CN104450611A - Primary separation and culture method of human amniotic mesenchymal stem cells - Google Patents
Primary separation and culture method of human amniotic mesenchymal stem cells Download PDFInfo
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Abstract
The invention relates to a primary separation and culture method of human amniotic mesenchymal stem cells. The primary separation and culture method of the human amniotic mesenchymal stem cells comprises the following steps of: providing a human amniotic tissue, washing with PBS (phosphate buffer saline), shearing into pieces, adding 0.1-0.3% (by mass) trypsinase to digest, adding I-type collagenase and basal high-glucose DMEM until the final concentration of the I-type collagenase is 0.1-0.2%, digesting, separating the digested human amniotic tissue centrifugally to obtain a precipitate, resuspending the precipitate with PBS, filtering, separating centrifugally, and removing the supernatant to obtain the human amniotic mesenchymal stem cells. Compared with the prior art, the primary separation and culture method provided by the invention has the advantages that because a small amount of trypsinase is used to pretreat and loosen the human amniotic tissue, and further the I-type collagenase with good tissue specificity is used to treat the human amniotic tissue, the digesting time can be shortened greatly, the primary separation steps can be simplified, the human amniotic mesenchymal stem cells can be obtained at a high yield, and the viability of the obtained human amniotic mesenchymal stem cells can be improved greatly compared with that of the human amniotic mesenchymal stem cells in the prior art.
Description
Technical field
The present invention relates to stem cells technology field, be specifically related to the primary separation of a kind of human amnion mesenchymal stem cell and cultural method.
Background technology
Mescenchymal stem cell (Mesenchymal Stem Cells, MSCs) be mesoderma origin there is height self-renewal capacity and the versatile stem cell of multi-lineage potential, extensively be present in whole body Various Tissues, amplification can be cultivated in vitro, and neurocyte, osteocyte, chondrocyte, muscle cell, adipocyte etc. can be divided under given conditions.MSCs is multipotential stem cell, has the ability of " transdifferentiationof " or " interdepartmental differentiation ", not only the growth of hematopoiesis support stem cell, under different inductive condition, can also be divided into Various Tissues cell in vitro.MSCs has wide potential applicability in clinical practice, is the first-selected seed cell of cell replacement therapy and organizational project.
Mesenchymal stem cells MSCs (Bone Marrow Derived Mesenchymal Stem Cells, BMSCs) be comparatively early by a kind of mescenchymal stem cell be familiar with, but because bone marrow extraction can bring great misery to patient, and with advancing age, cell quantity significantly declines, differentiation capability reduces gradually, thus more difficultly promotes the use of.From the histoorgans such as fat, spleen, bleeding of the umbilicus, umbilical cord, tire liver and tire kidney, isolate mescenchymal stem cell at present, but all have some limitations, as difficulty of drawing materials, by age limit, to relate to mescenchymal stem cell content in ethics problem, tissue few etc.So seek a kind of abundance, stem cell content is high, draws materials conveniently, and non-invasive and MSCs that is that do not limit by ethics originates and becomes study hotspot of today already.
Studies have found that recently, AMSCs has the phenotype similar to BMSCs, and has self and multi-lineage potential, and multiplication capacity is stronger than BMSCs.AMSCs low expression HLA-ABC, and not expression of HLA-DR, thus illustrate that AMSCs has reduced immunogenicity, after allogeneic, Xenogeneic, all immunological rejection can not occur, and AMSCs is without tumorigenicity, this is that the cell replacement therapy of various disease provides new selection.
People is amnion-derived in human placenta, without blood vessel, nerve and lymph, has certain elasticity, thick 0.02 ~ 0.5mm.Under Electronic Speculum, it is divided into 5 layers: epithelial lining, basilar membrane, tight zone, fibroblast layer and spongy layer.Amnion belongs to the waste after pregnant woman's production, the nearly 600cm of amnion area of a placenta
2, and amnion is easy to from ablatio placentae, so draw materials conveniently because amnion has, and the feature of raw material abundance and be considered to the best source of current mescenchymal stem cell.At present, the primary isolation cultivation method of hAMSCs can be divided into tissue mass cell culture and method of tissue block two kinds, tissue mass cell culture is not owing to being that each tissue block successfully can turn out cell, and be easily mixed into other heteroproteose cells, culture cycle is longer simultaneously, be unfavorable for that rapid, high volume obtains cell, be difficult to the demand meeting stem-cell research.Method of tissue block is the main flow of current amnion method for separating stem cell, but in view of amnion tissue compact structure, the type selecting of digestive ferment can have vital impact to the activity after stem cell separation.The technology that prior art utilizes II Collagenase Type to be separated has length consuming time, and cell must measure the weakness such as low and activity decrease.
Summary of the invention
The object of invention is the defect overcoming above-mentioned prior art, the primary separation of a kind of human amnion mesenchymal stem cell and cultural method are provided, the method utilizes a small amount of trypsinase pre-treatment amnion tissue, amnion tissue is loosened, and then utilize tissue specificity better type i collagen ferment treatment amnion, substantially reduce the time of digestion, simplify the step of primary separation, and higher yield of dried cell can be obtained, the stem cell vigor of acquisition also improves compared to existing technology greatly.
For achieving the above object, the present invention adopts following technical scheme:
A kind of primary separation of human amnion mesenchymal stem cell and cultural method, is characterized in that comprising the following steps:
(1) human amniotic tissue is provided;
(2) use PBS wash buffer, then amnion tissue is shredded;
(3) add mass concentration be 0.1 ~ 0.3% trypsinase digest;
(4) postdigestive amnion tissue PBS wash buffer, then shreds amnion tissue;
(5) adding type i collagen enzyme and DMEM in high glucose basic medium, is that 0.1 ~ 0.2% digestion to amnion tissue block melts to type i collagen enzyme final concentration;
(6) postdigestive tissue juice is diluted, centrifugation, taking precipitate with PBS damping fluid;
(7) throw out PBS solution is resuspended, centrifugation after filtering, and abandoning supernatant obtains human amnion mesenchymal stem cell;
(8) use the resuspended human amnion mesenchymal stem cell of serum free medium, adjustment cell density is (3 ~ 10) × 10
5/ mL is placed in the culture dish that with the addition of EGF, moves into CO
2change liquid after cultivating 48h in incubator, remove impurity, change a subculture every 2 ~ 3d.
Preferably, amnion tissue shreds to 6 × 6cm by step (2)
2be sub-packed in 50mL centrifuge tube, step (3) pancreatin addition is 45mL, and digests in constant-temperature table, and digestion time is 30min, and digestion temperature is 37 DEG C, and rotating speed is 150 ~ 300r/min.
Preferably, amnion tissue shreds to 1mm by step (4)
2after be transferred in 125mL liquid storage bottle, add type i collagen enzyme and DMEM in high glucose basic medium that 20mL mass concentration is 0.5%, be 0.1 ~ 0.2% to digest to type i collagen enzyme final concentration, digestion is carried out in constant-temperature table, digestion temperature is 37 DEG C, rotating speed is 250 ~ 300r/min, digests to tissue block and melts.
Preferably, described serum free medium is UltraCULTURE MEDIUM substratum.
Preferably, step (6) centrifugal speed is 1500 ~ 2000r/min, and centrifugation time is 5min.
Preferably, step (7) is through 100 μm, and centrifugal speed is 1500 ~ 2000r/min, and centrifugation time is 5min.
Preferably, step (8) adopts specification to be the culture dish of 10cm, and EGF addition is 100 μ L, and concentration is 1 μ g/mL.
Preferably, when primary human amnion mesenchymal stem cell grows to 80% fusion, namely digest with the EDTA-Trypsin of 0.25%, carry out Secondary Culture.
Compared with prior art, the invention has the beneficial effects as follows:
This patent utilizes a small amount of trypsinase pre-treatment amnion tissue, amnion tissue is loosened, and then utilizes tissue specificity better type i collagen ferment treatment amnion, thus shorten the time of digestion, improve output and cell viability.
1, the present invention explores enzymolysis kind, the order of enzymolysis, the condition of enzymolysis of the most applicable human amnion tissue feature, can by thorough for human amnion tissue enzymolysis by technical scheme of the present invention, human amnion mesenchymal stem cell is discharged, obtain the stem cell that quantity is more, purity is higher, the structure of stem cell can not be destroyed simultaneously, do not affect the activity of stem cell, ensure that the stem cell obtained has higher vigor, can mass propgation, meet Research Requirements.
2, in the present invention, early stage adopts a small amount of trypsinase to digest, under the consumption and digestion condition of the enzyme of regulation, amnion tissue can be made to be in just loosen, now the epithelial cell on amnion top layer is also just removed, the most applicable utilization removes enzymolysis, digestion to the better type i collagen enzyme of human amnion tissue specificity, digestive process is little to cell injury, Cell viability is high, the stem cell amount obtained after digestion is large and purity is high, can keep good cytoactive and multiplication capacity in Secondary Culture process afterwards.
3, to obtain cycle of human amnion mesenchymal stem cell short in the present invention, output and active high, and the amnion mesenchymal stem cell inoculation of separating can grow for latter three days merges to 80%-90%, is conducive to rapid, high volume and obtains stem cell, be applicable to large-scale production application
4, the present invention uses serum free medium (UltraCULTURE MEDIUM) to carry out cell cultures, can avoid having genotoxic potential effect and serum source contact scar containing serum free culture system basic weight serum to cell.
Accompanying drawing explanation
Fig. 1 is the expression figure of the surface standard thing of human amnion mesenchymal stem cell primary cell of the present invention;
Fig. 2 is the growth curve figure of human amnion mesenchymal stem cell P2 of the present invention for cell;
Fig. 3 is that human amnion mesenchymal stem cell P2 of the present invention is for cellular form variation diagram after cell adipogenic induction;
Wherein, A is negative control, the amnion mesenchymal stem cell aspect graph of not inducing; B is after adipogenic induction 10 days, without the cellular form figure of oil red O stain; C is adipogenic induction after 10 days, the cellular form figure of oil red O stain; D is adipogenic induction after 10 days, the cellular form figure of oil red O stain.
Fig. 4 is that human amnion mesenchymal stem cell P2 of the present invention is for cellular form variation diagram after cell osteogenic induction;
Wherein, A: negative control, the i.e. cellular form of non-induced osteogenesis differentiation; B: osteogenic induction is after 28 days, undyed cellular form; C: osteogenic induction is after 28 days, by the cellular form of Alizarin red staining.
Fig. 5 is the cellular form comparison diagram of the human amnion mesenchymal stem cell that human amnion mesenchymal stem cell of the present invention and prior art obtain;
Wherein, A1: the cellular form figure under primary cell of the present invention 2nd day 40 power microscopes,
A2: the cellular form figure under primary cell of the present invention 2nd day 100 power microscopes,
A3: the cellular form figure under primary cell of the present invention 4th day 40 power microscopes,
A4: the cellular form figure under primary cell of the present invention 4th day 100 power microscopes,
A5: P2 of the present invention for the cellular form figure under cell the 3rd day 40 power microscopes,
A6: P2 of the present invention for the cellular form figure under cell the 3rd day 100 power microscopes,
B1: the cellular form figure under prior art primary cell the 2nd day 40 power microscopes,
B2: the cellular form figure under prior art primary cell the 2nd day 100 power microscopes,
B3: the cellular form figure under prior art primary cell the 4th day 40 power microscopes,
B4: the cellular form figure under prior art primary cell the 4th day 100 power microscopes,
B5: prior art P2 for the cellular form figure under cell the 3rd day 40 power microscopes,
B6: prior art P2 for the cellular form figure under cell the 3rd day 100 power microscopes.
Embodiment
Below by specific embodiment, the present invention is described in further detail, understands the claimed technical scheme of the present invention so that clear.
Embodiment one
A kind of primary separation of human amnion mesenchymal stem cell and cultural method, specifically comprise the following steps:
1, the primary separation of human amnion mesenchymal stem cell
Aseptically, get the fresh human placenta of throwing aside postpartum in sterile tray, adopt instruments to be peeled off from placenta tissue by amnion, rinse for several times to remove residual bloodstain with PBS buffered soln.In 15cm glass dish, amnion tissue is cut into 6 × 6cm2, divides and be filled to (often pipe is containing amnion tissue 10 ~ 15cm) in 50mL centrifuge tube, add 0.1-0.3% pancreatin to 45mL.Centrifuge tube is transferred in constant-temperature table, 37 DEG C, 150-300r/min, digestion 30min, and every the violent shaken several times of 10min, to remove epithelial cell.Amnion tissue after trysinization is transferred in 250mL liquid storage bottle, adds 150mLPBS buffered soln, acutely shake, repeat this operation 2 times.Amnion tissue is transferred in 100mL small beaker, shreds to 1mm3.After be transferred in 125ml liquid storage bottle, adding 20mL0.5%I Collagenase Type and DMEM in high glucose basic medium, is 0.075-0.2% to collagenase final concentration.In constant-temperature table, 37 DEG C, 250r/min, digests to tissue block and substantially melts.Postdigestive tissue juice is diluted with PBS buffered soln, and 1500r/min, centrifugal 5min, collecting precipitation.After precipitation PBS buffered soln is resuspended, adopts 100 μm of filter screens to filter, filtrate 1500r/min, centrifugal 5min, obtain amnion mesenchymal stem cell.Use serum free medium re-suspended cell, platform is expected with countstar cell counter counting after blue dyeing, and adjustment cell density is that 3-10 × 105/mL carries out inoculation and is inoculated in 10cm culture dish, and every ware adds 100 μ L 1 μ g/mL EGF.Cell is moved in CO2 incubator, carry out changing liquid after cultivating 48h, remove not adherent hemocyte and other impurity.Every 2 ~ 3d changes a subculture afterwards.
2, human amnion mesenchymal stem cell Secondary Culture
When primary human amnion mesenchymal stem cell grows to 80% fusion, namely digest with the EDTA-Trypsin of 0.25%, carry out Secondary Culture.
Example two
Be separated the streaming qualification of amnion stem cell
When primary human amnion mesenchymal stem cell grows to 80% ~ 90%, collecting cell after trysinization, gets two 1.5mlEP pipes, often pipe 1x10
6individual cell, twice is washed with dye solution (10%FBS+90%PBS), often pipe adds 200 μ l dye solution, sample adds each 5 μ l of following four antibody CD45, CD59, HLADR, CD90, and negative control does not add antibody, hatches 20min for 4 DEG C, twice is washed with dye solution, after use 500 μ l 1640 substratum resuspended, gather 30000 samples with flow cytometer, detect the expression of its surface marker CD45, CD59, CD90, HLA-DR etc.The results are shown in accompanying drawing 1.
As can be seen from the result of accompanying drawing 1, sample expresses CD59, CD90; Low expression of HLA-DR, CD45, meet mescenchymal stem cell characteristic, also can find out that present method is primary and can obtain the higher mescenchymal stem cell of purity.
Example three
The growth curve of separate stem cells
The hAMSCs in collected by trypsinisation P2 generation, inoculating cell in 96 orifice plates, 2000/hole.At 37 DEG C, 5%CO
2cultured continuously 7d in incubator.Respectively at 1d, 3d, 5d, 7d, cell is detected.10ul staining agent is added, 37 DEG C, 5%CO in gaging hole to be checked
2cultivate 2h.Survey the light absorption value at 450nm place by microplate reader, each sample does 6 repetitions, the results are shown in Table 1.
Table 1
| 1 | 2 | 3 | 4 | 5 | 6 | Mean value | Variance | |
| 1d | 0.23 | 0.22 | 0.2 | 0.24 | 0.22 | 0.21 | 0.22 | 0.014142 |
| 3d | 0.36 | 0.38 | 0.38 | 0.37 | 0.39 | 0.36 | 0.373333 | 0.012111 |
| 5d | 0.72 | 0.69 | 0.71 | 0.68 | 0.69 | 0.7 | 0.698333 | 0.01472 |
| 7d | 0.82 | 0.84 | 0.91 | 0.88 | 0.87 | 0.9 | 0.87 | 0.034641 |
With OD value for ordinate zou, the time is X-coordinate, draws growth curve processed, as shown in Figure 2.
Can be found out by table 1 and accompanying drawing 2, the stem cell of separation and Culture gained of the present invention has stronger multiplication capacity.
Example four
The one-tenth fat differentiation capability of separate stem cells
Collect the human amnion mesenchymal stem cell in P2 generation, respectively with 10
5the cell density of individual/mL is inoculated in 6 orifice plates, when being cultured to 60 ~ 70% fusion, using adipogenic induction nutrient solution instead and cultivates, within every 2 days, change fresh inducing culture, adopt oil red O stain to be detected as fat differentiated result after 2 weeks.The results are shown in accompanying drawing 3.Accompanying drawing 3 is cellular form change (× 200) after hAMSCs adipogenic induction.
HAMSCs is in vitro in adipocyte Induction Process, cell is by original one-tenth threadiness, shorten and become short fusiformis, the rounded or Polygons of induction 10d visible most cells, and under high power lens, around visible cell, have the bright oil droplet cavity (Fig. 3-B) of different quantities.Induction 10d, oil red dyeing has red fat to drip (Fig. 3-C) as seen, and after 15d, visible most cells is full of red oil droplet cavity (Fig. 3-D).The cellular form of not inducing constant (Fig. 3-A).
Example five
The Osteoblast Differentiation ability of separate stem cells
Collect the human amnion mesenchymal stem cell in P2 generation, respectively with 10
5the cell density of individual/mL is inoculated in 6 orifice plates, when being cultured to 60 ~ 70% fusion, uses osteogenic induction nutrient solution instead, within every 2 days, changes fresh inducing culture, adopts Alizarin red staining to detect skeletonization differentiated result after 3 weeks.The results are shown in accompanying drawing 4.
As can be seen from accompanying drawing 4, hAMSCs is in vitro in Osteoinductive differentiation process, after induction, 7d starts to become polygon from spindle shape, cladding growth then, within about 2 weeks, forms the cellular nodules be dispersed in, there is calcareous infarct later, tubercle central authorities thicken gradually, until light tight, and visible significantly Mineral nodules during 21d, during 28d, Mineral nodules increases and merges (Fig. 4-B) mutually, and the calcium tubercle that can be formed with Alizarin red staining dyes redness (Fig. 4-C).And cellular control unit form constant be still spindle shape (Fig. 4-A).
Embodiment six
Human amnion mesenchymal stem cell is separated simultaneous test
Aseptically, get the placenta of health (HCV, HBV, HIV, syphilis are feminine gender) puerpera (pluripara's agreement) in sterile tray, adopt instruments to be peeled off from placenta tissue by amnion, rinse for several times to remove residual bloodstain with PBS buffered soln.In 15cm glass dish, amnion tissue is cut into 6 × 6cm
2, be divided into two parts, be respectively used to the test of group one and group two.
Group one: the amnion tissue after shredding is divided and is filled to (often pipe is containing amnion tissue 10 ~ 15cm) in two 50mL centrifuge tubes, add 0.1-0.3% pancreatin to 45mL.Centrifuge tube is transferred in constant-temperature table, 37 DEG C, 150-300r/min, digestion 30min, and every the violent shaken several times of 10min, to remove epithelial cell.Amnion tissue after trysinization is transferred in 250mL liquid storage bottle, adds 150mLPBS buffered soln, acutely shake, repeat this operation 2 times.Amnion tissue is transferred in 100mL small beaker, shreds to 1mm
3, after be divided into three parts.Every part adds 0.5%I Collagenase Type and DMEM in high glucose basic medium, is 0.1-0.2% to type i collagen enzyme final concentration; In constant-temperature table, 37 DEG C, 250r/min, digests to tissue block and substantially melts.Postdigestive tissue juice is diluted with PBS buffered soln, and 1500r/min, centrifugal 5min, collecting precipitation.After precipitation PBS buffered soln is resuspended, adopts 100 μm of filter screens to filter, filtrate 1500r/min, centrifugal 5min, obtain amnion mesenchymal stem cell.Use serum free medium re-suspended cell, count with countstar cell counter after platform expects blue dyeing, adjustment cell density is 3-10 × 10
5/ mL carries out inoculation and is inoculated in 10cm culture dish, and every ware adds 100 μ L 1 μ g/mL EGF.Cell is moved to CO
2in incubator, carry out changing liquid after cultivating 48h, remove not adherent hemocyte and other impurity.Every 2 ~ 3d changes a subculture afterwards.
Group two: by 37 DEG C, amnion tissue 2.5g/L trypsinase after shredding, 150-300r/min, digestion 10min; The DMEM added containing 5% calf serum stops digestion, adds the digestion of 1.0g/L II Collagenase Type, 37 DEG C, digestion 30min after crossing 200 order cell sieves; The DMEM added containing 5% calf serum stops digestion, after crossing 200 order cell sieves, and collecting cell; 1000r/min, centrifugal 5min, obtain human amnion mesenchymal stem cell.Count with countstar cell counter after platform expects blue dyeing, adjustment cell density is 1 × 10
6/ mL carries out inoculation and is inoculated in 10cm culture dish, and every ware adds 100 μ L 1 μ g/mL EGF.Cell is moved in CO2 incubator, carry out changing liquid after cultivating 48h, remove not adherent hemocyte and other impurity.Every 2 ~ 3d changes a subculture afterwards.When primary human amnion mesenchymal stem cell grows to 80% fusion, namely digest with the EDTA-Trypsin of 0.25%, carry out Secondary Culture.
1, cellular form is observed
After cell inoculation, observe under the microscope continuously, and take pictures to cell, the growing state of record cell and cellular form, the results are shown in Table 2 and accompanying drawing 5.
The primary separation of table 2
Accompanying drawing 5 have recorded group one and group two original cuiture two days later 40 times, cellular form under 100 power microscopes, original cuiture 40 times, cellular form under 100 power microscopes, and went down to posterity P2 culture after three days 40 times, cellular form under 100 power microscopes after four days.
As can be seen from table 2 and accompanying drawing 5, from the treatment time, cell viability, cell quantity and primary go down to posterity after the aspect such as cell state all can find out, the technique effect of technical scheme of the present invention is far superior to the technique effect of prior art.
The announcement of book and instruction according to the above description, those skilled in the art in the invention can also change above-mentioned embodiment and revise.Therefore, the present invention is not limited to embodiment disclosed and described above, also should fall in the protection domain of claim of the present invention modifications and changes more of the present invention.In addition, although employ some specific terms in this specification sheets, these terms just for convenience of description, do not form any restriction to the present invention.
Claims (8)
1. the primary separation of human amnion mesenchymal stem cell and a cultural method, is characterized in that comprising the following steps:
(1) human amniotic tissue is provided;
(2) use PBS wash buffer, then amnion tissue is shredded;
(3) add mass concentration be 0.1 ~ 0.3% trypsinase digest;
(4) postdigestive amnion tissue PBS wash buffer, then shreds amnion tissue;
(5) adding type i collagen enzyme and DMEM in high glucose basic medium, is that 0.1 ~ 0.2% digestion to amnion tissue block melts to type i collagen enzyme final concentration;
(6) postdigestive tissue juice is diluted, centrifugation, taking precipitate with PBS damping fluid;
(7) throw out PBS solution is resuspended, centrifugation after filtering, and abandoning supernatant obtains human amnion mesenchymal stem cell;
(8) use the resuspended human amnion mesenchymal stem cell of serum free medium, adjustment cell density is (3 ~ 10) × 10
5/ mL is placed in the culture dish that with the addition of EGF, moves into CO
2change liquid after cultivating 48h in incubator, remove impurity, change a subculture every 2 ~ 3d.
2. the primary separation of human amnion mesenchymal stem cell as claimed in claim 1 and cultural method, is characterized in that: amnion tissue shreds to 6 × 6cm by step (2)
2be sub-packed in 50mL centrifuge tube, step (3) pancreatin addition is 45mL, and digests in constant-temperature table, and digestion time is 30min, and digestion temperature is 37 DEG C, and rotating speed is 150 ~ 300r/min.
3. the primary separation of human amnion mesenchymal stem cell as claimed in claim 1 and cultural method, is characterized in that: amnion tissue shreds to 1mm by step (4)
2after be transferred in 125mL liquid storage bottle, add type i collagen enzyme and DMEM in high glucose basic medium that 20mL mass concentration is 0.5%, be 0.075 ~ 0.2% to digest to type i collagen enzyme final concentration, digestion is carried out in constant-temperature table, digestion temperature is 37 DEG C, rotating speed is 250 ~ 300r/min, digests to tissue block and melts.
4. the primary separation of human amnion mesenchymal stem cell as claimed in claim 1 and cultural method, is characterized in that: described serum free medium is UltraCULTURE MEDIUM substratum.
5. the primary separation of human amnion mesenchymal stem cell as claimed in claim 1 and cultural method, it is characterized in that: step (6) centrifugal speed is 1500 ~ 2000r/min, centrifugation time is 5min.
6. the primary separation of human amnion mesenchymal stem cell as claimed in claim 1 and cultural method, is characterized in that: step (7) is through 100 μm, and centrifugal speed is 1500 ~ 2000r/min, and centrifugation time is 5min.
7. the primary separation of human amnion mesenchymal stem cell as claimed in claim 1 and cultural method, is characterized in that: step (8) adopts specification to be the culture dish of 10cm, and EGF addition is 100 μ L, and concentration is 1 μ g/mL.
8. the primary separation of human amnion mesenchymal stem cell as claimed in claim 1 and cultural method, is characterized in that: when primary human amnion mesenchymal stem cell grows to 80% fusion, with the EDTA-Trypsin digestion of 0.25%, carries out Secondary Culture.
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| CN105062959A (en) * | 2015-09-20 | 2015-11-18 | 领航干细胞再生医学工程有限公司 | Isolated culture method of human amnia mesenchymal stem cells |
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