CN101928694A - Method for establishing human amniotic fluid stem cell line - Google Patents
Method for establishing human amniotic fluid stem cell line Download PDFInfo
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Abstract
The invention relates to a method for establishing a human amniotic fluid stem cell line. The method comprises the following steps of: 1) performing isolated culture on primary amniotic fluid stem cells; 2) separating and purifying the primary amniotic fluid stem cells subjected to isolated culture; and 3) performing monoclonal screening and amplification culture on the separated amniotic fluid stem cells so as to establish the amniotic fluid stem cell line. The invention provides a rapid, effective and safe method for establishing the human amniotic fluid stem cell line, can realize long-term amplification of the human amniotic fluid stem cells in vitro, can provide a novel seed cell source for tissue engineering research, and makes the human amniotic fluid stem cells serve as seed cells for clinical application.
Description
Technical field
The present invention relates to a kind of establishment method of clone, relate in particular to a kind of establishment method of human amniotic fluid stem cell line.
Background technology
Stem cell has the self ability, under certain inductive condition, can be divided into the cell with specific function.In the past between the more than ten years, separate and the success of cultivating embryonic stem cell (ES cell) provides new opportunity for fundamental research and cellular transplantation therapy.
Yet society and ethics problem have restricted the research and the application of ES cell.In addition, vitro culture ES cell is faced with great technological challenge, and the cultivation of ES cell needs feeder layer and expensive cytokine, and regular meeting's producer group sudden change in the vitro culture process.The more important thing is after the ES cells in vitro is transplanted to form tumour, still do not have terms of settlement at present.And limited amount after the mesenchymal stem cells MSCs adult stem cell separation and Culture such as (MSC), the in-vitro multiplication differentiation capability remains further to be proved.
Therefore, Many researchers all attempt seeking new source of human stem cell, attempts to set up a kind of stem cell line, and it is fast both to have had a rate of propagation, and the characteristics that the vitro differentiation ability is strong are broken away from ethics morals problem again.People's amniotic fluid is applied to antenatal diagnosis, and the history of seven more than ten years has been arranged.2003, Prusa etc. find the Oct-4 positive cell in amniotic fluid, may there be multipotential stem cell (Prusa AR in the prompting amniotic fluid, Marton E, Rosner M, et al.Oct-4-expressing cells in human amniotic fluid:a new source for stem cellresearch? Hum Reprod, 2003,18 (7): 1489-1493).In American Heart Association's meeting that hold in November, 2006, the Dr of Univ Zurich Switzerland.Simon announces that they cultivate the human heart valve with the stem cell of taking from amniotic fluid first.2007, reports such as Paolo separated the pluripotent cell that obtains having ES sample stem cell characteristic in people's second trimester amniotic fluid, its name is people's amniotic fluid derived stem cell (human AmnioticFluid Stem Cell, hAFS) [Paolo DC, 2007].This cell can and keep undifferentiated state in external long-term cultivation (above 2 years), express ES cell and derived adult stem cell labeling gene, the external evoked various cells that are divided into three germinal layers such as comprising ectoderm, mesoderm and entoderm, thus show that it has versatility.As the ES cell, the AFS cell can be external survival several years, and common somatocyte is external for some time of can surviving, subsequently just can be dead.AFS cell expressing Oct-4, and express part ES cell and derived adult stem cell labeling, therefore, the researchist thinks that the AFS cell is in the intermediateness of ES cell and adult stem cell.The advantage of AFS cell shows as, and compares easily with adult stem cell and obtains, and has stronger differentiation potential; The immunogenicity of comparing the AFS cell in embryonic stem cell is lower, more is applicable to researchs such as Transplanted cells.The AFS cell can be Tissue Engineering Study new seed cell source is provided, and AFS cells in vitro culture system is set up and improved to the differentiation mechanism of further investigation AFS cell, has important significance for theories and clinical value.
At present, the foreign scholar adopts immunological magnetic bead sorting method screening amniotic fluid stem cell more, the pair cell activity has certain influence, and higher (the Paolo DC of expense, Georg BJ, Anthony A, et al.Isolation of amnioticstem cell lines with potential for therapy[J] .Nat Biotechnol, 2007,25 (1): 100～106.).The many employings of domestic scholars simple direct former generation adherent method (Liu Hui, Liu Daqing, Yan Zhifeng, Li Baowei, Guan Lidong, Yue Wen, Lv Yang, Pei Xuetao, Li Yali. the separation and Culture of pregnant mature amniotic fluid stem cell [J]. Chinese Tissue Engineering Study and clinical rehabilitation, 2009,13 (14): 2733-2737.), or the method for mechanical selected clone is separated amniocyte (Li Xianduo, Geng Hongquan, Zhu Zhe waits separation and the cultivation and the biological characteristic research thereof of second trimester amniotic fluid source fetus mescenchymal stem cell.Chinese Medical Journal, 2006,86:4812483).But contain the cell of other types such as amniotic fluid specific cell, epithelioid cell in the cell mass that sub-elects, its purity is very low, and the multiplication capacity of cell is limited, and the back quantity that repeatedly goes down to posterity reduces, and external only can the amplification gone down to posterity about 10 generations.At present domestic nobody still sets up stable amniotic fluid stem cell line.
Summary of the invention
In order to solve the above-mentioned technical problem that exists in the background technology, the invention provides a kind of fast, effectively, safety, can realize the external long-term amplification of human amniotic fluid stem cell, can be Tissue Engineering Study new seed cell source and the establishment method that is expected to carry out as seed cell the human amniotic fluid stem cell line of clinical application are provided.
Technical solution of the present invention is: the invention provides a kind of establishment method of human amniotic fluid stem cell line, its special character is: this method may further comprise the steps:
1) former generation separation and Culture of amniotic fluid stem cell;
2) the former generation amniotic fluid stem cell with separation and Culture carries out the mechanical separation purifying;
3) amniotic fluid stem cell after the separation and purification is set up the amniotic fluid stem cell mono-clonal, after carry out enlarged culturing, set up amniotic fluid stem cell line, its specific implementation is:
3.1) cell of getting preliminary purification makes suspension, trypan blue dyeing back counting is measured viable cell concentrations;
3.2) with nutrient solution cell suspension is diluted to different concns, and in 37 ℃, 5%CO2 incubator, cultivate; The substratum composition is α-MEM+15%FBS+2% glutamine+1% mycillin;
3.3) treat cell attachment after, microscopically is observed and to be selected and mark only contains the hole of a cell, continues to cultivate;
3.4) between incubation period, judged whether that according to the changing conditions of pH value in the nutrient solution obvious mono-clonal occurs.If have, then digest monoclonal cell with 0.05% trypsinase+0.02%EDTA, carry out enlarged culturing respectively; If not, then abandon cultivating, no longer continue to change liquid;
3.5) when treating that cell reaches 70%-90% and merges, reach and continue amplification cultivation in the culture dish, the cell that obtain this moment promptly builds up amniotic fluid stem cell line more than 40 generations of continuous passage.
Above-mentioned steps 1) specific implementation is:
1.1) from obstetrics and gynecology hospital collect second trimester and pregnant late period amniotic fluid, 4 ℃ of preservations are taken back standby;
1.2) filter with 200 orders filters yarn, to remove bigger fragment of tissue, 1000r/min then, centrifugal 6min abandons supernatant, collecting cell;
1.3) with step 2) and in collected cell add and contain 1% pair of anti-PBS (-) solution, mixing is put 37 ℃ and handled 10min, and is centrifugal then, collecting cell repeats with 1% couple of anti-PBS (-) solution-treated 3 times.
Above-mentioned steps 2) specific implementation is:
2.1) isolated amniocyte is added former generation amniocyte nutrient solution, place 37 ℃, 5%CO2 incubator, and record adherent time of primary cell and attached cell quantity;
2.2) cultivate 4～12d after, under inverted microscope, observe and draw required cell growing state;
2.3) have the zone of epithelioid cell's type to push repeatedly with cell scraper in growth, make cell detachment, and carefully do not scrape the zone of mark;
2.4) with two anti-PBS (-) solution flushings 2～3 times, add nutrient solution and continue to cultivate;
2.5) when observing once more in unwanted cells when growth, arranged in the culture dish, still handle with aforesaid method, 2～3 times so repeatedly, cell can obtain preliminary purification;
2.6) cultured continuously is after 5～8 generations, the epithelioid cell reduces disappearance gradually, shows as fibroblast-like cells more than 90%.
Above-mentioned steps 2.2) in, is after cultivating 4～5d, under inverted microscope, observes and draw required cell growing state.
In above-mentioned former generation,, the amniocyte nutrient solution was the nutrient solution that contains α-MEM+10%FBS+20%ChangMedium+1% glutamine+1% mycillin.
Above-mentioned steps 2.6) composition of AFS cell culture fluid is α-MEM+10%FBS+1% glutamine+1% mycillin in.
Above-mentioned steps 3.1) in, be get first to ten generation preliminary purification cell make suspension.
Above-mentioned steps 3.1) in, be get the 6th generation preliminary purification cell make suspension.
Above-mentioned steps 3.1) in, be get the 6th generation preliminary purification the cell of logarithmic phase make suspension.
Advantage of the present invention is:
1, fast, effectively, safety.The present invention adopts former generation adherent method and mechanical phonograph recorder separation, separation and purification human amniotic fluid stem cell, and set up a kind of quick, effective, safe human amniotic fluid stem cell separation method.
2, can realize the external long-term amplification of human amniotic fluid stem cell.Establishment method according to human amniotic fluid stem cell line provided by the present invention, can utilize the culture system of different concns FBS and KSR, do not need the trophocyte, realized the external long-term amplification of human amniotic fluid stem cell, it is frozen to pass for 45 generations, show that through flow cytometry analysis caryogram was normal after isolating hAFS cell reached for 35 generations, frozen 1～1.5x10
8Individual seed cell, its stable performance, inherited character is good.And show that through immunocytochemistry, RT-PCR and flow cytometry evaluation the isolating human amniotic fluid stem cell of institute is expressed HLA-ABC, weak expression HLA-DR; Express ES cell sign gene Oct-4, hTERT, Nanog, SSEA-1, SSEA-4 and CD117, do not express SSEA-3; Express mesenchymal cell marker gene CD29, CD44, CD73, CD90 and CD105 etc.; Not expressing hematopoietic cells such as CD34, CD45 and CD133 is marker gene.Detect through alkaline phosphatase activities, the embryoid of formation is positive, and the RT-PCR analysis revealed has the potential of the various cells that are divided into the triploblastica source.
3, can be Tissue Engineering Study new seed cell source is provided.The present invention according to the requirement of setting up clone by the foundation of the definition success of clone human amniotic fluid stem cell line, can be Tissue Engineering Study new seed cell source is provided, not only avoided the ethics arguement of embryonic stem cell, and opened up new direction for stem-cell research from now on, be expected to carry out clinical application as seed cell.
Embodiment
The invention provides a kind of establishment method of human amniotic fluid stem cell line, this method may further comprise the steps:
1, amniotic fluid stem cell separating step: from obstetrics and gynecology hospital collect second trimester and pregnant late period amniotic fluid, the related datas such as size of conceived time of detail record, amniotic fluid outward appearance, amniotic fluid volume and centrifugal back cell mass, the laboratory is taken back in 4 ℃ of preservations.Earlier with the filtration of 200 orders filter yarn, to remove bigger fragment of tissue, 1000r/min then, centrifugal 6min abandons supernatant, collecting cell.Add and contain 1% pair of anti-PBS (-) solution, mixing is put 37 ℃ and handled 10min, and is centrifugal then, and collecting cell repeats with 1% couple of anti-PBS (-) solution-treated 3 times.
2, amniotic fluid stem cell culturing step: 1) the isolated amniocyte of back is added former generation amniocyte nutrient solution, culture medium prescription is: α-MEM+10%FBS+20%Chang Medium+1% glutamine+1% mycillin; In 37 ℃, 5%CO2 training incubator, carry out, and record adherent time of primary cell and attached cell quantity.Observation of cell growing state under the inverted microscope: cell attachment behind cultivation 4～12d can be observed the growth district that occurs epithelium sample, fibroblast-like cells in the culture dish, the zone of the observation and the required cell that draws under mirror.There is the zone of epithelioid cell's type to push repeatedly with cell scraper in growth, makes cell detachment, and carefully do not scrape the zone of mark, to reach the purpose of purifying cells.With two anti-PBS (-) solution flushing 2～3 times, add nutrient solution and continue to cultivate.When observing once more unwanted cells when growth arranged in the culture dish, still handle with aforesaid method, 2～3 times so repeatedly, cell can obtain preliminary purification.
Cultured continuously is after 5～8 generations, and the epithelioid cell reduces disappearance gradually, shows as fibroblast-like cells more than 90%.
3, the monoclonal foundation of amniotic fluid stem cell
3.1) get the 6th generation preliminary purification the cell of logarithmic phase make suspension, trypan blue dyeing back counting is measured viable cell concentrations.In ten generations, are all feasible with interior algebraically, if be higher than ten generations, then separating effect reduces.General algebra is low more good more, makes the establishment method that suspension can be realized human amniotic fluid stem cell line provided by the present invention such as the cell of the first-generation to the ten generations preliminary purification.
3.2) cell suspension is diluted in test tube, with nutrient solution with cell dilution to 50 cell/mL, 20 cell/mL, 10 cell/mL, 3 kinds of dilution cells are inoculated in respectively in 96 orifice plates, every hole is 0.1mL, add the 0.1mL nutrient solution respectively, in 37 ℃, 5%CO incubator, cultivate.The substratum composition here is α-MEM+15%FBS+2% glutamine+1% mycillin, promptly on the basis of AFS cell culture fluid (ACM), the concentration of FBS (foetal calf serum) is increased to 15%, and the concentration of glutamine is brought up to 2%.
3.3) after three days, treat cell attachment, under inverted microscope, observe the cell count in each hole of culture plate, select the hole that only contains a cell, carry out mark and continue to cultivate.
3.4) between incubation period, judged whether that according to the changing conditions of pH value in the nutrient solution obvious mono-clonal occurs.If have, then digest monoclonal cell with 0.05% trypsinase+0.02%EDTA, carry out enlarged culturing respectively; If not, then abandon cultivating, no longer continue to change liquid; When continuing to cultivate about 4-7 days, Kong Zhongyou obviously clones appearance, treats that cell grows to 1/3～1/2 of bottom surface, hole, with trypsin digestion the monoclonal cell in 96 holes is moved to respectively and carries out enlarged culturing in 24 orifice plates; If have mono-clonal to occur just digesting monoclonal cell, carry out enlarged culturing respectively with 0.05% trypsinase+0.02%EDTA; The hole that more than one clone occurs and the clone do not occur then abandons cultivating, and no longer continues to change liquid; Change liquid according to the variation (promptly observing the colour-change of nutrient solution) of pH value decision, when pH value changes, must change liquid, because cell can only grow under certain pH value, otherwise cell is understood death or old and feeble.Simultaneously, often change liquid and also can make the cell health growth.
3.5) when treating that cell reaches the 70%--90% fusion, reach in the 35mm culture dish and continue amplification cultivation.This moment, the cell that obtains was after continuous passage, i.e. amniotic fluid stem cell line.
After adopting aforesaid method to cultivate, have and see the cell growth in 58/192 hole, wherein being the living elder of mono-clonal has 8 holes.After individual cells in most of hole did not divide or splits into 2 cells, its form became greatly gradually, similar neurocyte form, and stretch out many projections gradually.When the cell that wherein is mono-clonal growth treats that it covers at the bottom of 96 holes, be transferred to amplification cultivation in 24 orifice plates or the 35mm culture dish, set up each mono-clonal ubcellular system.
In the process of subclone and amplification cultivation, the strain cell called after WHcs-07 that successfully goes down to posterity is arranged, this clone reached for 45 generations at present.
According to former generation of the present invention the amniocyte substratum become α-MEM+10%FBS+20%ChangMedium+1% glutamine+1% mycillin.
According to AFS cell culture fluid of the present invention (ACM) substratum composition is α-MEM+10%FBS+1% glutamine+1% mycillin.
Adopted serum and serum free medium to cultivate respectively to above-mentioned cell, when treating that cell grows to 80%～90% fusion, then gone down to posterity, added AFS cell culture fluid (ACM) and continue to cultivate.One strain cell was reached for 45 generations, and form still for becoming fiber-like, is whirlpool shape, radial arrangement, and frozen a large amount of seed cell; Such cell the 5th, 10,15 generation population doubling time is respectively 33,34,39h has measured its growth curve; AFS cell culture fluid (ACM) substratum composition is: α-MEM+10%FBS+1% glutamine+1% mycillin.
Show that through immunocytochemistry, RT-PCR and flow cytometry evaluation the isolating human amniotic fluid stem cell of institute is expressed HLA-ABC, weak expression HLA-DR; Express ES cell sign gene Oct-4, hTERT, Nanog, SSEA-1, SSEA-4 and CD117, do not express SSEA-3, prove that it has and the similar differentiation potential of ES cell; Express mesenchymal cell marker gene CD29, CD44, CD73, CD90 and CD105 etc.; Not expressing hematopoietic cells such as CD34, CD45 and CD133 is marker gene, has the numerous characteristics of adult stem cell.
Claims (9)
1. the establishment method of a human amniotic fluid stem cell line, it is characterized in that: this method may further comprise the steps:
1) former generation separation and Culture of amniotic fluid stem cell;
2) the former generation amniotic fluid stem cell with separation and Culture carries out the mechanical separation purifying;
3) amniotic fluid stem cell after the separation and purification is set up the amniotic fluid stem cell mono-clonal, after carry out enlarged culturing, set up amniotic fluid stem cell line, its specific implementation is:
3.1) cell of getting preliminary purification makes suspension, trypan blue dyeing back counting is measured viable cell concentrations;
3.2) with nutrient solution cell suspension is diluted to different concns, and in 37 ℃, 5%CO
2Cultivate in the incubator; The substratum composition is α-MEM+15%FBS+2% glutamine+1% mycillin;
3.3) treat cell attachment after, microscopically is observed and to be selected and mark only contains the hole of a cell, continues to cultivate;
3.4) between incubation period, judged whether that according to the changing conditions of pH value in the nutrient solution obvious mono-clonal occurs.If have, then digest monoclonal cell with 0.05% trypsinase+0.02%EDTA, carry out enlarged culturing respectively; If not, then abandon cultivating, no longer continue to change liquid;
3.5) when treating that cell reaches 70%-90% and merges, reach and continue amplification cultivation in the culture dish, the cell that obtain this moment promptly builds up amniotic fluid stem cell line more than 40 generations of continuous passage.
2. the establishment method of human amniotic fluid stem cell line according to claim 1, it is characterized in that: the specific implementation of described step 1) is:
1.1) from obstetrics and gynecology hospital collect second trimester and pregnant late period amniotic fluid, 4 ℃ of preservations are taken back standby;
1.2) filter with 200 orders filters yarn, to remove bigger fragment of tissue, 1000r/min then, centrifugal 6min abandons supernatant, collecting cell;
1.3) with step 2) and in collected cell add and contain 1% pair of anti-PBS (-) solution, mixing is put 37 ℃ and handled 10min, and is centrifugal then, collecting cell repeats with 1% couple of anti-PBS (-) solution-treated 3 times.
3. the establishment method of human amniotic fluid stem cell line according to claim 1, it is characterized in that: specific implementation described step 2) is:
2.1) isolated amniocyte is added former generation amniocyte nutrient solution, place 37 ℃, 5%CO2 incubator, and record adherent time of primary cell and attached cell quantity;
2.2) cultivate 4～12d after, under inverted microscope, observe and draw required cell growing state;
2.3) have the zone of epithelioid cell's type to push repeatedly with cell scraper in growth, make cell detachment, and carefully do not scrape the zone of mark;
2.4) with two anti-PBS (-) solution flushings 2～3 times, add nutrient solution and continue to cultivate;
2.5) when observing once more in unwanted cells when growth, arranged in the culture dish, still handle with aforesaid method, 2～3 times so repeatedly, cell can obtain preliminary purification;
2.6) cultured continuously is after 5～8 generations, the epithelioid cell reduces disappearance gradually, shows as fibroblast-like cells more than 90%.
4. the establishment method of human amniotic fluid stem cell line according to claim 3 is characterized in that: described step 2.2), be after cultivating 4～5d, observe and draw required cell growing state under inverted microscope.
5. the establishment method of human amniotic fluid stem cell line according to claim 4 is characterized in that: described former generation the amniocyte nutrient solution be the nutrient solution that contains α-MEM+10%FBS+20%Chang Medium+1% glutamine+1% mycillin.
6. the establishment method of human amniotic fluid stem cell line according to claim 4 is characterized in that: the composition of AFS cell culture fluid is α-MEM+10%FBS+1% glutamine+1% mycillin described step 2.6).
7. according to the establishment method of claim 1 or 2 or 3 or 4 or 5 or 6 described human amniotic fluid stem cell lines, it is characterized in that: described step 3.1), be get first to ten generation preliminary purification cell make suspension.
8. the establishment method of human amniotic fluid stem cell line according to claim 7 is characterized in that: described step 3.1), be get the 6th generation preliminary purification cell make suspension.
9. the establishment method of human amniotic fluid stem cell line according to claim 8 is characterized in that: described step 3.1), be get the 6th generation preliminary purification the cell of logarithmic phase make suspension.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107488628A (en) * | 2017-09-18 | 2017-12-19 | 佛山科学技术学院 | A kind of simple and efficient amniotic fluid stem cell cultural method |
| CN111759864A (en) * | 2020-07-14 | 2020-10-13 | 中国人民解放军总医院 | Application of amniotic fluid stem cells in preparation of medicine for treating lupus nephritis |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107488628A (en) * | 2017-09-18 | 2017-12-19 | 佛山科学技术学院 | A kind of simple and efficient amniotic fluid stem cell cultural method |
| CN111759864A (en) * | 2020-07-14 | 2020-10-13 | 中国人民解放军总医院 | Application of amniotic fluid stem cells in preparation of medicine for treating lupus nephritis |
| CN111759864B (en) * | 2020-07-14 | 2022-09-13 | 中国人民解放军总医院 | Application of amniotic fluid stem cells in preparation of medicine for treating lupus nephritis |
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Application publication date: 20101229 |