and the evaluation of multidirectional differentiation potential of cell.
The biological characteristics of cell is identified and has been carried out cell growth characteristic and the evaluation of morphology characteristics, flow cytometry identification of M SC surface marker, the analysis of placenta MSC cell cycle, the drafting of placenta MSC growth curve and the mensuration of logarithmic phase doubling time.
The multidirectional differentiation potential of placenta MSC identify carried out that one-tenth fat is induced, osteogenic induction with become chondrocyte induction.
The experimental result explanation, isolating attached cell has identical biological characteristics and multidirectional differentiation capability with the mesenchymal stem cells MSCs of reporting in the past from placenta, i.e. successfully separation from people's placenta, purifying, acquisition mescenchymal stem cell.
MSC mainly adopts modus operandi to extract donor marrow at present, and density gradient centrifugation is separated mononuclearcell, is inoculated in to cultivate in the MSC special culture media to obtain.This method complex operation, donor is being got the possibility that infection is all arranged after marrow is got in the marrow neutralization.The contriver utilizes the special anatomical structure of placenta summing up on the basis of chorista cell in the past, adopts perfusion method, and success separates in placenta and obtains the higher MSC of purity.
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 be with a wide range of applications clinically.
Description of drawings
Fig. 1 observes under the cell growth morphology mirror.Wherein
A is for cultivating the attached cell that as seen is dispersed in behind the 3d
B is that 7～10d forms the clone
C is for to form fine and close attached cell through screening and cloning
D is the dyeing of Rui Shi Ji's nurse Sa
Fig. 2 is flow cytometry identification of M SC surface marker result.
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 stationary phase (G0/G1 phase, 96.66%), and 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 stationary phase (G0/G1 phase, 96.35%), and few cell is in the propagation phase (S phase, 2.54%).
Fig. 4 is cell growth curve figure.
Fig. 5 is the one-tenth fat inducing cell figure that the multidirectional differentiation potential of placenta MSC is identified.
Wherein A is for inducing 3d, the cellular control unit form
B is for inducing 3d, and the experimental group cellular form changes
C-D is for inducing 7d, and visible endochylema lactones drips formation
E-F is that oil red dyeing fat drips by specificity and dyes redness
Fig. 6 is the osteogenic induction cytological map that the multidirectional differentiation potential of placenta MSC is identified.
Wherein A is for inducing 1w, the cellular control unit form
B is for inducing 1w, and the experimental group cellular form changes
C is for inducing 2w, and original position dyeing shows the alkaline phosphatase expression of enzymes positive
D is for inducing 2w, and smear staining shows the alkaline phosphatase expression of enzymes positive
E-F is for inducing 4w, and von Kossa dyeing shows that the bone tubercle forms
Fig. 7 is the one-tenth chondrocyte induction cytological map that the multidirectional differentiation potential of placenta MSC is identified.
Wherein A is for inducing 2w, cellular control unit Alcian blue dyeing
B is for inducing 2w, experimental group cell Alcian blue dyeing
C-D is that visible II Collagen Type VI formation extracellular matrix is dyed blueness by specificity under the high power lens
Embodiment
Embodiment one placenta MSC isolation cultivation method
According to the anatomical structure of placenta, under aseptic condition, insert polyethylene catheter from umbilical blood vessels (2 Umbilical artery and 1 umbilical vein) and form the circulating liquid perfusion system, with the DMEM that contains heparin or IMDM substratum as perfusate.Residual blood was washed out in 0.5～2.5h in postpartum by the arteriovenous circulation with perfusate earlier, hatch 12～24h for 20～25 ℃ with 100～250ml perfusate then.Adopt density gradient centrifugation from perfusate, to separate mononuclearcell, wash back with the MSC substratum suspension cell that obtains, 37 ℃, 5%CO
2After cultivating 24～48h under the complete wet condition, change liquid and remove not adherent suspension cell, continue to cultivate, change liquid once every 3～4d.After treating that early stage disseminated cell forms the clone, each clone cell chosen with the MSC substratum cultivate respectively, after cell 80～90% fusions, use 0.25% trysinization, went down to posterity in 1: 3 or 1: 4, the cell that reaches more than 3 generations is used for following experiment, and frozen part cell is for future use in liquid nitrogen.
The biological characteristics of embodiment two placenta MSC is identified
One, cell growth characteristic and morphology characteristics
Cultivate by embodiment one method, need 3d approximately, as seen be dispersed under the mirror and be the fusiform attached cell, form radial clone about 7～10d, each clone is chosen respectively to 24 orifice plate single culture.Treat cell purification to 3 after generation, be used for characteristics of cell biology and detect.In the culturing process, find the relative homogeneous of this cellular form, rate of growth is fast, and adherent speed is fast, easily by trysinization, is passaged to more than 15 generations, and its form and growth characteristic also do not have obvious change.See Fig. 1.
Two, flow cytometry identification of M SC surface marker
Get respectively the 3rd, 6,9,12,15 generation cell, the Flow cytometry surface marker dynamic observes the variation of surface marker in the culturing process.The digestion collecting cell gets 5 * 10 behind the counting
6Individual, packing 10 pipes; PBS washes once, the centrifugal 10min of 1500rpm; Abandon supernatant, residual 100～200 μ l, piping and druming mixing cell; Add CD29, each 10 μ l of CD44 antibody of CD45, CD105, HLA-DR, UEA-1 antibody and indirect labelling of CD34, CD73, CD166 antibody and the FITC mark of PE mark, and establish a pipe and be blank, a pipe is two anti-contrasts; 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 the 3d.The cell of indirect labelling repeats to go up machine testing after the above-mentioned steps after need resisting reaction with two.
Flow cytometer detects the surface marker of cell, dynamic observe derive from each clone the 3rd, 6,9,12,15 generation cell, do not have obviously and change.Not expressing the hematopoietic cell surface marker is that CD34 (HSPC and the endotheliocyte positive), CD45 (the white corpuscle positive), HLA-DR (MHC-II quasi-molecule) continue feminine gender, CD29 and the 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) 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%.See Fig. 2.
Three, the analysis of placenta MSC cell cycle
When cell grows to the 80-90% fusion, 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 RNaseI 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.
After measured the 3rd generation and the 6th generation cell dna content, cell cycle analysis, G0/G1 phase, S phase and G2M phase proportion are respectively 96.35%, 96.66%, 1.11%, 0.09% and 2.54%, 3.25%.The cell of results suggest vitro culture has typical stem cells hyperplasia characteristics, promptly has only a few cell to be in the active propagation phase (1.11%, 0.09%), and most cell is in quiescent stage (96.35%, 96.66%).See Fig. 3.
Four, the drafting of placenta MSC growth curve and the mensuration of logarithmic phase doubling time
The cell in vegetative period of taking the logarithm, the digestion counting is made cell suspension (2 * 10 with the LG-DMEM substratum of 10%FBS
4/ ml), every hole inoculation 0.5ml in 24 orifice plates, 37 ℃, 5%CO
2, cultivate under the saturated humidity.Get 3 multiple holes every day, trypan blue dyeing back living cell counting number, calculating mean value is observed 7d 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 by every day, calculates the doubling time.By cell growth curve as can be seen, cell is in exponential phase of growth at 2-4d.According to formula calculate the 5th generation cell be respectively 22.6h in the doubling time of exponential phase of growth.See Fig. 4.
The multidirectional differentiation potential of embodiment three placenta MSC is identified
One, become fat to induce
Above MSC of 3 generations is by 1 * 10
5/ hole is inoculated in six orifice plates, standard medium is cultivated to use instead behind the 24h and is contained the 10% high sugared DMEM through screening FBS, and adds dexamethasone 1 μ M, INDOMETHACIN 200 μ M, IBMX 0.5mM, Regular Insulin 10 μ g/ml, and every 3d half amount is changed liquid, coinduction 2w, oil red dyeing identifies that fat drips formation.
Containing 10% DMEM-HG through screening FBS, adding dexamethasone 1 μ M, INDOMETHACIN 200 μ M, IBMX 0.5mM, Regular Insulin 10 μ g/ml cultivate 3d, form promptly takes place and changes in cell, is shunk gradually by fusiform inoblast sample to shorten, and 90% above cell becomes cube or polygon; Cultured continuously 7d, 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 merge, and when cultivating 2w, as seen merges agglomerating fat and drips and be full of whole cell.The fat that produces in the oil red O stain visible cell is dyed redness by specificity.See Fig. 5.
Two, osteogenic induction
Above MSC of 3 generations is by 1 * 10
5Six orifice plates are inoculated in/hole, cultivate to use instead behind the 24h with standard medium to contain 10% through the DMEM-HG of screening FBS and add dexamethasone 0.1 μ M, ascorbyl phosphate 50 μ M, β-phospho-glycerol 10mM, and amount was changed liquid, coinduction 2-4 week in per 3 days half.Alkaline phosphatase staining identifies that scleroblast forms, and Von Kossa dyeing identifies that the bone tubercle forms.
Containing 10% DMEM-HG through screening FBS, adding dexamethasone 0.1 μ M, ascorbyl phosphate 50 μ M, β-phospho-glycerol 10mM cultivate 1w, cellular form takes place significantly to change, become polygon by fusiform inoblast sample, be similar to the neuronal cell sample, it is outstanding that the long filament shape appears in the cell periphery, and can extend towards periphery.Continue to cultivate 2w above after, calcified plaque appears in the cell matrix, mineralizer engenders, and begins to form the little junction structure of multilayer, after cultivating 4w, visible obviously calcification tubercle.The 2w alkaline phosphatase staining is strong positive reaction, reaches more than 95%, and the inductive control group is then most of not negative in addition, 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.See Fig. 6.
Three, become chondrocyte induction
3 generations above cell, low-speed centrifugal makes cell form micelle in test tube, adds each 6.25 μ g/ml of Regular Insulin, Transferrins,iron complexes, Sodium Selenite in containing the DMEM-HG of 2.5%FBS, BSA1.25 μ g/ml, Sodium.alpha.-ketopropionate 1mM/L, xitix phosphoric acid 37.5 μ g/ml, TGF-β
150ng/ml, every 3d half amount is changed liquid, cultured continuously 2w.
After inducing 2w the cell micelle is broken up smear, dye visible II Collagen Type VI of Alcian blue forms extracellular matrix and is blue, and control group does not have indigo plant and dyes.See Fig. 7.
Claims (10)
1, a kind of method of separation of human placenta mesenchyma stem cell may further comprise the steps:
(1) under aseptic condition, inserts conduit from umbilical blood vessels and form the circulating liquid perfusion system;
(2) residual blood is washed out by the arteriovenous circulation with perfusate earlier, hatch with perfusate then;
(3) from perfusate, separate mononuclearcell, cultivate;
(4) go down to posterity with trysinization and identify.
2, method according to claim 1 is characterized in that described perfusate is the substratum that contains antithrombotics.
3, method according to claim 2 is characterized in that described antithrombotics is a heparin.
4, method according to claim 2 is characterized in that described substratum is DMEM or IMDM substratum.
5, method according to claim 1 is characterized in that separating mononuclearcell and adopts centrifugation method to realize from perfusion liquid.
6, method according to claim 5 is characterized in that separating mononuclearcell and adopts the method for density gradient centrifugation to realize from perfusion liquid.
7, method according to claim 1 is characterized in that isolating mononuclearcell is cultivated with the MSC substratum from perfusion liquid.
8, method according to claim 1 is characterized in that the isolated cells evaluation comprises the biological characteristics evaluation and multidirectional differentiation potential is identified.
9, method according to claim 8, the biological characteristics evaluation that it is characterized in that isolated cells comprises cell growth characteristic and the evaluation of morphology characteristics, flow cytometry identification of M SC surface marker, the analysis of placenta MSC cell cycle, the drafting of placenta MSC growth curve and the mensuration of logarithmic phase doubling time.
10, method according to claim 8, the multidirectional differentiation potential that it is characterized in that isolated cells identify comprise into that fat is induced, osteogenic induction with become chondrocyte induction.
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