CN104450611B - A kind of primary isolation and culture method of human amnion mesenchymal stem cell - Google Patents
A kind of primary isolation and culture method of human amnion mesenchymal stem cell Download PDFInfo
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Abstract
The present invention relates to a kind of primary isolation and culture methods of human amnion mesenchymal stem cell, comprise the following steps：There is provided human amniotic tissue, with PBS buffer solution rinse then shred, adds in mass concentration be 0.1~0.3% trypsase digested, add in type i collagen enzyme and DMEM in high glucose minimal medium to type i collagen enzyme it is final concentration of 0.1~0.2% digestion, centrifugation, taking precipitate, sediment are resuspended with PBS solution, it is centrifuged after filtering, abandoning supernatant obtains human amnion mesenchymal stem cell.Compared with prior art, the present invention pre-processes amnion tissue using a small amount of trypsase, make amnion tissue loose, and then utilize the better type i collagen enzymatic treatment amnion of tissue specificity, substantially reduce the time of digestion, the step of simplifying primary separation, and higher yield of dried cell can be obtained, the stem cell vigor of acquisition also greatly improves compared with prior art.
Description
Technical field
The present invention relates to stem cells technology fields, and in particular to a kind of primary isolation and culture of human amnion mesenchymal stem cell
Method.
Background technology
Mescenchymal stem cell (Mesenchymal Stem Cells, MSCs) be mesoderma origin have height self more
The versatile stem cell of new ability and multi-lineage potential, is widely present in whole body Various Tissues, can cultivate amplification in vitro,
And nerve cell, osteocyte, cartilage cell, muscle cell, adipocyte etc. can be divided under given conditions.MSCs is multipotency
Stem cell has the growth of the ability, not only hematopoiesis support stem cell of " transdifferentiationof " or " interdepartmental differentiation ", can also be in difference
Under inductive condition, Various Tissues cell is divided into vitro.MSCs has wide potential applicability in clinical practice, is cell replacement therapy
With the preferred seed cell of organizational project.
Mesenchymal stem cell (Bone Marrow Derived Mesenchymal Stem Cells, BMSCs) is
A kind of mescenchymal stem cell being more early realized, but since bone marrow extraction can bring great pain to patient, and with year
The growth in age, cell quantity are remarkably decreased, and differentiation capability continuously decreases, and are promoted the use of so as to more difficult.At present from fat,
Mescenchymal stem cell is isolated in the histoorgans such as spleen, bleeding of the umbilicus, umbilical cord, tire liver and tire kidney, but is had some limitations,
Such as draw materials it is difficult, by age limit, to be related to mescenchymal stem cell content in ethics problem, tissue few.So seek a kind of next
Source is enriched, and stem cell content is high, convenient material drawing, and non-invasive and from ethics limitation MSCs sources already become of today
Research hotspot.
Studies have found that, AMSCs has the phenotype similar to BMSCs, and dives with self-renewing and Multidirectional Differentiation recently
Can, and multiplication capacity is more stronger than BMSCs.AMSCs low expression HLA-ABC, without expression of HLA-DR, so as to illustrate that AMSCs has
After allogeneic, Xenogeneic, immunological rejection will not occur for low immunogenicity, and AMSCs is without tumorigenesis
Property, this provides new selection for the cell replacement therapy of various diseases.
People is amnion-derived in human placenta, no blood vessel, nerve and lymph, has certain elasticity, and thickness 0.02~
0.5mm.Under Electronic Speculum, it is divided into 5 layers：Epithelial layer, basilar memebrane, compacted zone, fibroblast layer and spongy layer.Amnion belongs to pregnant
Discarded object after woman's production, the amnion area about 600cm of a placenta2, and amnion is easy to from separation of placenta, so because
Amnion is considered as the best source of current mescenchymal stem cell with the characteristics of convenient material drawing, raw material is sufficient.At present,
The primary isolated culture methods of hAMSCs can be divided into two kinds of tissue mass cell culture and method of tissue block, and tissue mass cell culture is not due to
It is that each tissue block can successfully turn out cell, and is easily mixed into other heteroproteose cells, while cultivation cycle is longer, is unfavorable for fast
Fast a large amount of acquisition cells, it is difficult to meet the needs of stem-cell research.Method of tissue block is current amnion method for separating stem cell
Mainstream, but in view of amnion tissue compact structure, the type selection of digestive ferment stem cell can be separated after activity have to
Close important influence.The prior art has that time-consuming using the separated technology of II Collagenase Types, and cell must be measured under low and activity
The weakness such as drop.
The content of the invention
The defects of aiming to overcome that the above-mentioned prior art of invention, provides a kind of primary point of human amnion mesenchymal stem cell
From and cultural method, this method pre-process amnion tissue using a small amount of trypsase, make amnion tissue loose, and then using tissue
The step of specific preferably type i collagen enzymatic treatment amnion substantially reduces the time of digestion, simplifies primary separation, and
Higher yield of dried cell can be obtained, the stem cell vigor of acquisition also greatly improves compared with prior art.
To achieve the above object, the present invention adopts the following technical scheme that：
A kind of primary isolation and culture method of human amnion mesenchymal stem cell, it is characterised in that comprise the following steps：
(1) human amniotic tissue is provided；
(2) rinsed with PBS buffer solution, then shred amnion tissue；
(3) trypsase that mass concentration is 0.1~0.3% is added in be digested；
(4) postdigestive amnion tissue is rinsed with PBS buffer solution, then shreds amnion tissue；
(5) type i collagen enzyme and DMEM in high glucose basal medium are added in, until type i collagen enzyme final concentration of 0.1~0.2% disappears
Change to amnion tissue block and melt；
(6) postdigestive tissue fluid is diluted with PBS buffer solution, centrifuged, taking precipitate；
(7) sediment is resuspended with PBS solution, is centrifuged after filtering, abandoning supernatant, is obtained human amnion mesenchymal and is done carefully
Born of the same parents；
(8) human amnion mesenchymal stem cell is resuspended using serum free medium, adjustment cell density is (3~10) × 105/
ML is placed in the culture dish for being with the addition of EGF, moves into CO2Liquid is changed after 48h is cultivated in incubator, impurity is removed, one is changed every 2~3d
Subculture.
Preferably, step (2) shreds amnion tissue to 6 × 6cm2It is sub-packed in 50mL centrifuge tubes, step (3) pancreatin adds
Dosage is 45mL, and is digested in constant-temperature table, digestion time 30min, and digestion temperature is 37 DEG C, and rotating speed is 150~300r/
min。
Preferably, step (4) shreds amnion tissue to 1mm2After be transferred in 125mL liquid storage bottles, add in 20mL mass
Type i collagen enzyme and the DMEM in high glucose basal medium that concentration is 0.5%, until type i collagen enzyme final concentration of 0.1~0.2% carries out
Digestion, digests and is carried out in constant-temperature table, and digestion temperature is 37 DEG C, and rotating speed is 250~300r/min, digests to tissue block and melts
Change.
Preferably, the serum free medium is UltraCULTURE MEDIUM culture mediums.
Preferably, step (6) centrifugal speed is 1500~2000r/min, centrifugation time 5min.
Preferably, for step (7) through 100 μm, centrifugal speed is 1500~2000r/min, centrifugation time 5min.
Preferably, step (8) uses culture dish of the specification for 10cm, and EGF additive amounts are 100 μ L, and concentration is 1 μ g/mL.
Preferably, when primary human amnion mesenchymal stem cell grows to 80% fusion, i.e., with 0.25% EDTA-
Trypsin digests, and carries out secondary culture.
Compared with prior art, the beneficial effects of the invention are as follows：
This patent pre-processes amnion tissue using a small amount of trypsase, makes amnion tissue loose, and then utilizes organizing specific
Property better type i collagen enzymatic treatment amnion, so as to shorten the time of digestion, improve yield and cell viability.
1st, the present invention explores the enzymolysis species of most suitable human amnion tissue feature, the order of enzymolysis, the condition of enzymolysis, leads to
It crosses technical scheme human amnion tissue can thoroughly to be digested, human amnion mesenchymal stem cell is released, obtain
The stem cell that quantity is more, purity is higher, while the structure of stem cell will not be destroyed, the activity of stem cell is not influenced, ensures to obtain
The stem cell obtained has higher vigor, can meet Research Requirements with mass propgation.
2nd, in the present invention, early period is digested using a small amount of trypsase, in the dosage of defined enzyme and digestion item
Under part, amnion tissue can be made to be in just loose, the epithelial cell on amnion surface layer is also just removed at this time, is most suitable for utilizing
To human amnion tissue specificity, preferably type i collagen enzyme removes enzymolysis, digestion, and digestion process is small to cellular damage, and Cell viability is high,
The stem cell amount obtained after digestion is big and purity is high, can be kept during secondary culture afterwards good cytoactive and
Multiplication capacity.
3rd, the cycle of present invention acquisition human amnion mesenchymal stem cell is short, and yield and activity are high, between the amnion separated
It can be grown within three days after mesenchymal stem cells inoculation and be fused to 80%-90%, be conducive to rapid, high volume and obtain stem cell, be suitble to extensive
Change production application
4th, the present invention using serum free medium (UltraCULTURE MEDIUM) carry out cell culture, can to avoid containing
Serum free culture system base weight serum has genotoxic potential effect and serum source contact scar to cell.
Description of the drawings
Fig. 1 is the expression figure of the surface standard object of the human amnion mesenchymal stem cell primary cell of the present invention；
Fig. 2 is growth curve figures of the human amnion mesenchymal stem cell P2 for cell of the present invention；
Fig. 3 is the human amnion mesenchymal stem cell P2 of the present invention for cellular morphology variation diagram after cell adipogenic induction；
Wherein, A is negative control, the amnion mesenchymal stem cell aspect graph not induced；B is nothing 10 days after adipogenic induction
The cellular morphology figure of oil red O stain；C is adipogenic induction after 10 days, the cellular morphology figure of oil red O stain；D is adipogenic induction 10 days
Afterwards, the cellular morphology figure of oil red O stain.
Fig. 4 is the human amnion mesenchymal stem cell P2 of the present invention for cellular morphology variation diagram after cell osteogenic induction；
Wherein, A:Negative control, i.e., the cellular morphology of non-induced osteogenesis differentiation；B:It is undyed after osteogenic induction 28 days
Cellular morphology；C:After osteogenic induction 28 days, with the cellular morphology of Alizarin red staining.
Fig. 5 be the present invention human amnion mesenchymal stem cell and the human amnion mesenchymal stem cell that obtains of the prior art it is thin
Born of the same parents' form comparison diagram；
Wherein, A1：Cellular morphology figure under 2nd day 40 power microscopes of primary cell of the present invention,
A2：Cellular morphology figure under 2nd day 100 power microscopes of primary cell of the present invention,
A3：Cellular morphology figure under 4th day 40 power microscopes of primary cell of the present invention,
A4：Cellular morphology figure under 4th day 100 power microscopes of primary cell of the present invention,
A5：P2 of the present invention for the cellular morphology figure under the 3rd day 40 power microscopes of cell,
A6:P2 of the present invention for the cellular morphology figure under the 3rd day 100 power microscopes of cell,
B1:Cellular morphology figure under the 2nd day 40 power microscopes of prior art primary cell,
B2：Cellular morphology figure under the 2nd day 100 power microscopes of prior art primary cell,
B3：Cellular morphology figure under the 4th day 40 power microscopes of prior art primary cell,
B4：Cellular morphology figure under the 4th day 100 power microscopes of prior art primary cell,
B5：Prior art P2 for the cellular morphology figure under the 3rd day 40 power microscopes of cell,
B6:Prior art P2 is for the cellular morphology figure under the 3rd day 100 power microscopes of cell.
Specific embodiment
The present invention is described in further detail below by specific embodiment, to be clearly understood that the present invention to be protected
The technical solution of shield.
Embodiment one
A kind of primary isolation and culture method of human amnion mesenchymal stem cell, specifically includes following steps：
1st, the primary separation of human amnion mesenchymal stem cell
Aseptically, the fresh human placenta that postpartum throws aside is taken in sterile tray, using surgical instrument by amnion from tire
Disk tissue is removed, and is rinsed with PBS buffer solutions and is remained bloodstain for several times to remove.Amnion tissue is cut in 15cm glass dishes
Into 6 × 6cm2, (often pipe contains 10~15cm of amnion tissue) is dispensed into 50mL centrifuge tubes, adds 0.1-0.3% pancreatin to 45mL.
Centrifuge tube is transferred in constant-temperature table, 37 DEG C, 150-300r/min, digests 30min, and every the violent shaken several times of 10min,
To remove epithelial cell.The postdigestive amnion tissue of pancreatin is transferred in 250mL liquid storage bottles, it is molten to add in 150mLPBS bufferings
Liquid is aggressively shaken, and repeats this operation 2 times.Amnion tissue is transferred in 100mL small beakers, is shredded to 1mm3.After be transferred to
In 125ml liquid storage bottles, 20mL0.5%I Collagenase Types and DMEM in high glucose basal medium are added in, until clostridiopetidase A is final concentration of
0.075-0.2%.In constant-temperature table, 37 DEG C, 250r/min is digested to tissue block and melted substantially.It is diluted with PBS buffer solutions
Postdigestive tissue fluid, and 1500r/min centrifuge 5min, collect precipitation.After precipitation is resuspended with PBS buffer solutions, using 100 μ
M filter screens filter, filtrate 1500r/min, centrifuge 5min, obtain amnion mesenchymal stem cell.It is resuspended with serum free medium
Cell, platform are counted after expecting blue dyeing with countstar cell counters, and adjustment cell density is inoculated with for 3-10 × 105/mL
It is inoculated in 10cm culture dishes, 100 μ L, 1 μ g/mL EGF is added per ware.Cell is moved in CO2 incubators, culture 48h is laggard
Row changes liquid, the not adherent haemocyte of removal and other impurities.Every 2~3d changes a subculture afterwards.
2nd, human amnion mesenchymal stem cell secondary culture
When primary human amnion mesenchymal stem cell grows to 80% fusion, i.e., disappeared with 0.25% EDTA-Trypsin
Change, carry out secondary culture.
Example two
Separate the streaming identification of amnion stem cell
When primary human amnion mesenchymal stem cell grows to 80%~90%, cell is collected after pancreatin digestion, takes two
1.5mlEP is managed, often pipe 1x106A cell is washed twice with dye solution (10%FBS+90%PBS), often pipe addition 200 μ l dyes
Color buffer solution, sample add in following four antibody each 5 μ l of CD45, CD59, HLADR, CD90, and negative control is not added with antibody, and 4 DEG C incubate
20min is educated, is washed twice with dye solution, it is rear to be resuspended with 500 μ l, 1640 culture mediums, gather 30000 samples with flow cytometer
This, detects the expression of its surface marker CD45, CD59, CD90, HLA-DR etc..The result is shown in attached drawings 1.
Sample expression CD59, CD90 are can be seen that from the result of attached drawing 1；Low expression HLA-DR, CD45 meets mesenchyma
Stem cell properties, it is also seen that this method is primary can to obtain the higher mescenchymal stem cell of purity.
Example three
Separate the growth curve of stem cell
The hAMSCs in collected by trypsinisation P2 generations, inoculating cell is in 96 orifice plates, 2000/hole.At 37 DEG C, 5%CO2Training
It supports in case and continuously cultivates 7d.Respectively in 1d, 3d, 5d, 7d, cell is detected.10ul coloring agents are added in gaging hole to be checked,
37 DEG C, 5%CO2Cultivate 2h.The light absorption value at 450nm is surveyed with microplate reader, each sample does 6 repetitions, the results are shown in Table 1.
Table 1
| 1 | 2 | 3 | 4 | 5 | 6 | Average 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 |
Using OD values as ordinate, the time is abscissa, draws growth curve processed, as shown in Figure 2.
It can be seen that by table 1 and attached drawing 2, the stem cell that the present invention is separately cultured gained has stronger multiplication capacity.
Example four
Separate stem cell into fat differentiation capability
The human amnion mesenchymal stem cell in P2 generations is collected, respectively with 105The cell density of a/mL is inoculated in 6 orifice plates, training
It supports to adipogenic induction culture solution culture during 60~70% fusion, is used instead, replaces within every 2 days fresh inducing culture, adopted after 2 weeks
It is detected with oil red O stain into fat differentiated result.The result is shown in attached drawings 3.Attached drawing 3 is cellular morphology variation after hAMSCs adipogenic inductions
(×200)。
HAMSCs is in vitro into adipocyte Induction Process, and for cell by original into threadiness, shortening becomes short fusiformis,
It induces the visible most cells of 10d rounded or polygon, and has the bright of different number around visible cell under high power lens
Bright oil droplet vacuole (Fig. 3-B).Induce 10d, oil red dyeing is visible there are a red fat drips (Fig. 3-C), visible most of thin after 15d
Born of the same parents are full of red oil droplet vacuole (Fig. 3-D).The cellular morphology not induced is constant (Fig. 3-A).
Example five
Separate the Osteoblast Differentiation ability of stem cell
The human amnion mesenchymal stem cell in P2 generations is collected, respectively with 105The cell density of a/mL is inoculated in 6 orifice plates, training
It supports to osteogenic induction culture solution during 60~70% fusion, is used instead, fresh inducing culture is replaced within every 2 days, using alizarin after 3 weeks
Plain red colouring detects Osteoblast Differentiation result.The result is shown in attached drawings 4.
During hAMSCs it can be seen from attached drawing 4 in vitro Osteoinductive differentiation, 7d starts by long shuttle after induction
Shape becomes polygonal, and then cladding is grown, and forms the cellular nodules being dispersed within 2 weeks or so, occurs calcareous infarct, tubercle center later
Gradually thicken, until light tight, visible apparent Mineral nodules during 21d, during 28d Mineral nodules increase and merge into each other (Fig. 4-
B), the calcium tubercle that can be formed with Alizarin red staining is dyed red (Fig. 4-C).And it is still long shuttle that cellular control unit form is constant
Shape (Fig. 4-A).
Embodiment six
Human amnion mesenchymal stem cell separates contrast test
Aseptically, take the placenta of healthy (HCV, HBV, HIV, syphilis are feminine gender) puerpera (multipara's agreement) in
In sterile tray, amnion is removed from placenta tissue using surgical instrument, is rinsed with PBS buffer solutions and is remained for several times with removing
Bloodstain.Amnion tissue is cut into 6 × 6cm in 15cm glass dishes2, it is divided into two parts, is respectively used to the experiment of group one and group two.
Group one：Amnion tissue after shredding is dispensed into two 50mL centrifuge tubes (often pipe containing amnion tissue 10~
15cm), 0.1-0.3% pancreatin is added to 45mL.Centrifuge tube is transferred in constant-temperature table, 37 DEG C, 150-300r/min, digested
30min, and every the violent shaken several times of 10min, to remove epithelial cell.The postdigestive amnion tissue of pancreatin is transferred to
In 250mL liquid storage bottles, 150mLPBS buffer solutions are added in, are aggressively shaken, repeat this operation 2 times.Amnion tissue is transferred to
In 100mL small beakers, shred to 1mm3, after be divided into three parts.Every part adds in 0.5%I Collagenase Types and DMEM in high glucose basis
Culture medium, until the final concentration of 0.1-0.2% of type i collagen enzyme；In constant-temperature table, 37 DEG C, 250r/min, digest to tissue block base
This thawing.Postdigestive tissue fluid, and 1500r/min are diluted with PBS buffer solutions, 5min is centrifuged, collects precipitation.Precipitation is used
It after PBS buffer solutions are resuspended, is filtered using 100 μm of filter screens, filtrate 1500r/min, centrifuges 5min, obtain amnion mesenchymal
Stem cell.Cell is resuspended with serum free medium, platform is counted after expecting blue dyeing with countstar cell counters, adjusts cell
Density is 3-10 × 105/ mL be inoculated in 10cm culture dishes, and 100 μ L, 1 μ g/mL EGF are added per ware.By cell
Move to CO2In incubator, carry out changing liquid, the not adherent haemocyte of removal and other impurities after cultivating 48h.Every 2~3d is changed afterwards
One subculture.
Group two：By 37 DEG C, 150-300r/min of 2.5g/L trypsase of the amnion tissue after shredding, 10min is digested；Add
The DMEM for entering to contain 5% calf serum terminates digestion, and addition 1.0g/L II Collagenase Types digest after crossing 200 mesh cell sieves, 37 DEG C,
Digest 30min；It adds in the DMEM containing 5% calf serum and terminates digestion, after crossing 200 mesh cell sieves, collect cell；1000r/min,
5min is centrifuged, obtains human amnion mesenchymal stem cell.Platform is counted after expecting blue dyeing with countstar cell counters, and adjustment is thin
Born of the same parents' density is 1 × 106/ mL be inoculated in 10cm culture dishes, and 100 μ L, 1 μ g/mL EGF are added per ware.Cell is moved
Into CO2 incubators, carry out changing liquid, the not adherent haemocyte of removal and other impurities after cultivating 48h.Every 2~3d changes one afterwards
Subculture.When primary human amnion mesenchymal stem cell grows to 80% fusion, i.e., disappeared with 0.25% EDTA-Trypsin
Change, carry out secondary culture.
1st, cellular morphology is observed
After cell inoculation, continuously cell is observed under the microscope, and is taken pictures, record cell growing state and
Cellular morphology the results are shown in Table 2 and attached drawing 5.
2 primary separation of table
Attached drawing 5 has recorded group one and two original cuitures of the group cellular morphology under 40 times, 100 power microscopes two days later, primary
Culture four days after under 40 times, 100 power microscopes cellular morphology and passage P2 be commissioned to train foster three days after 40 times, 100 times it is micro-
Cellular morphology under mirror.
It can be seen that from table 2 and attached drawing 5 from cellular after processing time, cell viability, cell quantity and primary passage
The technique effect that state etc. can be seen that technical scheme is far superior to the technique effect of prior art.
According to the disclosure and teachings of the above specification, those skilled in the art in the invention can also be to above-mentioned embodiment party
Formula is changed and changed.Therefore, the invention is not limited in specific embodiment disclosed and described above, to the present invention's
Some modifications and changes should also be as falling into the scope of the claims of the present invention.In addition, it although is used in this specification
Some specific terms, but these terms are merely for convenience of description, do not limit the present invention in any way.
Claims (4)
- A kind of 1. primary isolation and culture method of human amnion mesenchymal stem cell, it is characterised in that comprise the following steps：(1) human amniotic tissue is provided；(2) rinsed with PBS buffer solution, then shred amnion tissue；(3) trypsase that mass concentration is 0.1~0.3% is added in be digested；(4) postdigestive amnion tissue is rinsed with PBS buffer solution, then shreds amnion tissue；(5) amnion tissue is shredded to 1mm2After be transferred in 125mL liquid storage bottles, add in the I types that 20mL mass concentrations are 0.5% Clostridiopetidase A and DMEM in high glucose basal medium until type i collagen enzyme final concentration of 0.075~0.2% is digested, are digested in constant temperature It is carried out in shaking table, digestion temperature is 37 DEG C, and rotating speed is 250~300r/min, digests to tissue block and melts；(6) postdigestive tissue fluid is diluted with PBS buffer solution, centrifuged, taking precipitate；(7) sediment is resuspended with PBS solution, is centrifuged after filtering, abandoning supernatant, obtains human amnion mesenchymal stem cell；(8) human amnion mesenchymal stem cell is resuspended using serum free medium, adjustment cell density is (3~10) × 105/ mL is put In being with the addition of in the culture dish of EGF, CO is moved into2Liquid is changed after 48h is cultivated in incubator, impurity is removed, changes every 2~3d and once train Support base；Wherein, the serum free medium is UltraCULTURE MEDIUM culture mediums；Step (2) shreds amnion tissue to 6 ×6cm2It is sub-packed in 50mL centrifuge tubes, step (3) pancreatin additive amount is 45mL, and is digested in constant-temperature table, digestion time For 30min, digestion temperature is 37 DEG C, and rotating speed is 150~300r/min；Step (8) uses culture dish of the specification for 10cm, EGF Additive amount is 100 μ L, and concentration is 1 μ g/mL.
- 2. the primary isolation and culture method of human amnion mesenchymal stem cell as described in claim 1, it is characterised in that：Step (6) centrifugal speed is 1500~2000r/min, centrifugation time 5min.
- 3. the primary isolation and culture method of human amnion mesenchymal stem cell as described in claim 1, it is characterised in that：Step (7) filtered through 100 μm of filter screens, centrifugal speed is 1500~2000r/min, centrifugation time 5min.
- 4. the primary isolation and culture method of human amnion mesenchymal stem cell as described in claim 1, it is characterised in that：It treats primary When human amnion mesenchymal stem cell grows to 80% fusion, digested with 0.25% EDTA-Trypsin, carry 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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| CN103789258A (en) * | 2012-11-30 | 2014-05-14 | 陆华 | Separation method for human amniotic mesenchymal stem cells |
| CN103013913A (en) * | 2012-11-30 | 2013-04-03 | 陆华 | Method for culturing human amniotic mesenchymal stem cells by enzyme digestion and EGF (epidermal growth factor) |
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