CN1810959A - Separating and culturing process of human amnion mesenchyme stem cell and its medical composition - Google Patents
Separating and culturing process of human amnion mesenchyme stem cell and its medical composition Download PDFInfo
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- CN1810959A CN1810959A CNA2006100330079A CN200610033007A CN1810959A CN 1810959 A CN1810959 A CN 1810959A CN A2006100330079 A CNA2006100330079 A CN A2006100330079A CN 200610033007 A CN200610033007 A CN 200610033007A CN 1810959 A CN1810959 A CN 1810959A
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Abstract
The present invention is separating and culturing process of human amnion mesenchyme stem cell and its medical composition. The separating and culturing process includes digesting human amnion successively with trypsin, collagenase and deoxyribonuclease, and filtering to prepare single cell suspension; culturing in DMEM/F12 culture medium with VDMEM and VF12 in the equal ratio and containing ox embryo blood serum in 10-20 vol% and basic fibroblast growth factor of ultimate concentration 10-20 ng/ml inside a culture box at 37 deg.c, saturated humidity and CO2 in 5 vol%; and replacing liquid and culture passage to proliferate and purify human amnion mesenchyme stem cell. The process has wide material source no ethnic limitation and wide application foreground. The medical composition may be used in various kinds of treatment.
Description
Technical field
The invention belongs to the separation and the cultural method of mescenchymal stem cell, what be specifically related to is a kind of separation and extracorporeal culturing method of human amnion mesenchymal stem cell, and contains the medical composition of human amnion mesenchymal stem cell as effective constituent.
Background technology
In recent years, the research of stem cell has obtained important breakthrough .1999 and 2000,10 big sciences that continuous 2 years of the most authoritative U.S. " Science " magazine in the world is classified stem cell and the Human Genome Project then as are broken through, especially in 1999 even classify stem-cell research as first.Stem cell causes revolutionary advancement at medical field probably, thereby have immeasurable medical value and cause global extensive concern and research, U.S.'s " epoch " weekly thinks that the stem cell and the Human Genome Project will become the field that the new millennium has development and application prospect most simultaneously.
Mescenchymal stem cell (mesenchymal stem cells, be called for short MSCs) be the multipotential stem cell that derives from the mature tissue, having height self ability and multidirectional differentiation potential, is the cenospecies daughter cell of Transplanted cells and organizational project, has broad application prospects.Wherein, research MSCs the most widely is the mescenchymal stem cell that derives from marrow, reduces although its ethics problem is compared with embryonic stem cell greatly, and medullary cell must obtain by the approach of invading, and with advancing age, stem cell population significantly reduces.Many research institutions search out multipotential stem cell in other tissue, comprise peripheral blood, bleeding of the umbilicus, deciduous teeth, the umbilical cord mesenchyma cell of mobilization, yet the cell quantity that obtains from these tissues is very limited.
Summary of the invention
First purpose of the present invention is at existing source of human stem cell or is subjected to ethics restriction or a limited number of problem, a kind of separation and cultural method that is not subjected to the human amnion mesenchymal stem cell of ethics restriction and wide material sources is provided.
Second purpose of the present invention is the human amnion mesenchymal stem cell that utilizes method of the present invention to make to provide a kind of medical composition.
First purpose of the present invention is achieved in that the step that the present invention includes following order:
(1) gets people's amnion and use earlier tryptic digestion, with collagenase, deoxyribonuclease digestion, obtain cell suspension then;
(2) cell suspension with the previous step gained filters, and makes single cell suspension;
(3) with (2) step gained cell inoculation in substratum, placing 37 ℃, saturated humidity, volume fraction is 5% CO
2Cultivate in the incubator, and by changing liquid and going down to posterity, make human amnion mesenchymal stem cell obtain amplification and purifying gradually, described substratum adopts V
DMEM: V
F12=1: 1 DMEM/F12 substratum, wherein containing foetal calf serum and the final concentration that volumn concentration is 10%-20% is the Basic Fibroblast Growth Factor of 10-20ng/ml.
In above-mentioned (1) step, preferred employing weight percentage is the trypsinase of 0.125-0.25%, room temperature digestion 30-60 minute, 2-4 time altogether, with containing the DMEM/F12 nutrient solution termination trypsinase that volumn concentration is the 10%-20% foetal calf serum, adopt the V that contains 0.5-2.0g/L collagenase and 0.05-0.20g/L deoxyribonuclease then
DMEM: V
F12=1: 1 DMEM/F12 nutrient solution, 37 ℃ digested 1-2 hour.
In above-mentioned (3) step, preferably go on foot the gained cell with 1 * 10 with (2)
5-1 * 10
6/ ml density is inoculated in the substratum cultivates.
The process of cell amplification and purifying is preferably as follows: after (3) step beginning cell cultures of the present invention, cultivated 48～72 hours, change nutrient solution, discard not adherent cell, according to the cell growing state, full dose was changed liquid once in per 3～4 days, treated that cell reaches 80%～90% when merging, and was 0.25% tryptic digestion with weight percentage, then in the ratio of 1: 2 ratio or 1: 3 inoculation culture that goes down to posterity, and being designated as P1 generation, per 3 days full doses are changed liquid in the culturing process that goes down to posterity, and merge each other until attached cell, at the bottom of being paved with bottle, repeat the aforesaid operations cultivation of going down to posterity, and be designated as P2 generation, continue the above-mentioned culturing process that goes down to posterity.
Medical composition of the present invention comprises the human amnion mesenchymal stem cell that makes by method of the present invention, preferably includes the medical composition of human amnion mesenchymal stem cell and Fibrin Glue.Medical composition of the present invention can be used for neural replacement therapy, is used for the treatment of myocardial infarction, is used for treatment of diabetes, is used for bone tissue engineer, is applied to plastic surgery.
The invention has the advantages that: people's amnion is the later waste of fetus birth, the present invention is from amnion separation, vitro culture human amnion mesenchymal stem cell (Human Amniotic-derivedmesenchymal stem cells, be called for short HADMSCs), have wide material sources, be not subjected to superiority such as ethics restriction; Medical composition of the present invention contains the human amnion mesenchymal stem cell of the mature tissue that can be divided into mesoderm and other germinal layer, is widely used.
Description of drawings
Fig. 1 is the Photomicrograph of 100 times of amplifications of cultivating the HADMSCs of different time among the embodiment one.
Fig. 2 is flow cytometry figure as a result among the embodiment one.
Fig. 3 is that HADMSCs is through inducing the cell photo of neuralward cytodifferentiation among the embodiment two, and wherein A is the photo of inducing 4 days 100 times of amplifications; B is the photo of inducing 7 days; C is the photo of cell expressing nestin before inducing, amplifies 200 times; D is the photo of inducing 7 days cell expressing NSE, amplifies 200 times.
Fig. 4 is a photo of transplanting back neurocyte expression specificity mark among the embodiment three, wherein A, B, C, D are the HADMSCs transplantation group, amplify 400 times, A, B express the photo of GFAP for 2 weeks, 4 all transplanted cells after transplanting, and C, D are for transplanting the photo of 2 weeks of back, 4 all transplanted cells expression NSE; E, F, G, H are Fibrin Glue and HADMSCs mixed transplantation group, amplify 400 times, and E, F express the photo of GFAP for 2 weeks, 4 all transplanted cells after transplanting, and G, H are for transplanting the photo of 2 weeks of back, 4 all transplanted cells expression NSE.
Embodiment
Embodiment one: the separation of human amnion mesenchymal stem cell, cultivation, amplification and purifying
1, the separation of human amniotic mesenchymal cell:
Under aseptic condition, get people's placenta of normal mature c-section fetus, passivity is separated the amnion of placenta umbilical cord face, with fully flushing of phosphoric acid buffer (PBS), amnion is cut into the sheet of 1.0cm * 1.0cm size, adding weight percentage by every gram tissue is 0.25% trypsinase 2ml, room temperature digestion 30 minutes, totally 3 times, then with containing the DMEM/F12 nutrient solution termination trypsinase that volumn concentration is 10% foetal calf serum (FBS), to remove epithelial cell as far as possible.Then amnion is shredded as far as possible, add the V that contains 1.0g/L collagenase and 0.10g/L deoxyribonuclease with every gram tissue
DMEM: V
F12=1: 1 DMEM/F12 nutrient solution 2ml, 37 ℃ of digestion get cell suspension after 1 hour.Filter with stainless (steel) wire then the cell that digests is made single cell suspension, centrifugal 10 minutes of 1000r/m washes 2 times with PBS, and with the blue dyeing of platform dish, living cell counting is with 2 * 10
5The density of/ml with the gained cell inoculation in the 75ml culturing bottle.The cell density of the present invention when inoculation can adopt 1 * 10
5-1 * 10
6/ ml.
2, the cultivation of human amnion mesenchymal stem cell, purifying and amplification:
Substratum adopts V
DMEM: V
F12=1: 1 DMEM/F12 substratum, wherein added volumn concentration and be the Basic Fibroblast Growth Factor (bFGF) that 10% foetal calf serum (FBS) and final concentration are 20ng/ml, the human amniotic mesenchymal cell of a last process gained is sub-packed in the 75ml culturing bottle, and placing 37 ℃, saturated humidity, volume fraction is 5% CO
2Cultivate in the incubator after 48-72 hour, change nutrient solution, discard not adherent cell, according to the cell growing state, every 3-4 days full dose is changed liquid once.When treating that cell reaches 80%-90% and merges, be 0.25% tryptic digestion,, and be designated as P1 generation then in the ratio of the ratio of 1: 2 or 1: 3 inoculation culture that goes down to posterity with weight percentage.Per 3 days full doses are changed liquid in the culturing process that goes down to posterity, and merge each other until attached cell, at the bottom of being paved with bottle, repeat aforesaid operations again and go down to posterity, and this goes down to posterity to cultivate and is designated as P2 generation, continues the above-mentioned culturing process that goes down to posterity then.Cell began adherent in 24 hours, observed in 72 hours, see that cell is rounded, fusiformis, star, variforms such as polygon, change liquid by full dose and remove not attached cell gradually, this moment, attached cell was the clone of single dispersion or several cells, cellular form is even gradually after 10 days, be spindle shape, pseudopodium is arranged, visible two kinds of cell clones about 14 days: epithelioid cell clone and fibroblast-like cells clone, the former is a heteroproteose cell, the latter is human amnion mesenchymal stem cell HADMSCs, and each clone contains 200～300 cells approximately, is cultured to 14～18 days cells and reaches 80%～90% fusion, see that fibroblast-like cells is radial or the whirlpool shape distributes, the epithelioid cell is the paving stone sample and arranges.It is adherent fully in the cell 24 hours of back to go down to posterity, and reaches fully in 7～10 days to merge, and reaches for 3 generations, and cellular form is very even, and the epithelioid cell clones disappearance.Along with the carrying out of going down to posterity, the cell space of HADMSCs increases gradually, the roomy flat cell of part occurs, lose propagation and differentiation capability, and most of cell is still kept elongated fusiformis, keeps hyperplasia and differentiation capability.Vitro culture is after 10 generations, and the rate of propagation of cell obviously slows down, and catabiosis appears in cell.
Cellular form in process is gone down to posterity in above-mentioned cultivation can be the figure that cultivated 8 days referring to Fig. 1: A, and cell mixes, and sees variforms such as circle, star, polygon, fusiformis; B, C are the figure that cultivated 13 days, and wherein B is inoblast sample clone, and C is epithelial cell sample clone; D is the figure in 3 days the 1st generations, and E is the figure in 3 days the 3rd generations, and F is the figure in 6 days the 3rd generations, and G is the figure in 3 days the 6th generations, and H is the figure in 10 days the 8th generations.
3, fluidic cell detects:
The human amnion mesenchymal stem cell weight percentage of cultivating 4-6 generation is behind 0.25% the tryptic digestion, with containing PBS adjustment cell to 5 * 10 that volumn concentration is 1% bSA
5/ 50ul.Add following mouse-anti human monoclonal antibodies then respectively: CD29-PE, CD34-FITC, CD45-FITC, CD44-FITC, HLA-ABC, HLA-DR, putting 4 ℃ hatched 30 minutes, wash 1 time with PBS, straight mark monoclonal antibody directly upflowing cell instrument detects, and that a mark monoclonal antibody adds the anti-mouse FITC of rabbit two again is anti-, put 4 ℃ and hatched 30 minutes, after PBS washing 2 times, carry out flow cytometry analysis.Analytical results shows CD29, CD44, the HLA-ABC positive, CD34, CD45, HLA-DR feminine gender as shown in the figure.Above-mentioned mouse-anti human monoclonal antibodies CD29-PE, CD34-FITC, CD45-FITC, CD44-FITC, HLA-ABC, HLA-DR are available from Santa Cruz Biontechnology Ins. company.
In the 1st separating step that goes on foot of present embodiment, tryptic weight percentage can be 0.125-0.25%, and digestion time can be 30-60 minute, and number of times is 2-4 time; The volumn concentration of ending foetal calf serum in the tryptic DMEM/F12 nutrient solution can be 10%-20%; When collagenase, deoxyribonuclease digestion, V
DMEM: V
F12The concentration of collagenase can adopt 0.5-2.0g/L in=1: 1 the DMEM/F12 nutrient solution, and deoxyribonuclease can be 0.05-0.20g/L, and digestion time can be 1-2 hour.
Embodiment two: the like cell differentiation of directional induction HADMSCs neuralward unit
1, directional induction: get reached for the 3rd generation HADMSCs by 4 * 10
5/ L density is inoculated in preparation cell climbing sheet in six orifice plates that are placed with the disinfection cap slide in advance, when treating that cell reaches 80% fusion, to contain 30 μ mol/L all-trans-retinoic acid (Retinic acid, available from sigma company), the DMEM/F12 substratum of 20ng/mlbFGF, 10%FBS induced 7 days, control group does not add any inductor.The result: the human amnion mesenchymal stem cell of cultivation is expressed the mark people neural nest albumen (nestin) of neural stem cell, cell expressing nestin after inducing and neuronic mark human neure specificity olefinic alcohol enzyme (NSE) are not expressed the mark glial fibrillary acidic protein (GFAP) of neurogliocyte.
2, inverted microscope is observed down: HADMSCs neuralward cell induce differentiation, after adding induced liquid 2 days, part cell generation metamorphosis is arranged promptly, the cell space retraction, projection attenuates elongated, presents the neuron cell form gradually, can be observed the part cell and takes off wall death; After inducing 4 days, most of cell generation form changes, more neuron cell flocks together, adjacent iuntercellular aixs cylinder is coupled to netted, inductive neuron cell form differs, and simple Beale's ganglion cells is arranged, and complicated multipolar cell is also arranged, form does not take place and changes in small part cell all the time, still keeps roomy flats; After 7 days, most of cell is the neuron form.HADMSCs induces cellular form in the atomization referring to Fig. 3 above-mentioned.
3, immunocytochemistry detects: getting the cover glass that growth has cell, adopting SP immunohistochemical methods method to carry out immunocytochemical stain, the cell cover glass is with 80% acetone fixed 10
Minute, 3%H
2O
2Sealed 10 minutes, lowlenthal serum sealing 15 minutes, add rabbit anti human nerve nidogen, mouse anti human neuronspecific enolase and glial fibrillary acidic protein monoclonal antibody working fluid respectively after removing serum, and establish negative control, replace one to resist with PBS, place under 4 ℃ in the wet box and spend the night, PBS washing 3 times, drip two anti-working fluids of goat-anti rabbit and sheep anti mouse respectively, room temperature 30 minutes adds horseradish peroxidase-labeled strepto-avidin again, incubated at room 30 minutes, with developer DAB colour developing, neutral gum mounting.Get 10 non-overlapped visuals field (amplifying 100 times) under the mirror at random, calculate the ratio that nestin and NSE and GFAP positive cell account for total cell.The result shows that inducing the nestin expression rate of the preceding HADMSCs of differentiation is 82.0 ± 3.3; Inducing 4 days NSE expression rates is 40.4 ± 2.0, induces that the NSE expression rate is 78.1 ± 3.4 after 7 days, induce compared with 7 days in 4 days NSE express variant statistically, t=-30.52, P＜0.05, up-regulated; Do not express GFAP.
Embodiment three: contain the application of composition in the treatment nervous system disorders of HADMSCs
HADMSCs can be divided into neurocyte, for new cell source has been opened up in neural transplantation.Can by topical application or, approach such as lumbar puncture and venoclysis transplants human body with HADMSCs and treats nervous system disorderss such as brain injury, parkinsonism, cerebral apoplexy, Spinal injury and peripheral nerve injury.
1, animal grouping and Modelling:
80 of Wistar rats, male and female are not limit, and are divided into 4 groups at random: the A group is damage group (Sham), and 20, not transplanted cells of cerebral tissue is not hit in a craniotomy drill hole, as negative control group.The B group is false transplantation group (TBI+NS), and 20, strike cerebral tissue injecting normal saline 10 μ l after 1 day in craniotomy drill hole are as intervening contrast.The C group is transplantation group (TBI+HADMSCs), and 20, the craniotomy drill hole is hit cerebral tissue and injected the physiological saline 10 μ l that contain HADMSCs after 1 day, as the HADMSCs transplantation group.D group is Fibrin Glue transplantation group (TBI+FG+HADMSCs), 20, the craniotomy drill hole hit cerebral tissue after 1 day with HADMSCs with 1 * 10
6The component main gel and the catalyst mix of/ml density and Fibrin Glue are injected mixed solution 100 μ l in damage zone with Fibrin Glue special syringe single-point, as Fibrin Glue Transplanted cells group.Fibrin Glue is produced by the doubly elegant Bioisystech Co., Ltd in Guangzhou.
B, C, D organize three groups and adopt freely falling body epidural bump legal system to make the rat brain trauma model, and the damage device has base plate, fixed support, and vertical guide stem, the 20g counterweight, polyethylene bump circular cone is formed.Bump circular cone head end diameter 4mm, high 2.5mm.10% Chloral Hydrate intraperitoneal injection anesthetized rat, injection volume is the 3ml/kg body weight, and rat head is fixed on the operating table, deduct the top fur, sterilization is along midline incision skin and periosteum, 3mm behind the bregma of left side, a left side is other opens 2mm, bores the ox-eye that a diameter is 5mm, to clash into the circular cone head end and put into the bone window, counterweight vertically falls from the 30cm eminence, and bump places epidural circular cone, causes the moderate brain injury, sew up periosteum and skin, put back to recovery.
2, immunohistochemical methods:
2 weeks, 4 weeks are put to death rat with the excessive injecting anesthetic of 10% Chloral Hydrate intraperitoneal after the wound, every group each 5, open chest immediately through the heart aortic cannulation, pour into fast with 1% heparin-saline 100ml in 1 minute, use 4% paraformaldehyde solution drip irrigation 2 hours then, cerebral tissue is taken out in perfusion back, puts into 4 ℃, 4% paraformaldehyde solution and fixedly spends the night.Gradient alcohol dehydration, dimethylbenzene are transparent, waxdip and make wax stone, are 10 of center sections with the damage zone, and section is attached on the slide that poly-lysine handles in order to immunohistochemical staining.
Use immunohistochemistrySP SP: with paraffin section de-waxing and aquation; With the PBS flushing of PH7.4, wash 3 times, each 5 minutes, 3%H
2O
2Incubated at room 5-10 minute, to eliminate the activity of endogenous peroxydase; PBS flushing 3 times, each 5 minutes; Coctoantigen is repaired: antigen retrieval liquid is put in section, and electric furnace is heated to 96～100 ℃, continues 20 minutes; Room temperature cooling back is washed 5 minutes * 3 times with PBS; The sealing of 10% normal goats serum, incubated at room 20 minutes, the serum deprivation that inclines is not washed; Drip mouse-anti people GFAP working fluid, mouse-anti people NSE working fluid, 4 ℃ of wet boxes spend the night; PBS flushing 3 minutes * 3 times; It is biotin labeling mountain sheep anti-mouse igg working fluid that dropping two resists, and hatches under the room temperature 30 minutes; PBS flushing 3 minutes * 3 times; It is horseradish enzyme labelling strepto-avidin-superoxide enzyme solution that dropping three resists, and hatches 30 minutes for 37 ℃; PBS flushing 3 minutes * 3 times; Add freshly prepared DAB solution, microscopically was observed 5～10 minutes; Tap water fully washes; Hematorylin is redyed; The differentiation of acid alcohol; Gradient alcohol dehydration, dimethylbenzene is transparent, the neutral gum mounting.The result shows: 2 weeks of animal intracerebral transplantation, 4 weeks are all expressed NSE and GFAP, referring to Fig. 4.Show that HADMSCs is divided into neurone and neurogliocyte in vivo, HADMSCs can survive in Fibrin Glue and break up.
Embodiment four: Fibrin Glue is as the application of timbering material in organizational project of HADMSCs
Fibrin Glue is to extract the biomaterial for medical purpose that relevant composition such as Fibrinogen, the XIII factor, zymoplasm etc. are made from blood, can be used as the bonding repair tissue damage of tackiness agent, and its histocompatibility and biological degradability are good, can be organized fully to absorb.We mix Fibrin Glue and HADMSCs, and transplant in the injured brain tissue of rat, 2 the week and all find that it is divided into neurone and neurogliocyte 4 weeks, the HADMSCs that shows transplanting can be good at existence in Fibrin Glue, and under the influence of local microenvironment, can be divided into neurocyte.Graft area is not seen cell infiltration, shows the excellent compatibility of itself and cerebral tissue.The Fibrin Glue agglomerate is not found in organizing in the HE section of 2 weeks, and Fibrin Glue dissolving fully is described, its degradation property is good.And with the support of Fibrin Glue, not only can play local hemostatic effect, can also prevent flowing of cell, make the local bigger cell concn that keeps, more help the reparation that damages as Transplanted cells.Simultaneously, Fibrin Glue also can be used for other organizational projects such as bone tissue engineer as the timbering material of HADMSCs.
From above embodiment as seen: human amnion mesenchymal stem cell be separated, be turned out to the inventive method can successfully from amnion, this cell can be at external successfully amplification and purifying, detect through fluidic cell, this cell expressing CD29, CD44, HLA-ABC do not express CD34, CD45, HLA-DR feminine gender; Human amnion mesenchymal stem cell is expressed the mark nestin of neural stem cell, can be divided into neurone through external evoked this cell, and the district can be divided into neurone and neurogliocyte at the damage brain.
Embodiment five: the medical composition that contains HADMSCs is used for the treatment of myocardial infarction
HADMSCs has the universal feature of mescenchymal stem cell, can be divided into the mature tissue of mesoderm and other germinal layer.Can it be transplanted in site of myocardial infarction by approach such as intervention means or venoclysises, substitute infarcted myocardium, recover heart function.
Embodiment six: the medical composition that contains HADMSCs is used for treatment of diabetes
Diabetes mainly are due to the islet function depletion, and HADMSCs is transplanted in pancreas islet, and the differentiation regulation and control by certain make it be divided into functional islet cells, substitute islet function, the treatment diabetes.
Embodiment seven: the medical composition that contains HADMSCs is used for bone tissue engineer
HADMSCs is divided into osseous tissue under certain inductive condition, HADMSCs can be transplanted in the damaged place of bone, repairing bone defect.
Embodiment eight: the medical composition that contains HADMSCs is in orthopedic application
HADMSCs is induced by the suitable system of inducing, be divided into adipocyte, be used for health plasticity.
Claims (6)
1. the separation of a human amnion mesenchymal stem cell and cultural method is characterized in that comprising the step of following order:
(1) gets people's amnion and use earlier tryptic digestion, with collagenase, deoxyribonuclease digestion, obtain cell suspension then;
(2) cell suspension with the previous step gained filters, and makes single cell suspension;
(3) with (2) step gained cell inoculation in substratum, placing 37 ℃, saturated humidity, volume fraction is 5% CO
2Cultivate in the incubator, and by changing liquid and going down to posterity, make human amnion mesenchymal stem cell obtain amplification and purifying gradually, described substratum adopts V
DMEM: V
F12=1: 1 DMEM/F12 substratum, wherein containing foetal calf serum and the final concentration that volumn concentration is 10%-20% is the Basic Fibroblast Growth Factor of 10-20ng/ml.
2. the separation of human amnion mesenchymal stem cell according to claim 1 and cultural method, it is characterized in that: in (1) step, adopting weight percentage is the trypsinase of 0.125-0.25%, room temperature digestion 30-60 minute, 2-4 time altogether, with containing the DMEM/F12 nutrient solution termination trypsinase that volumn concentration is the 10%-20% foetal calf serum, adopt the V that contains 0.5-2.0g/L collagenase and 0.05-0.20g/L deoxyribonuclease then
DMEM: V
F12=1: 1 DMEM/F12 nutrient solution, 37 ℃ digested 1-2 hour.
3. the separation of human amnion mesenchymal stem cell according to claim 1 and cultural method is characterized in that: go on foot the gained cell with 1 * 10 with (2)
5-1 * 10
6/ ml density is inoculated in the substratum cultivates.
4. according to the separation and the cultural method of described any one human amnion mesenchymal stem cell of claim 1 to 3, it is characterized in that: after (3) step beginning cell cultures, cultivated 48～72 hours, change nutrient solution, discard not adherent cell, according to the cell growing state, full dose was changed liquid once in per 3～4 days, when treating that cell reaches 80%～90% fusion, with weight percentage is 0.25% tryptic digestion, then in the ratio of 1: 2 ratio or 1: 3 inoculation culture that goes down to posterity, and is designated as P1 generation, per 3 days full doses are changed liquid in the culturing process that goes down to posterity, merge each other until attached cell, at the bottom of being paved with bottle, repeat the aforesaid operations cultivation of going down to posterity, and be designated as P2 generation, continue the above-mentioned culturing process that goes down to posterity.
5. a medical composition comprises the human amnion mesenchymal stem cell that makes by described any one method of claim 1 to 3.
6. medical composition according to claim 5 is characterized in that: also comprise Fibrin Glue.
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