CN105062959A - Isolated culture method of human amnia mesenchymal stem cells - Google Patents
Isolated culture method of human amnia mesenchymal stem cells Download PDFInfo
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- CN105062959A CN105062959A CN201510597698.4A CN201510597698A CN105062959A CN 105062959 A CN105062959 A CN 105062959A CN 201510597698 A CN201510597698 A CN 201510597698A CN 105062959 A CN105062959 A CN 105062959A
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Abstract
The invention discloses an isolated culture method of human amnia mesenchymal stem cells, which comprises the following steps: carrying out human placenta amnia tissue acquisition, amnia tissue disinfection, washing, shredding and mixed collagenase digestion to obtain mesenchymal stem cells, and carrying out mesenchymal stem cell in-vitro culture. The method can obtain abundant mesenchymal stem cells, which have the advantages of uniform and stable phenotype and high multiplication capacity and have the multidirectional differentiation potential, from the human amnia tissues; and the culture medium in-vitro amplification is performed to purify the cells, thereby satisfying the quantity for cryopreservation or clinical use. The method has the advantages of short cell culture period and high repetitiveness. The stem cells obtained by the method have the phenotypes of CD105<+>, CD90<+> and CD73<+>.
Description
Technical field
The present invention relates to biomedicine field, specifically a kind of isolation cultivation method of human amnion mesenchymal stem cell.
Background technology
Mescenchymal stem cell (MSC) is that a class has self-renewal capacity and under applicable microenvironment, has the initiating cell of polyphyly differentiation potential.First it find in marrow, subsequently respectively at fat, bone, muscle, lung, liver, pancreas and separate in umbilical cord, Cord blood, placenta tissue.Because it has powerful multiplication capacity, stronger differentiation capability, reduced immunogenicity and immunoloregulation function, its using value clinically is more and more paid close attention to by people.
As the cellular product that will be applied to clinical cytology treatment, should have tissue sampling convenient, wide material sources, do not limit by ethics; The features such as sepn process is simple, and the MSC of bone marrow has difficulty of drawing materials, cell proliferation and differentiation capability limit by donor age, the shortcomings such as heteroplastic transplantation immunological rejection is strong, 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, the mescenchymal stem cell (hAMSCs) in people's amnion source has self and multi-lineage potential, there is reduced immunogenicity, after allogeneic, Xenogeneic, immunological rejection can not be there is, without tumorigenicity, this is that the cell replacement therapy of various disease provides new selection.
Amnion-derived in the placenta tissue in pregnant woman's postpartum, without blood vessel, nerve and Lymphoid tissue, have certain elasticity, be easy to peel off from placenta tissue, therefore amnion is acknowledged as one of mescenchymal stem cell best source.At present, separation method block culture method and the enzyme elimination two kinds in a organized way of hAMSCs, it is long that tissue mass cell culture has culture cycle, the shortcomings such as cell purity is low are difficult to the needs meeting stem cell clinical study, enzyme digestion improves on the basis of uterus tissue pieces, there is cell culture period short, cell purity advantages of higher, but the type selecting of digestive ferment has very crucial effect to the separation of cell and activity, prior art utilizes the separation method of pancreatin and the process of collagenase branch to have length consuming time, complex steps, the weakness such as cell viability is low; And add the animal derived materials such as foetal calf serum in cultivation body, add toxigenic capacity, have also been introduced exogeneous animal albumen, make cultured cells be unfavorable for clinical application.
Summary of the invention
The object of the present invention is to provide a kind of isolation cultivation method of homogeneous, stable, human amnion mesenchymal stem cell that growth cycle is short, to solve the problem proposed in above-mentioned background technology.
For achieving the above object, the invention provides following technical scheme:
An isolation cultivation method for human amnion mesenchymal stem cell, comprises the following steps:
(1) collection of Human plactnta amnion tissue: be immersed in after Human plactnta takes off in placenta conserving liquid, maintains in the environment of 2-8 DEG C and preserves;
(2) amnion tissue sterilization: placenta is taken out from placenta conserving liquid, lies on sterile tray, the one side containing amnion tissue upwards, with the clockwise wiping of 60-90% alcohol swab, repeat 2-3 time, from placenta centered by umbilical cord denuded amniotic membrane tissue;
(3) wash: amnion tissue is put into sterile petri dish, with tissue samples washings washing 2-3 time, every minor tick 3-8min, then use brine 2-3 time;
(4) shred: amnion tissue is put into centrifuge tube, add-on is 4.8-5.2ml/ pipe, is cut into 1-3mm with a head scissors
3fritter;
(5) collagenase digesting: add isopyknic epoxy glue protoenzyme and fully mix, 35-40 DEG C, 150-200rpm concussion digestion 8-30min; Effective constituent in epoxy glue protoenzyme is 0.1-0.5% according to the add-on of mass volume ratio m/v;
(6) acquisition of mescenchymal stem cell: by digestion product through screen filtration, collecting cell just suspension, by cell, just suspension is after three subgradients are centrifugal, and getting the centrifugal precipitation obtained, to add blood serum medium resuspended, obtains the mesenchyma stem cell suspension be separated;
(7) derived mesenchymal stem cells in vitro cultivate: the stem cell of separation is inoculated in culturing bottle, and adds substratum, be then placed in 37 DEG C, saturated humidity, CO
2volume fraction is the interior cultivation of incubator of 5%, liquid is changed first in 24-48h, remove the hemocyte of suspension and not adherent heteroproteose cell, within three days afterwards, full dose changes liquid, when cell grows to 80%-90% degrees of fusion, namely obtain the dry primary cell of human amnion mesenchymal, now carry out Secondary Culture to cell and reach 80% degree of converging, namely obtain the human amnion mesenchymal stem cell of vitro culture, then carry out frozen to Human plactnta mescenchymal stem cell or continue amplification of going down to posterity.
As the further scheme of the present invention: the concrete steps of Secondary Culture: outwell the substratum in culturing bottle, with the brine cell surface twice that massfraction is 0.9%, add the trypsinase that 10ml concentration is 0.075% ~ 0.125%, 37 DEG C of digestion, basis of microscopic observation starts to become circle to cell, add nutrient solution and stop digestion, cell is blown and beaten with transfer pipet, cell is come off completely, the cell come off and nutrient solution are transferred in centrifuge tube, the centrifugal 6-10min of 1000-1800rpm, by resuspended for the blood serum medium of the precipitation of centrifugal gained, by 1-2 × 10
5/ ml density is inoculated in culturing bottle and carries out P1 culture, and every bottle adds 15ml substratum, and within 2-3 days, namely cell reaches 80% degree of converging.
As the further scheme of the present invention: the shelf time that Human plactnta maintains in the environment of 2-8 DEG C is no more than 24h.Pregnant woman HBV antigen, HCV antigen/antibody combination, the test items such as ANTI-HIV DRUGS, anti-syphilis helicoid antibody, ALT, mycoplasma are feminine gender.
As the further scheme of the present invention: the composition of placenta conserving liquid is DMEM/F12,0.3-0.8% serum substitute, 45000-55000IU/ml Cephalexin Monohydrate Micro/Compacted, 75000-85000U/ml gentamicin, 25-35ug/ml amphotericin B; Use NaHCO
3adjusted to ph is 7.0-7.2.Placenta conserving liquid and cleaning process add the Cephalexin Monohydrate Micro/Compacted of wide spectrum, gentamicin and amphotericin B, to very effective with the amnion process of mould, bacterium in birth process, can greatly reduce the underproof probability of mescenchymal stem cell separation and Culture because mould, bacterial contamination cause.
As the further scheme of the present invention: in tissue digestion process, the concentration of epoxy glue protoenzyme is 0.25%, digestion time is 23min.
As the further scheme of the present invention: described epoxy glue protoenzyme is the mixture of collagenase P and type i collagen enzyme, the mass ratio of collagenase P and type i collagen enzyme is 3:2.Collagenase can be hydrolyzed the three-dimensional spiral structure of natural collagen protein specifically under physiological pH and temperature condition, only has digestion and little to impact cell to intercellular substance, compare pancreatin action temperature and; And after traditional amnion cell is separated and all adopts pancreatin pre-treatment, then digest by collagenase I type or II type, not only complex steps, and digestion time is long, and cell injury is large.
As the further scheme of the present invention: blood serum medium end user AB serum.
As the further scheme of the present invention: three subgradients are successively centrifugal: the centrifugal 8min of 2000rpm centrifugal 10min, 1800rpm centrifugal 10min, 1500rpm.
As the further scheme of the present invention: tissue samples washings is the physiological saline containing 450-550IU/ml Cephalexin Monohydrate Micro/Compacted, 700-900U/ml gentamicin, 2-5ug/ml amphotericin.
As the further scheme of the present invention: the order number of screen cloth is 150 orders.
Compared with prior art, the invention has the beneficial effects as follows:
Stem cell isolation techniques of the present invention, in conjunction with special substratum, makes 5ml human amnion tissue just can obtain 2.0*10 through once going down to posterity
7stem cell seed, the original cuiture cycle is 6-7 days, and the cycle of going down to posterity is 2-3 days.The present invention can obtain a large amount of phenotype stable homogeneous from human amnion tissue, and multiplication capacity is strong, and has the mescenchymal stem cell of multi-lineage potential, and by substratum amplification in vitro, purifying cells, meets quantity that is frozen or Clinical practice.Cell culture period is short, and repeatability is strong.The stem cell that this method obtains has CD105
+, CD90
+, CD73
+phenotype.
The present invention optimizes the separation method of human amnion mesenchymal stem cell further in actually operating, adopt collagenase P and type i collagen enzyme simultaneous digestion method that amnion tissue is digested more thorough, reticular tissue is removed by screen filtration, significantly improve the purity of cell, adopt human serum substratum effectively can maintain propagation and the stem cell properties of mescenchymal stem cell, the cell of acquisition more can be applicable to clinical.
Accompanying drawing explanation
Fig. 1 is schema of the present invention.
Fig. 2 changes cell attachment figure after liquid human amnion mesenchymal stem cell first time.
Fig. 3 is that primary cell reaches 80% degree of converging figure.
Fig. 4 is fluidic cell figure, CD73.
Fig. 5 is fluidic cell figure, CD90.
Fig. 6 is fluidic cell figure, CD105.
Fig. 7 is fluidic cell figure, HLA-DR.
Fig. 8 is fluidic cell figure, CD34.
Embodiment
Below in conjunction with the embodiment of the present invention, be clearly and completely described the technical scheme in the embodiment of the present invention, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
Embodiment 1
Refer to Fig. 1-8, in the embodiment of the present invention, a kind of isolation cultivation method of human amnion mesenchymal stem cell, comprises the following steps:
One, the preparation of mescenchymal stem cell
(1) collection of Human plactnta amnion tissue: be immersed in after Human plactnta takes off in placenta conserving liquid, maintain in the environment of 2-8 DEG C and preserve, the shelf time is no more than 24h.
(2) amnion tissue sterilization: taken out from placenta conserving liquid by placenta, lie on sterile tray, the one side containing amnion tissue upwards, with the clockwise wiping of 75% alcohol swab, repeats 2 times, peels off the amnion tissue disinfected from placenta centered by umbilical cord.
(3) wash: sterile petri dish amnion tissue being put into 90cm, wash 2 times with tissue samples washings, every minor tick 5min, then use brine 2 times; Wherein tissue samples washings is the physiological saline containing 500IU/ml Cephalexin Monohydrate Micro/Compacted, 800U/ml gentamicin, 3ug/ml amphotericin.
(4) shred: amnion tissue is put into 10ml centrifuge tube, 4.8-5.2ml/ manages, and is cut into 1-3mm with a head scissors
3fritter.
(5) mix collagenase digesting: add isopyknic 0.1-0.5%(m/v) epoxy glue protoenzyme fully mix, 37 DEG C, 180rpm concussion digestion 8-30min.
(6) acquisition of mescenchymal stem cell: digestion product is filtered through 150 eye mesh screens, collecting cell is suspension just, by first for cell suspension after 2000rpm centrifugal 10min, 1800rpm centrifugal 10min, 1500rpm centrifugal 8min tri-subgradient is centrifugal, getting the centrifugal precipitation obtained, to add blood serum medium resuspended, obtains the mesenchyma stem cell suspension be separated.
Two: derived mesenchymal stem cells in vitro is cultivated and streaming Phenotypic examination
By the single stem cell of separation with 0.5-1 × 10
4/ cm
2density be inoculated in T75 culturing bottle, and add substratum, be then placed in 37 DEG C, saturated humidity, CO
2volume fraction is the interior cultivation of incubator of 5%, liquid is changed first in 24-48h, remove the hemocyte of suspension and not adherent heteroproteose cell, within three days afterwards, full dose changes liquid, within 7-10 days, cell can grow to 80%-90% degrees of fusion, now need to go down to posterity to cell: outwell the substratum in sterile culture flask, brine cell surface with 0.9% twice, add the trypsinase that 10ml concentration is 0.075% ~ 0.125%, after 37 DEG C of digestion 1-3min, basis of microscopic observation starts to become circle to cell, add appropriate nutrient solution and stop digestion, cell is blown and beaten with transfer pipet, cell is come off completely, the cell come off and nutrient solution are transferred in centrifuge tube, the centrifugal 6-10min of 1000-1800rpm, by resuspended for the blood serum medium of the precipitation of centrifugal gained, by 1-2 × 10
5/ ml density is inoculated in T75 culturing bottle and carries out P1 culture, and every bottle adds 15ml substratum, and within 2-3 days, namely cell reaches 80% degree of converging, and then carries out frozen to cell or continues amplification of going down to posterity.
Flow cytometer Phenotypic examination: get P1 for cell, digest centrifugal after, cell precipitation DPBS is washed 2 times, gets 0.8 ~ 1 × 10
6, 100ul cell suspension is put in EP pipe, adds primary antibodie 15ul(blank tube and does not add primary antibodie), 4 DEG C of lucifuges hatch 30min, add 400ulDPBS, the centrifugal 6min of 1500rpm, supernatant discarded, add 10ul two resist, and after 4 DEG C of lucifuges hatch 30min, add 400ulDPBS, the centrifugal 6min of 1500rpm, supernatant discarded, transfers in streaming in machine pipe after adding 500ulDPBS mixing, upper machine testing.
Flow cytomery result showed cell expresses CD73, CD90, CD105; And do not express CD34, HLA-DR.
To those skilled in the art, obviously the invention is not restricted to the details of above-mentioned one exemplary embodiment, and when not deviating from spirit of the present invention or essential characteristic, the present invention can be realized in other specific forms.Therefore, no matter from which point, all should embodiment be regarded as exemplary, and be nonrestrictive, scope of the present invention is limited by claims instead of above-mentioned explanation, and all changes be therefore intended in the implication of the equivalency by dropping on claim and scope are included in the present invention.
In addition, be to be understood that, although this specification sheets is described according to embodiment, but not each embodiment only comprises an independently technical scheme, this narrating mode of specification sheets is only for clarity sake, those skilled in the art should by specification sheets integrally, and the technical scheme in each embodiment also through appropriately combined, can form other embodiments that it will be appreciated by those skilled in the art that.
Claims (10)
1. an isolation cultivation method for human amnion mesenchymal stem cell, is characterized in that, comprises the following steps:
(1) collection of Human plactnta amnion tissue: be immersed in after Human plactnta takes off in placenta conserving liquid, maintains in the environment of 2-8 DEG C and preserves;
(2) amnion tissue sterilization: placenta is taken out from placenta conserving liquid, lies on sterile tray, the one side containing amnion tissue upwards, with the clockwise wiping of 60-90% alcohol swab, repeat 2-3 time, from placenta centered by umbilical cord denuded amniotic membrane tissue;
(3) wash: amnion tissue is put into sterile petri dish, with tissue samples washings washing 2-3 time, every minor tick 3-8min, then use brine 2-3 time;
(4) shred: amnion tissue is put into centrifuge tube, add-on is 4.8-5.2ml/ pipe, is cut into 1-3mm with a head scissors
3fritter;
(5) collagenase digesting: add isopyknic epoxy glue protoenzyme and fully mix, 35-40 DEG C, 150-200rpm concussion digestion 8-30min; Effective constituent in epoxy glue protoenzyme is 0.1-0.5% according to the add-on of mass volume ratio m/v;
(6) acquisition of mescenchymal stem cell: by digestion product through screen filtration, collecting cell just suspension, by cell, just suspension is after three subgradients are centrifugal, and getting the centrifugal precipitation obtained, to add blood serum medium resuspended, obtains the mesenchyma stem cell suspension be separated;
(7) derived mesenchymal stem cells in vitro cultivate: the stem cell of separation is inoculated in culturing bottle, and adds substratum, be then placed in 37 DEG C, saturated humidity, CO
2volume fraction is the interior cultivation of incubator of 5%, liquid is changed first in 24-48h, remove the hemocyte of suspension and not adherent heteroproteose cell, within three days afterwards, full dose changes liquid, when cell grows to 80%-90% degrees of fusion, namely obtain the dry primary cell of human amnion mesenchymal, now carry out Secondary Culture to cell and reach 80% degree of converging, namely obtain the human amnion mesenchymal stem cell of vitro culture, then carry out frozen to Human plactnta mescenchymal stem cell or continue amplification of going down to posterity.
2. the isolation cultivation method of human amnion mesenchymal stem cell according to claim 1, it is characterized in that, the concrete steps of Secondary Culture: outwell the substratum in culturing bottle, with the brine cell surface twice that massfraction is 0.9%, add the trypsinase that 10ml concentration is 0.075% ~ 0.125%, 37 DEG C of digestion, basis of microscopic observation starts to become circle to cell, add nutrient solution and stop digestion, cell is blown and beaten with transfer pipet, cell is come off completely, the cell come off and nutrient solution are transferred in centrifuge tube, the centrifugal 6-10min of 1000-1800rpm, by resuspended for the blood serum medium of the precipitation of centrifugal gained, by 1-2 × 10
5/ ml density is inoculated in culturing bottle and carries out P1 culture, and every bottle adds 15ml substratum, and within 2-3 days, namely cell reaches 80% degree of converging.
3., according to the isolation cultivation method of the arbitrary described human amnion mesenchymal stem cell of claim 1-2, it is characterized in that, the shelf time that Human plactnta maintains in the environment of 2-8 DEG C is no more than 24h.
4. according to the isolation cultivation method of the arbitrary described human amnion mesenchymal stem cell of claim 1-2, it is characterized in that, the composition of placenta conserving liquid is DMEM/F12,0.3-0.8% serum substitute, 45000-55000IU/ml Cephalexin Monohydrate Micro/Compacted, 75000-85000U/ml gentamicin, 25-35ug/ml amphotericin B; Use NaHCO
3adjusted to ph is 7.0-7.2.
5., according to the isolation cultivation method of the arbitrary described human amnion mesenchymal stem cell of claim 1-2, it is characterized in that, in tissue digestion process, the concentration of epoxy glue protoenzyme is 0.25%, and digestion time is 23min.
6. the isolation cultivation method of human amnion mesenchymal stem cell according to claim 5, is characterized in that, described epoxy glue protoenzyme is the mixture of collagenase P and type i collagen enzyme, and the mass ratio of collagenase P and type i collagen enzyme is 3:2.
7., according to the isolation cultivation method of the arbitrary described human amnion mesenchymal stem cell of claim 1-2, it is characterized in that, blood serum medium end user AB serum.
8., according to the isolation cultivation method of the arbitrary described human amnion mesenchymal stem cell of claim 1-2, it is characterized in that, three subgradients are successively centrifugal: the centrifugal 8min of 2000rpm centrifugal 10min, 1800rpm centrifugal 10min, 1500rpm.
9. according to the isolation cultivation method of the arbitrary described human amnion mesenchymal stem cell of claim 1-2, it is characterized in that, tissue samples washings is the physiological saline containing 450-550IU/ml Cephalexin Monohydrate Micro/Compacted, 700-900U/ml gentamicin, 2-5ug/ml amphotericin.
10., according to the isolation cultivation method of the arbitrary described human amnion mesenchymal stem cell of claim 1-2, it is characterized in that, the order number of screen cloth is 150 orders.
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| CN106754674A (en) * | 2016-12-09 | 2017-05-31 | 博雅干细胞科技有限公司 | Method and its application of amnion mesenchymal stem cell are prepared from Human plactnta amnion |
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