CN109161522A - A kind of amnion mesenchymal stem cell isolated culture method - Google Patents
A kind of amnion mesenchymal stem cell isolated culture method Download PDFInfo
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- CN109161522A CN109161522A CN201811105128.9A CN201811105128A CN109161522A CN 109161522 A CN109161522 A CN 109161522A CN 201811105128 A CN201811105128 A CN 201811105128A CN 109161522 A CN109161522 A CN 109161522A
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Abstract
The present invention provides a kind of amnion mesenchymal stem cell separation methods, belong to field of biotechnology.The described method includes: the pretreatment of (one) amnion；(2) the first digestion of amnion；(3) amnion again digests；(4) collection of amnion mesenchymal stem cell；(5) culture of amnion mesenchymal stem cell.This method can obtain the amnion mesenchymal stem cell of more yield, the problems such as solving many disease source of human stem cell in short supply and immunological rejection, as ideal stem-cell therapy substitute, have broad application prospects in stem-cell therapy field, organizational project and regenerative medicine field.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of amnion mesenchymal stem cell isolated culture method.
Background technique
Amnion is generally exactly that layer of transparent membrane for wrapping up amniotic fluid, a mature amnion area up to 2 square metres,
Have accumulated 300,000,000 amnion cells.As the protective layer of embryo, amnion is not only that development of fetus provides nutrition and a blockage
The marvellous barrier that parent and fetus Liang Ge allosome tissue do not repel mutually.
The amnion-derived stem cell of people includes human amnion membrane, human amnion mesenchymal stem cell.Research in recent years proves, people
Amnion mesenchymal stem cell has phenotypic spectrum similar with mesenchymal stem cell, has multilineage differentiated potential, oriented
The potential of triploblastica cell differentiation: ectoderm (nerve), mesoderm (skeletal muscle, cardiac muscle, endothelium etc.), entoderm (pancreas, liver),
And its amplification ability is better than mesenchymal stem cell, while from a wealth of sources.A large amount of experimental study also demonstrates people's amnion
Mescenchymal stem cell has stronger proliferative capacity and stem cell properties than mesenchymal stem cell.
Researcher is by transplanting after human amnion mesenchymal stem cell is direct or oriented induction in various disease (bones at present
The diseases such as damage, diabetes, muscular dystrophy, Parkinson, nerve, bone marrow injury) carry out scientific research.It is filled between people's amnion
Matter stem cell promotes and applies the problems such as undoubtedly can solve many disease source of human stem cell in short supply and immunological rejection, becomes ideal
Stem-cell therapy substitute, this is not only that hope is brought in stem-cell therapy field, but will be organizational project and regenerative medicine field
Open up new prospect.
Summary of the invention
In view of this, can efficiently divide it is an object of the invention to a kind of amnion mesenchymal stem cell isolated culture method
From amnion mesenchymal stem cell, more aim cells are obtained, to be amnion mesenchymal stem cell transplantation treatment related disease
Research and application enough raw material sources are provided.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
Amnion mesenchymal stem cell separation method of the invention specifically successively includes amnion pretreatment, just digests, again
The step of digestion, amnion mesenchymal stem cell are collected and are cultivated.
In embodiment of the present invention, the pretreated step of amnion can be with are as follows: in the toilet of A grades of cleanliness,
The amnion is cleaned three times with sterile buffer, eliminates bloodstain；Then the spongy layer of the amnion is removed, stripping process will be residual
It stays blood and minute blood vessel to remove, is finally cleaned again with sterile buffer primary.In preferred embodiments, the amnion
Pretreatment will carry out sterile working in Biohazard Safety Equipment；Preferably, the sterile buffer includes PBS and physiological saline.
In embodiment of the present invention, the step of amnion just digests, can be with are as follows: cuts the pretreated amnion
It is broken, it is placed in sterile chamber, trypsase is added and carries out digestion process.In preferred embodiments, the amnion just digests
The step of are as follows: the trypsase is added to the sterile chamber equipped with amnion fragment, in 37 DEG C of constant temperature removals after digestion 30 minutes
Clear liquid then proceedes to that the trypsase is added, and 37 DEG C of constant temperature digest 30 minutes, and pancreatin terminate liquid is added and carries out termination digestion.
Preferably, the pancreatin terminate liquid is without animal blood serum terminate liquid.
In the preferred embodiments of the invention, the step of amnion digests again, can be with are as follows: the amnion is gone after just digesting
Except supernatant, Collagenase is added, 37 DEG C of constant temperature digest 60 minutes.
In the preferred embodiments of the invention, the step of amnion mesenchymal stem cell is collected, can be with are as follows: the amnion
Amnion tissue suspension is obtained after digesting again, is filtered by 100 μm of steril cell sieve, filtrate is centrifuged through centrifuge
Afterwards, amnion mesenchymal stem cell is collected.Preferably, the centrifugal force of the centrifugation is 500g, centrifugation time is 6 minutes.
It can be in the preferred embodiments of the invention, the step of the amnion mesenchymal stem cell culture are as follows: filter centrifugation
Amnion tissue suspension afterwards, removes supernatant, and precipitating is placed in 37 DEG C, 5% dioxy with the resuspension of amnion mesenchymal stem cell culture medium
Change and is cultivated under carbocyclic ring border.Preferably, the amnion mesenchymal stem cell culture medium is serum free medium.In preferred embodiment party
In case, the step of the amnion mesenchymal stem cell culture are as follows: the amnion tissue suspension after filter centrifugation removes supernatant, sinks
It forms sediment with the resuspension of amnion mesenchymal stem cell culture medium, is placed in 37 DEG C, cultivates under 5% carbon dioxide environment；Culture 24 hours is resuspended
After remove supernatant, remove contaminating cell and cell out of order, then add the amnion mesenchymal stem cell culture medium
Continue to cultivate, be passed on when cells grown degrees of fusion is 70%-90%.In a more preferred embodiment, the amnion
The step of mescenchymal stem cell culture are as follows: the amnion tissue suspension after filter centrifugation removes supernatant, precipitates with amnion mesenchymal
Stem cell media is resuspended, and is placed in 37 DEG C, cultivates under 5% carbon dioxide environment；Supernatant is removed after culture being resuspended 24 hours, is removed
Contaminating cell and cell out of order, then add the amnion mesenchymal stem cell culture medium and continue to cultivate, to cell
Growth degrees of fusion is passed on when being 75%-85%.In a more preferred embodiment, the amnion mesenchymal stem cell culture
The step of are as follows: the amnion tissue suspension after filter centrifugation removes supernatant, precipitates with amnion mesenchymal stem cell culture base weight
It is outstanding, it is placed in 37 DEG C, cultivates under 5% carbon dioxide environment；Supernatant is removed after culture being resuspended 24 hours, removes contaminating cell and state
Then bad cell adds the amnion mesenchymal stem cell culture medium and continues to cultivate, is to cells grown degrees of fusion
It is passed on when 80% or so.
The beneficial effects of the present invention are: more yield can be obtained by above-mentioned amnion mesenchymal stem cell separation method
Amnion mesenchymal stem cell, amnion mesenchymal stem cell source is abundant, with mescenchymal stem cell Biological characteristics and function
Can, and proliferative capacity is stronger, the problems such as solving many disease source of human stem cell in short supply and immunological rejection, becomes ideal stem cell
Substitute is treated, is had broad application prospects in stem-cell therapy field, organizational project and regenerative medicine field.
Detailed description of the invention
Fig. 1 is the amnion tissue pattern being placed in culture dish；
Fig. 2 is the pattern after amnion tissue struts.
Specific embodiment
The technical solution that the invention will now be described in detail with reference to the accompanying drawings, but protection scope of the present invention is not limited to following realities
Apply example.
Embodiment
Amnion mesenchymal stem cell separation method is the acquisition of amnion first, will cut open (in operating room) in an aseptic environment
Amnion tissue is removed (referring to shown in attached Fig. 1 and 2) from nearly fetus face by the placenta in abdomen postpartum, is then transported to toilet and is carried out sheep
Film mescenchymal stem cell is separately cultured.Specific embodiment is as follows:
(1) pretreatment of amnion
1, sterile PBS (gentamicin containing 100U/ml) cleaning amnion three times, eliminates bloodstain.
2, carefully the spongy layer of amnion is removed, pays attention to distinguishing amnion mesenchymal stem cell face, stripping process will be as far as possible
Remaining blood and minute blood vessel are removed.
3, sterile PBS (gentamicin containing 100U/ml) is cleaned once again.
(2) the first digestion of amnion
1, it after shredding amnion as far as possible, is placed in 50ml centrifuge tube.
2, with 0.25% trypsase that total amount is 100ml (containing 0.02%EDTA) to the amnion tissue shredded in 37 DEG C of perseverances
It is digested in two times in incubator.Digestion 30 minutes for the first time remove supernatant.It is vibrated in second of digestion process, every 5 points
Clock vibrates once manually.Whole process about 60 minutes, depending on Different Individual amnion, disintegrate down cell from basilar memebrane.
3, the effect of trypsase is terminated with 10ml serum-free pancreatin inhibitor.
(3) amnion again digests
1, postdigestive cell suspension will be terminated to be centrifuged through 500g, 6 minutes, remove supernatant.
2, the 1mg/ml Collagenase for being 100ml with total amount digests amnion fragment again.In 37 DEG C of insulating boxs,
60 minutes, depending on Different Individual amnion, digest amnion fragment sufficiently.
(4) collection of amnion mesenchymal stem cell
1, amnion tissue suspension is taken into filtrate by 100 μm of cell screen clothes filterings, is centrifuged, collects through 500g, 6 minutes
Amnion mesenchymal stem cell under dissociation.
2, DMEM/F12 culture medium is added and removes supernatant after 500g, centrifugation in 6 minutes to 100ml.
3, sedimentation cell is suspended again with serum-free amnion mesenchymal stem cell culture medium.
4, calculated yield and survival rate.
(5) culture of amnion mesenchymal stem cell
Cell suspension is centrifuged again through 500g, 6min, the cell being collected by centrifugation serum-free amnion mesenchymal stem cell
Culture medium is resuspended, and is placed in 37 DEG C, 5%CO2Carbon dioxide incubator culture.After being resuspended culture 24 hours in removal culture
Clearly, to remove contaminating cell and cell out of order, new amnion mesenchymal stem cell culture medium is then added and continues to cultivate, to
Cells grown degrees of fusion is passed on for 80% or so.
Claims (10)
1. a kind of amnion mesenchymal stem cell separation method, which is characterized in that successively include amnion pretreatment, just digestion, disappear again
The step of change, amnion mesenchymal stem cell are collected and are cultivated.
2. amnion mesenchymal stem cell separation method according to claim 1, which is characterized in that the pretreated step of amnion
Suddenly are as follows: in the toilet of A grades of cleanliness, clean the amnion three times with sterile buffer, eliminate bloodstain；Then by the sheep
The spongy layer of film is removed, and remained blood and minute blood vessel are removed, then is cleaned again once with the sterile buffer.
3. amnion mesenchymal stem cell separation method according to claim 1 or claim 2, which is characterized in that the amnion pretreatment
Sterile working is carried out in Biohazard Safety Equipment；The sterile buffer includes PBS and physiological saline.
4. amnion mesenchymal stem cell separation method according to claim 1, which is characterized in that the step that the amnion just digests
Suddenly are as follows: the pretreated amnion is shredded, is placed in sterile chamber, trypsase is added and carries out digestion process.
5. amnion mesenchymal stem cell separation method according to claim 4, which is characterized in that equipped with after the pretreatment
The sterile chamber of amnion fragment be added the trypsase, 37 DEG C of constant temperature remove supernatant after digestion 30 minutes, then proceed to
The trypsase is added, 37 DEG C of constant temperature digest 30 minutes, and pancreatin terminate liquid is added and carries out termination digestion；Wherein, the pancreatin
Terminate liquid is without animal blood serum terminate liquid.
6. amnion mesenchymal stem cell separation method according to claim 1, which is characterized in that the step that the amnion digests again
Suddenly are as follows: the amnion removes supernatant after just digesting, Collagenase is added, and 37 DEG C of constant temperature digest 60 minutes.
7. amnion mesenchymal stem cell separation method according to claim 1, which is characterized in that the amnion mesenchymal is dry thin
The step of born of the same parents collect are as follows: the amnion obtains amnion tissue suspension after digesting again, carries out by 100 μm of steril cell sieves
Filtering, filtrate collect amnion mesenchymal stem cell after centrifuge is centrifuged；Wherein, the centrifugal force of the centrifugation is 500g, is centrifuged
Time is 6 minutes.
8. amnion mesenchymal stem cell separation method according to claim 1, which is characterized in that the amnion mesenchymal is dry thin
The step of born of the same parents cultivate are as follows: the amnion tissue suspension after filter centrifugation removes supernatant, precipitates with amnion mesenchymal stem cell culture
Base weight is outstanding, is placed in 37 DEG C, cultivates under 5% carbon dioxide environment；Wherein, the amnion mesenchymal stem cell culture medium is serum-free
Culture medium.
9. amnion mesenchymal stem cell separation method according to claim 8, which is characterized in that the amnion group after filter centrifugation
Suspension removal supernatant is knitted, precipitating is placed in 37 DEG C, under 5% carbon dioxide environment with the resuspension of amnion mesenchymal stem cell culture medium
Culture；Above-mentioned resuspension culture removes culture supernatant after 24 hours, remove contaminating cell and cell out of order, then add
The amnion mesenchymal stem cell culture medium continues that culture is resuspended, and is passed on when cells grown degrees of fusion is 70%-90%.
10. amnion mesenchymal stem cell separation method according to claim 9, which is characterized in that the amnion after filter centrifugation
Tissue suspension removes supernatant, and precipitating is placed in 37 DEG C, 5% carbon dioxide environment with the resuspension of amnion mesenchymal stem cell culture medium
Lower culture；Above-mentioned resuspension culture removes culture supernatant after 24 hours, remove contaminating cell and cell out of order, then again plus
Enter the amnion mesenchymal stem cell culture medium to continue that culture is resuspended, be passed on when cells grown degrees of fusion is 80%.
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Cited By (1)
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Effective date of registration: 20200325 Address after: Room fiII (201-202) - 32, torch Park, high tech Zone, 1789 Xinfei Avenue, Xinxiang City, Henan Province Applicant after: Huayang Biotechnology Co.,Ltd. Applicant after: Hangzhou Zhenyi Intelligent Technology Co.,Ltd. Applicant after: LUOYANG WEIYANG BIOTECHNOLOGY Co.,Ltd. Address before: Room 2006, Luoyang Zhongke Science and Technology Park, No. 6 Huaxia Road, Luoyang High-tech Zone, Luoyang Free Trade Experimental Zone, Henan Province Applicant before: LUOYANG WEIYANG BIOTECHNOLOGY Co.,Ltd. Applicant before: SHANGHAI ANKU SHENGYI BIOTECHNOLOGY Co.,Ltd. |
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Application publication date: 20190108 |