CN107488628A - A kind of simple and efficient amniotic fluid stem cell cultural method - Google Patents
A kind of simple and efficient amniotic fluid stem cell cultural method Download PDFInfo
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- CN107488628A CN107488628A CN201710839832.6A CN201710839832A CN107488628A CN 107488628 A CN107488628 A CN 107488628A CN 201710839832 A CN201710839832 A CN 201710839832A CN 107488628 A CN107488628 A CN 107488628A
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- C12N5/06—Animal cells or tissues; Human cells or tissues
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- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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Abstract
The invention provides a kind of simple and efficient amniotic fluid stem cell cultural method, including by amniotic fluid samples and the PBS 1 added with EDTA：1 dilution；1200r/min centrifuges 5min；Supernatant is abandoned, amniotic fluid special culture media is added and blows and beats mixing repeatedly；It is seeded in the coated culture dish of gelatin；With 37 DEG C, 5%CO2Environmental condition culture 45 days amniotic fluid stem cell growth can be observed.The present invention has broken traditional method, simplifies laboratory operating procedures, saves the time, reduces experimental cost；Reduce the risk of the cell contamination caused by experimental procedure is cumbersome；The cell cultivated still has due biological characteristics.
Description
Technical field
The present invention relates to technical field of bioengineering, more particularly to a kind of simple and efficient amniotic fluid stem cell cultural method.
Background technology
Cell therapy, latest developments in regenerative medicine and stem-cell research are significant to be added to various types of appearances
The demand of the cell suspending liquid easily obtained, particularly to pluripotency stroma cell (mesenchymal stem cells, MSCs)
Needs.The main reason for MSCs is welcome in Current medical practice is their unique immuno-modulating properties.But MSCs
Growth ability and differentiation capability increase with the age and reduce, such as fat, corium, bone, synovial membrane and marrow in adult tissue
The cytoactive of the cell of middle acquisition, differentiation capability etc. can not show a candle to the cell of fetal origin, and these adult stem cells point
From, purifying and maintenance on acquire a certain degree of difficulty, it is difficult to reach quantity required during graft application.Therefore fetal tissue's such as umbilical cord
Blood, Dun Shi glue is irrigated, amnion and amniotic fluid are considered as significantly more efficient source.
Human amniotic fluid stem cell (human Amniotic Fluid-derived Stem Cells, hAFSCs) is easy to obtain
Take, can be extracted from a small amount of amniotic fluid of pre-natal diagnosis；It can also be separated from the amniotic fluid in postpartum.And extraction amniotic fluid will not damage
Evil parent, will not also be impacted to embryo, and ethics and moral problem just also is not present.Cell in amniotic fluid is at a group
In the cell mass of embryonic development early stage, the human amniotic fluid stem cell cell therefrom separated is between embryonic stem cell (Embryonic
Stem cells, ES) cell type between adult stem cell, the mark of ES cells and adult stem cell is expressed, there is phase
As multi-lineage potential, under the stimulation of different growth factors, culture medium etc., three germinal layer sources can be divided into not
Same cell.Unlike embryonic stem cell, AFSCs does not form tumour or teratoma in vivo, and tissue phase is not present in AFSCs
The problems such as capacitive and immunological rejection., will be advantageously using AFSCs in regenerative medicine and research in future therapeutic application.
The sorting purification process of amniotic fluid stem cell mainly has following four at present:The adherent separating method of mechanical phonograph recorder separation, difference,
Immunological magnetic bead sorting method and selected by flow cytometry apoptosis method, but waste time and energy, step is relatively complicated to be caused in incubation as needed
When cell shifts, instrument consumables cost needed for contaminated possibility increase and these methods is higher.Therefore search out easy to be fast
Prompt amniotic fluid stem cell cultural method turns into an important topic.
The content of the invention
In order to overcome existing amniotic fluid stem cell incubation step is cumbersome to be inconvenient to operate and cost is too high, or there may be
The problem of unknown pathogen body infection risk, the present invention ensures to obtain under easier, cost-effective operation has good growth
Activity and the holding due biological characteristics of amniotic fluid stem cell, have a extensive future.
The present invention is achieved by the following technical solutions：
A kind of simple and efficient amniotic fluid stem cell cultural method, comprises the following steps：
(1) original cuiture of amniotic fluid stem cell：
By amniotic fluid and the PBS 1 added with EDTA：1 dilution, then centrifuges 5min with 1200r/min, sheep is added after abandoning supernatant
Water special culture media blows and beats mixing repeatedly；Then the coated culture dish of gelatin is seeded in, with 37 DEG C, 5%CO2Environmental condition training
Support 4-5 days；
(2) Secondary Culture of amniotic fluid stem cell：
When the amniotic fluid stem cell of original cuiture, which reaches 80~90%, to be converged, cell culture fluid is discarded；Three are washed with PBS
It is secondary；Cell is dissociated with the 0.25% trypsase covering cell containing 0.02%EDTA；Treat cell retraction be rounded and from
When being started shedding off on culture dish digestion is terminated with the complete medium containing 10% hyclone；Mixed liquor is blown and beaten repeatedly until
Attached cell comes off to be formed under cell suspension 1200rpm from culture dish to be centrifuged 5 minutes；Addition culture medium is soft after discarding liquid
Blowing even makes cell scatter.
Compared with prior art, the beneficial effects of the invention are as follows：
(1) it is thin to have broken the adherent separating method of traditional mechanical phonograph recorder separation, difference, immunological magnetic bead sorting method and streaming by the present invention
Born of the same parents' instrument separating method, simplifies laboratory operating procedures, saves the time, reduces experimental cost；
(2) present invention, avoiding experimental procedure it is cumbersome when, shift sample in caused by pollution；
(3) cell turned out in the present invention is still with good activity and with due biological characteristics.
Brief description of the drawings
Fig. 1 is the growth curve chart of the amniotic fluid stem cell Secondary Culture of embodiment 2；
Fig. 2 is the identified by immunofluorescence result figure of the amniotic fluid stem cell surface marker of embodiment 3；
Fig. 3 is the Osteoblast Differentiation result figure of the amniotic fluid stem cell of embodiment 4；
Fig. 4 is the amniotic fluid stem cell of embodiment 4 into fat differentiated result figure；
Fig. 5 is the results of comparison figure of the amniotic fluid stem cell of embodiment 4；
Fig. 6 is the flow cytometry figure of the amniotic fluid stem cell of embodiment 5.
Embodiment
The present invention is described in further detail below by embodiment combination accompanying drawing.But those skilled in the art
It will be understood that the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.It is unreceipted in embodiment
Particular technique or condition person, carried out according to the technology described by document in the art or condition or according to product description.
Agents useful for same or the unreceipted production firm person of instrument, being can be by the conventional products of acquisition purchased in market.
Embodiment 1：Amniotic fluid stem cell is separately cultured
(1) original cuiture of amniotic fluid stem cell：
Amniotic fluid samples and the PBS 1 added with EDTA：1 dilution；1200r/min centrifuges 5min；Supernatant is abandoned, it is special to add amniotic fluid
Culture medium blows and beats mixing repeatedly；It is seeded in the coated culture dish of gelatin；With 37 DEG C, 5%CO2Environmental condition culture be within 4-5 days
Amniotic fluid stem cell growth can be observed.
(2) Secondary Culture of amniotic fluid stem cell：
When the amniotic fluid stem cell of original cuiture, which reaches 80~90%, to be converged, cell culture fluid is discarded；Three are washed with PBS
It is secondary；Cell is dissociated with the 0.25% trypsase covering cell containing 0.02%EDTA；Treat cell retraction be rounded and from
When being started shedding off on culture dish digestion is terminated with the complete medium containing 10% hyclone；Mixed liquor is blown and beaten repeatedly until
Attached cell comes off to be formed under cell suspension 1200rpm from culture dish to be centrifuged 5 minutes；Addition culture medium is soft after discarding liquid
Blow it is even cell is scattered, so as to carry out Secondary Culture to the amniotic fluid stem cell of culture.
The amniotic fluid special culture media is purchased from BI companies；Described described pancreatin is purchased from Gibco companies.
Embodiment 2：The drafting of amniotic fluid stem cell growth curve
The amniotic fluid stem cell in the 3rd generation, the 6th generation is obtained by Secondary Culture, is counted, and is drawn and given birth to according to the data obtained
Long curve, with 1 × 104It is seeded in 24 orifice plates, took three holes at interval of 24 hours, and write down average.Obtain as shown in Figure 1
Amniotic fluid stem cell growth curve chart.
Embodiment 3：The identified by immunofluorescence of amniotic fluid stem cell
(1) amniotic fluid stem cell in the 3rd generation is obtained by Secondary Culture, is seeded on coated 24 orifice plate of gelatin, it is to be achieved
During 70% fusion, nutrient solution is discarded, is washed 2 times with PBS；
(2) 3.7% paraformaldehyde solution is added in 24 orifice plates and fixes 30 minutes, and is washed with the PBS containing 5%FBS
Cell is three times (washing 10 minutes every time)；
(3) permeabilization agent (0.1%TritonX-100, PBS) is added in 24 orifice plates, is discarded after being incubated at room temperature 15min, is used
PBS is washed three times, each 5min；
(4) confining liquid (10%FBS is added in 24 orifice plates:PBS) discarded after 37 DEG C of closing 1h, PBS washes three times, every time
5min；
(5) with being directed to CD34, CD44 and CD90 (1 at 4 DEG C:200, Santa Cruz, CA) primary antibody be incubated overnight, remove
After removing primary antibody, cell (washing 10 minutes every time) three times is washed with the PBS containing 5%FBS；
(6) at room temperature in the dark, the secondary antibody (1 marked with FITC is added：500, SantaCruz, CA) 1 hour, use PBS
Wash three times, each 5min；
(7) finally sample is incubated 5 minutes under the conditions of lucifuge with DAPI, washed three times with PBS, each 5min；
(8) identified by immunofluorescence figure as shown in Figure 2 is obtained under fluorescence microscope after adding anti-fluorescence quenching, it is known that
CD34 is not expressed, and CD44, CD90 can be expressed.
The DAPI is purchased from Invitrogen companies.
Embodiment 4：The Multidirectional Differentiation of amniotic fluid stem cell
By Secondary Culture, wherein in three holes of six orifice plates are seeded to equal densities when passing on for the 3rd time, mark 1,
2nd, 3, wherein 1,2 holes are experimental group, 3 holes are control, when cell growth to 80% is when converging, remove culture medium, Ran Houyong
PBS washings cell 3 times.
(1) Adipogenesis induction culture medium is added in 1 hole；
(2) Osteoblast Differentiation inducing culture is added in 2 holes；
(3) will be added in complete medium in 3 hole cellular control units.
Culture medium is every to be changed for 3-4 days completely.After induction 17 days, it was observed that leading to during obvious a large amount of calcium tubercles occurs in 1 hole
Alizarin red staining measure osteogenic ability is crossed, is commented when producing a large amount of greases during 2 holes are observed after inducing 17 days by oil red O stain
Valency fat generative capacity.Observe and take pictures under an optical microscope, obtain differentiated result figure as shown in Figure 3,4, and Fig. 5 institutes
The control group shown.
The Adipogenesis induction culture medium (ADM) and Osteoblast Differentiation inducing culture (ODM) are purchased from Gibco public affairs
Department；The oil red O, Alizarin red staining liquid are purchased from Solarbio companies.
Embodiment 5：Flow cytometry
(1) take the 3rd generation amniotic fluid stem cell (P3), 0.25% Trypsin Induced collect cell, regulation cell concentration 1 ×
106Individual/ml；
(2) 1500r/min, centrifuges 5min, and cell precipitation is resuspended in 100 μ l PBS；
(3) corresponding detection cell surface antibodies HLA-ABC, HLA-DR and corresponding Isotype control IgG1, IgG2 are added,
Room temperature avoid light place 30min；
(4) PBS 2 times, 1000r/min, 5min is centrifuged, abandons supernatant, added 500 μ l PBS and cell is resuspended, streaming is thin
Born of the same parents' instrument detects.
The HLA-ABC, HLA-DR, Isotype control IgG1, IgG2 antibody are purchased from BD companies.
As shown in Figure 6, the results showed that amniotic fluid stem cell has an immunosuppressive action, and its low expression HLA-ABC and
HLA-DR surface antigens, show low immunogenicity and immunosuppressive action, it was demonstrated that amniotic fluid stem cell is ideal is immunized
Absolve cell.
Claims (2)
- A kind of 1. simple and efficient amniotic fluid stem cell cultural method, it is characterised in that the original cuiture including amniotic fluid stem cell：Will Amniotic fluid and the PBS 1 added with EDTA：1 dilution, then centrifuges 5min with 1200r/min, and the special culture of amniotic fluid is added after abandoning supernatant Base blows and beats mixing repeatedly；Then the coated culture dish of gelatin is seeded in, with 37 DEG C, 5%CO2Environmental condition culture 4-5 days.
- 2. simple and efficient amniotic fluid stem cell cultural method according to claim 1, it is characterised in that also done including amniotic fluid The Secondary Culture of cell：When the amniotic fluid stem cell of original cuiture, which reaches 80~90%, to be converged, cell culture fluid is discarded；Use PBS Washing is three times；Cell is dissociated with the 0.25% trypsase covering cell containing 0.02%EDTA；Treat that cell retraction becomes When justifying and being started shedding off from culture dish digestion is terminated with the complete medium containing 10% hyclone；Mixed liquor is blown repeatedly Beat until attached cell comes off to be formed under cell suspension 1200rpm from culture dish and centrifuged 5 minutes；Culture is added after discarding liquid Base, which softly blows, even makes cell scatter.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108094406A (en) * | 2017-12-28 | 2018-06-01 | 重庆斯德姆生物技术有限公司 | A kind of cell freezing, rapid fluid resuscitation and the method for culture |
| CN108624649A (en) * | 2018-04-09 | 2018-10-09 | 佛山科学技术学院 | A kind of culture medium of the secretion containing amniotic fluid stem cell and its application |
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| CN104450610A (en) * | 2014-11-28 | 2015-03-25 | 广州赛莱拉干细胞科技股份有限公司 | Subculture method of human amniotic mesenchymal stem cells |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108094406A (en) * | 2017-12-28 | 2018-06-01 | 重庆斯德姆生物技术有限公司 | A kind of cell freezing, rapid fluid resuscitation and the method for culture |
| CN108624649A (en) * | 2018-04-09 | 2018-10-09 | 佛山科学技术学院 | A kind of culture medium of the secretion containing amniotic fluid stem cell and its application |
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