CN102161980A - Method for culturing induced pluripotent stem cells by using human mesenchymal stem cells as trophoblast - Google Patents
Method for culturing induced pluripotent stem cells by using human mesenchymal stem cells as trophoblast Download PDFInfo
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Abstract
The invention provides a method for culturing human induced pluripotent stem cells (iPSCs) by using human bone marrow mesenchymal stem cells as a trophoblast. The method comprises the following steps of: obtaining the human iPSCs from child foreskin fibroblasts by using third to fifth generation human bone marrow mesenchymal stem cells obtained through subculture and culture after thawing from low-temperature freezing, performing amplification culture, and inoculating into trophoblast cells to obtain the human iPSCs. In the method, the human iPSCs are cultured by using the human bone marrow mesenchymal stem cells as the trophoblast cells instead of mouse embryonic fibroblasts in the conventional method, so that the pollution of heterologous cells in a culture system is reduced, that the culture system can make the human iPSCs amplified in vitro is proved, biological characteristics and multipotency of the human iPSCs are maintained for a long time, and the possibility of clinical application of the human iPSCs is provided.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of method of cultivating induced multi-potent stem cells with human marrow mesenchymal stem cell as trophoderm.
Background technology
(Induced Pluripotent Stem Cells iPSCs) is the breakthrough that has milestone significance in the stem-cell research field in recent years to induced multi-potent stem cells.It is by ectopic expression several with keep embryonic stem cell (Embryonic Stem Cells, ESCs) the relevant key nuclear factor of multipotency, make somatocyte generation reprogrammed, the multipotential cell of a kind of similar ESCs that obtains can be divided into tridermic all histocytes in vivo and in vitro.The iPSCs that is come by reprogramming of somatic cells obtains the multipotential stem cell consistent with patient self genetic background to have increased a new way, and no longer use human body early embryo and ovocyte, so ethical arguement will be calmed down thereupon, nuclear transfer technology lacks ovocyte and the numerous and diverse awkward situation of technology has also obtained rational solution.Be the desirable seed cell of organizational project and regenerative medicine, disease model, drug screening, therefore have wide clinical application background.At present, the investigator induces differentiation with people iPSCs to various histocytes external, as hematopoietic stem, red corpuscle, T cell, B cell, myocardial cell, beta Cell of islet, liver cell etc.; Set up multiple heredopathia people iPSCs disease model, be used for Study on Pathogenesis, and on animal model, attempted by the iPSCs that hereditary defect is arranged being carried out genetic modification, the treatment heredopathia; Set up kinds of tumors iPSCs disease model, be used for Study on Pathogenesis and drug screening.
The vitro culture of people iPSCs needs the interaction of cell and cell, various cytokine, matrix to wait to keep its self and multipotency.Its classical training mode is that (Mouse Embryonic fibroblasts MEF) is trophoderm, with containing bFGF, serum substitute (serum replacement, nutrient solution cultivation SR) with mouse embryo fibroblasts.But before people iPSCs entered clinical application, heterologous protein, cell contamination were problems that must solve in the removal culture system.Therefore, after people iPSCs occurred, the investigator just began to attempt improving its culture system, when keeping its self and multipotency, reduces the pollution of heterologous protein, cell.As 2010, Christian etc. used the human skin fibroblast of immortalization as trophoderm, and cultivator iPSCs finds that this humanized trophoderm can produce and keep the cultivation of people iPSCs, but its incubation time is lacked (the cultivation algebraically of report was 7 generations); The same year, Kristiina etc. are with a kind of RegES nutrient solution cultivator iPSCs of no heterologous protein of definite ingredients, find its for a long time (＞80 generation) keep self and the multipotency of iPSCs, but proof is not in the reprogrammed process, and it is iPSCs that this nutrient solution can make the human body cell reprogrammed.Ruth etc. are with the mTeSRTM nutrient solution suspension culture people iPSCs of another kind of definite ingredients, after finding to cultivate for 17 generations, still can keep its self and multipotency, but this culture system costs an arm and a leg, and same proof is not in the reprogrammed process, and it is iPSCs that this nutrient solution can make the human body cell reprogrammed.Therefore, further the humanized culture system of development research people iPSCs comprises the reprogrammed process and keeps cultivation, and to the influence of iPSCs biological characteristics, is that people iPSCs moves towards premise for clinical application.
Summary of the invention
The purpose of this invention is to provide a kind of with human marrow mesenchymal stem cell (Mesenchymal Stem Cells MSCs) as the method for trophoderm cultivator induced multi-potent stem cells (iPSCs), is achieved through the following technical solutions:
(1) trophocyte's preparation: the human marrow mesenchymal stem cell of using the 3rd ~ the 5th generation of cultivating behind go down to posterity cultivation, the low temperature cryopreservation resuscitation is as the trophocyte.Human marrow mesenchymal stem cell adopts Ficoll-paque density gradient centrifugation commonly used at present to cultivate in conjunction with adherent screening and separating and obtains.During as trophoderm, on six orifice plates of cell inoculation after handle with 0.1%gelatin, the cytogamy degree reaches 80%-90%(about 2 * 10
4/ cm
2), and handle deactivation in 2 hours with the ametycin of 10ug/ml, the cell after the deactivation used in 24 hours;
(2) acquisition of people iPSCs: get 2-3 year children foreskin inoblast, with twice of the retrovirus transfection that carries Klf4, Sox2, Oct4 and c-Myc transcription factor.Second day vitaminize C 25ug/ml in culture system added VPA2 μ M(and used altogether 5 days on the 5th day), the inoblast after the transfection was inoculated on the trophocyte in the 6th day, the 7th day, use the people ESCs nutrient solution that contains bFGF, alternative serum instead.Change liquid subsequently every day, the clone up to class ESCs occurring selects, amplification cultivation;
(3) amplification cultivation of people iPSCs: microscopically is selected the clone of class ESCs, after 5-10 minute, dispels into little agglomerate with the enzymic digestion of IV Collagen Type VI gently, is inoculated on the ready trophocyte.Change liquid every day.Went down to posterity once in every 7-8 days.Cell after the amplification goes down to posterity with mechanical process, and promptly the glass dropper with 5ml scrapes cell from trophoderm gently, dispels into little agglomerate subsequently gently, is inoculated on the ready trophocyte;
(4) keeping of the biological characteristics of people iPSCs and multipotency: be that amplification cultivation is identified the ESCs specific gene expression of amplification cultivation descendant iPSCs after 14 generations on the trophoderm with the RT-PCR method with the human marrow mesenchymal stem cell; Whether detect it with EB forming method and immunodeficient mouse teratoma forming method simultaneously can break up to triploblastica in vivo and in vitro.
The invention provides a kind of is the method for human marrow mesenchymal stem cell as trophoderm acquisition cultivator iPSCs with the humanization cell, but biological property and the multipotency of this humanized trophocyte's long term maintenance people iPSCs; Thus obtained people iPSCs does not have the pollution of allos cell, for its clinical application provides possibility.End user's mesenchymal stem cells MSCs of the present invention replaces using in the traditional method mouse embryo fibroblasts as the trophocyte, the cultivator induced multi-potent stem cells, reduced the pollution of allos cell in the culture system, and proved that this culture system can make people's induced multi-potent stem cells at amplification in vitro (seeing embodiment 4), and kept its biological characteristics and multipotency (seeing embodiment 5), provide possibility for people's induced multi-potent stem cells enters clinical application.
Description of drawings
Fig. 1 is technological line figure.
Fig. 2 is the 4th generation human marrow mesenchyme stem cell.Before A is deactivation; After B is the ametycin deactivation.
The people iPSCs of Fig. 3 for obtaining as trophoderm with human marrow mesenchymal stem cell.A is inoblast transfection after 21-25 days, the granuloma sample of appearance clone (scale is 100 μ m), and B is the iPSCs clone (scale is 100 μ m) who forms after the amplification cultivation, C is clone's local cells form (scale is 20 μ m).
To be 8-11 with the 4th generation human marrow mesenchyme stem cell be growth curve in the trophoblastic culture system for people iPSCs to Fig. 4.
Fig. 5 is the 9th, 10 generation people iPSCs at different donors the 4th generation human marrow mesenchyme stem cell of originating is amplification times (hMSC1 and hMSC2 are women's donor source, and hMSC3 originates for male sex's donor) in the trophoblastic culture system.
Fig. 6 for RT-PCR detect people iPS/MSC7 human marrow mesenchymal stem cell be in the trophoblastic culture system 14 generations of amplification cultivation with the expression of descendant ESCs specific gene.
Fig. 7 iPS/MSC7 that behaves is that amplification cultivation is broken up situation at external formation EB to triploblastica after 14 generations in the trophoblastic culture system at human marrow mesenchymal stem cell.A is that the suspension EB(scale that the EB cultivation formed after 8 days is 50 μ m); B-D cultivates the adherent all kinds cell that is divided into of EB (scale is 100 μ m) after 14 days; E is that PT-PCT detects EB differentiation back pluripotency gene (SOX2 and OCT4) and tridermic marker gene (FOXA2 and AFP are the entoderm marker gene; MSX1 and BRACHYURY are the mesoderm marker gene; MAP2 and PAX6 are the ectoderm marker gene) expression (U represents not break up, and D represents the differentiation back).
Fig. 8 iPS/MSC7 that behaves is that amplification cultivation forms the teratoma situation in the trophoblastic culture system in NOD-SCID mouse body after 14 generations at human marrow mesenchymal stem cell.A is for growing teratomatous mouse; B is the adenoid tissue (HE dyeing, scale is 20 μ m) that forms in the teratoma; C is the muscle sample tissue (HE dyeing, scale is 20 μ m) that forms in the teratoma.
Embodiment
The present invention is further described in conjunction with the accompanying drawings and embodiments.
Embodiment 1: human marrow mesenchymal stem cell separation and Culture, cryopreservation resuscitation
(1) human marrow mesenchymal stem cell separation and Culture
Aseptic condition is gathered the marrow of 3 parts of healthy donors down, and anticoagulant heparin is with Ficoll-paque(proportion 1.077) density gradient centrifugation collection mononuclearcell, with 5 * 10
5Individual cell/cm
2Density inoculation, nutrient solution is for containing 10%(V/V) the low sugar DMEM(LG-DMEM of foetal calf serum and 1% glutamine) nutrient solution, place 37
0C, 5%CO2 incubator are cultivated.Change nutrient solution after 24 hours, discard non-adherent cell, as seen the fusiformis attached cell is arranged.Changed liquid 1 time in per afterwards 3 days, the adherent fusion of left and right sides primary cell in 14 days reaches 90%-95%, is arranged with obvious directivity, becomes whirlpool shape, netted, radial (seeing Fig. 2 A).With 0.25% trypsinase-1 mmol EDTA digestion, divide the bottle cultivation of going down to posterity in the 1:3 ratio, and be labeled as the P1 cell.Changed liquid once in per 3 days in the culturing process that goes down to posterity, cell reaches 90% and merges the back had digestive transfer culture, and is labeled as P2, by that analogy.
(2) the frozen and recovery of human marrow mesenchymal stem cell
When frozen, will be in the mescenchymal stem cell digestion of logarithmic phase after, with frozen protection liquid (60% LG-DMEM, 30% foetal calf serum, 10% DMSO) re-suspended cell, final concentration of cells is 5 * 10
6/ ml divides to change in the frozen pipe cell title, algebraically, frozen time and frozen person on the mark over to.After immediately frozen pipe being put into the Virahol freezing storing box, put into-80
0C, the back of spending the night moves into-196
0The C liquid nitrogen container is preserved.During cell recovery, from liquid nitrogen, take out cell and put into 37
0C constant water bath box quick-thawing treats that frozen storing liquid thaws to little ice crystal, moves into super clean bench.Under the aseptic condition, cell suspension is moved into the 15ml centrifuge tube, dropwise add the nutrient solution 10ml of precooling, behind the centrifugal 5min of 200g, supernatant discarded.With cell mass with the resuspended cultivation of an amount of preheating nutrient solution.Adherent fully behind the recovery survivaling cell inoculation 6h, cellular form becomes fusiformis gradually.Cell is bred rapidly through beginning after 1-2 days resting stages.Cellular form after the recovery and frozen preceding basically identical.
Embodiment 2: human marrow mesenchymal stem cell is as trophocyte's preparation
Get the 3-5 generation human marrow mesenchyme stem cell as trophoblastic cell.Use 0.1%gelatin 37 in advance 6 orifice plates
0C is hatched 1h to spending the night.Human marrow mesenchymal stem cell is with 2 * 10
4/ cm
2Density is inoculated on six orifice plates after gelatin handles, makes second day cytogamy degree reach 80%-90%.Ametycin with 10ug/ml is handled deactivation in 2 hours, basically identical (see figure 2) before cellular form after the deactivation and the deactivation.Cell after the deactivation used in 24 hours.
Embodiment 3: with human marrow mesenchymal stem cell is inducing of trophoblastic people iPSCs
Get third generation children (2-3 year) foreskin inoblast, with twice of the retrovirus transfection that carries Klf4, Sox2, Oct4 and c-Myc transcription factor.Transfection finish the back in culture system vitaminize C 25ug/ml up to selected clone, adding VPA2 μ M(on the 5th day used 5 days altogether), inoblast after the transfection was inoculated into on the human marrow mesenchymal stem cell trophocyte in the 6th day, used the people ESCs nutrient solution of the bFGF, 1% glutamine, 1% non-essential amino acid, 100 μ M and 20% alternative serum that contain 8ng/ml on the 7th day instead.Change liquid subsequently every day, up to the clone who class ESCs occurs (seeing Fig. 3 A), clear border, cell densification.Microscopically is selected one by one, amplification cultivation.Technological line is seen Fig. 1
Embodiment 4: be the amplification cultivation of trophoblastic people iPSCs with human marrow mesenchymal stem cell
(1) people iPSCs is being the cellular form after the amplification cultivation in the trophoblastic culture system with human marrow mesenchymal stem cell
Microscopically is selected the clone of class ESCs, and the IV Collagen Type VI enzymic digestion of using 1mg/ml dispels into little agglomerate after 5-10 minute gently, is inoculated on the ready trophocyte, and personnel selection ESCs nutrient solution is 37
0C, 5%CO
2The middle cultivation.Change liquid every day.Went down to posterity once in the every 7-8 of iPSCs days.Cell after the amplification goes down to posterity with mechanical process, and promptly the glass dropper with 5ml scrapes cell from trophoderm gently, dispels into little agglomerate subsequently gently, is inoculated on the ready trophocyte with 1:2-1:3.People iPSCs is being that the amplification back forms typical people ESCs sample clone in the trophoblastic culture system with human marrow mesenchymal stem cell: clear border, and the cell densification, most of cell is circular, and kernel is obvious, and the border is class fibroblast-like cells (seeing Fig. 3 B, C).
(2) people iPSCs with human marrow mesenchymal stem cell is being growth kinetics in the trophoblastic culture system
Three strain people iPSCs(iPS/MSC1, iPS/MSC7 and iPS/MSC10 with the 8th generation) with 5 * 10
4Cells/well inoculation (with little agglomerate inoculation) is in trophoblastic 12 orifice plates to being covered with human marrow mesenchymal stem cell, establishes three multiple holes.Cultivate after 7 days, people iPSCs is scraped from trophoderm, get the part counting, calculate amplification times; Getting simultaneously and wherein partly being inoculated into the new human marrow mesenchymal stem cell that is covered with is in trophoblastic 12 orifice plates, establishes three multiple holes, continues to cultivate 7 days, and counting is the calculating amplification times also.By that analogy, cultivate the three generations altogether, calculate amplification times.People iPSCs is being sustainable amplification in the trophoblastic culture system with human marrow mesenchymal stem cell, 2.32 ± 0.05 times of (see figure 4)s of average per generation amplification.
(3) human marrow mesenchymal stem cell of people iPSCs in different donors source is the growing state in the trophoblastic culture system
Three strain people iPSCs(iPS/MSC1, iPS/MSC7 and iPS/MSC10 with the 9th, 10 generations) with 5 * 10
4Cells/well inoculation (with little agglomerate inoculation) is to the human marrow mesenchymal stem cell (hMSC1, hMSC2, the hMSC3 that are covered with different donors source, wherein hMSC1 and hMSC2 are women's donor, hMSC3 is male sex's donor) in trophoblastic 12 orifice plates, establish three multiple holes.Cultivate after 7 days, people iPSCs is scraped from trophoderm, get the part counting, calculate amplification times.The mesenchymal stem cells MSCs in different donors source is trophoblastic culture system does not have the significant difference (see figure 5) to the amplification of people iPSCs cell.
Embodiment 5: people iPSCs is the situation of keeping of amplification cultivation biological characteristics and multipotency after 14 generations in the trophoblastic culture system at human marrow mesenchymal stem cell
(1) people iPSCs expressing human ESCs specific gene situation
Being collected in human marrow mesenchymal stem cell is the people iPSC/MSC7 cell mass of amplification cultivation after 14 generations in the trophoblastic culture system, extract total RNA with TRIZOL, carry out RT-PCR subsequently, detect the expression of people ESCs specific gene: comprise endogenous KLF4, SOX2, c-Myc, OCT4 and NANOG, DNMT3B, DPPA4, hTERT, NODAL, REX13, TDGF1 etc.Be contrast with H1 people ESCs and children's foreskin inoblast simultaneously.Detected result shows that the expression level of these genes in people iPSCs is similar to H1 people ESCs.
(2) people iPSCs breaks up situation at external formation EB to triploblastica
At human marrow mesenchymal stem cell is that the people iPSC/MSC7 of amplification cultivation after 14 generations scrapes from trophoderm in the trophoblastic culture system, is seeded in ultralow adhesion 6 orifice plates with cell mass, and nutrient solution is for not containing the people ESCs nutrient solution of bFGF, 37
0C, 5%CO
2The middle cultivation changed liquid every other day; Cultivate after 8 days, be inoculated in 6 orifice plates after 0.1%gelatin handles, continue with the people ESCs nutrient solution that does not contain bFGF, 37
0C, 5%CO
2Middle adherent culture 8 days.Experimental result shows that the people iPSCs after the amplification can see Fig. 7 A at external formation EB(), after these EB adherent culture, can be divided into various types of cells (seeing Fig. 7 B-D).Collect the cell of these differentiation, detect triploblastica mark expression of gene situation with RT-PCR, the result shows that (FOXA2 and AFP are the entoderm marker gene for cell triploblastica marker gene after the differentiation; MSX1 and BRACHYURY are the mesoderm marker gene; MAP2 and PAX6 are the ectoderm marker gene) obviously increase of expression.Show at human marrow mesenchymal stem cell to be that the people iPSCs of amplification cultivation after 14 generations still kept its multidirectional differentiation potential in the trophoblastic culture system.
(3) people iPSCs forms teratoma in vivo and breaks up situation to triploblastica
To be that the people iPSC/MSC7 of amplification cultivation after 14 generations scrapes from trophoderm in the trophoblastic culture system at human marrow mesenchymal stem cell, with twice of the PBS buffer solution for cleaning that does not contain calcium ions and magnesium ions, with PBS that people iPSCs is resuspended, get in the back leg root muscle that 200ul is expelled to the NOD-SCID mouse, the cell quantity of each injection point is 2-3 * 10
6Observe into the knurl situation after the injection.After treating that teratoma forms, take out teratoma row paraffin section and HE dyeing, observe it and break up situation to triploblastica.Experimental result shows that the people iPSCs after the amplification can form teratoma (seeing Fig. 8 A) in the immunodeficient mouse body, see after the HE dyeing muscle sample is arranged in the knurl, adenoid tissue forms (seeing Fig. 8 B, C).Being illustrated in human marrow mesenchymal stem cell is that the people iPSCs of amplification cultivation after 14 generations still kept its multidirectional differentiation potential in the trophoblastic culture system.
Claims (2)
1. one kind with the method for human marrow mesenchymal stem cell as trophoderm cultivator inductive pluripotent stem cells, is achieved through the following technical solutions:
(1) trophocyte's preparation: the human marrow mesenchymal stem cell of using the 3rd ~ the 5th generation of cultivating behind go down to posterity cultivation, the low temperature cryopreservation resuscitation is as the trophocyte, on six orifice plates of cell inoculation after handle with 0.1%gelatin, cytogamy degree 80%-90%, and handle deactivation in 2 hours with the ametycin of 10ug/ml, the cell after the deactivation used in 24 hours;
(2) acquisition of people's inductive pluripotent stem cells: get 2-3 year children's foreskin inoblast, with twice of the retrovirus transfection that carries Klf4, Sox2, Oct4 and c-Myc transcription factor, second day vitaminize C 25ug/ml in culture system, added VPA2 μ M on the 5th day, inoblast after the transfection was inoculated on the trophocyte in the 6th day, the 7th day, use the people ESCs nutrient solution that contains bFGF, alternative serum instead, change liquid subsequently every day, clone up to class ESCs occurring selects, amplification cultivation;
(3) amplification cultivation of people's inductive pluripotent stem cells: microscopically is selected the clone of class ESCs, with the enzymic digestion of IV Collagen Type VI after 5-10 minute, dispel into little agglomerate gently, be inoculated on the ready trophocyte, change liquid every day, went down to posterity once in every 7-8 days, the cell after the amplification goes down to posterity with mechanical process;
(4) keeping of the biological characteristics of people's inductive pluripotent stem cells and multipotency: with the human marrow mesenchymal stem cell be amplification cultivation after 14 generations on the trophoderm, whether the ESCs specific gene expression with RT-PCR method evaluation amplification cultivation descendant inductive pluripotent stem cells detects it with EB forming method and immunodeficient mouse teratoma forming method simultaneously and can break up to triploblastica in vivo and in vitro.
2. according to claim 1 a kind of with the method for human marrow mesenchymal stem cell as trophoderm cultivator inductive pluripotent stem cells, it is characterized in that, the described amplification cultivation of step (3) is that the glass dropper with 5ml scrapes cell from trophoderm gently, dispel into little agglomerate subsequently gently, be inoculated on the ready trophocyte.
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| CN102286532A (en) * | 2011-09-05 | 2011-12-21 | 浙江大学 | Method for obtaining inductive pluripotent stem cell |
| CN102965393A (en) * | 2012-11-06 | 2013-03-13 | 上海交通大学 | Method for preparing human induced pluripotent stem cells and use of human induced pluripotent stem cells |
| CN103589686A (en) * | 2013-11-06 | 2014-02-19 | 四川新生命干细胞科技股份有限公司 | Method of utilizing human umbilical cord mesenchymal stem cells as feed layer to culture human induced pluripotent stem cells |
| CN104140951A (en) * | 2014-08-06 | 2014-11-12 | 深圳市科晖瑞生物医药有限公司 | Method for developing and cultivating human induced pluripotent stem cells |
| CN106381282A (en) * | 2016-10-12 | 2017-02-08 | 广东艾时代生物科技有限责任公司 | Induced pluripotent stem cell subculture method |
| CN107236703A (en) * | 2017-05-25 | 2017-10-10 | 佛山科学技术学院 | A kind of feeder cells, cultural method and application |
| CN109975264A (en) * | 2019-04-18 | 2019-07-05 | 安徽农业大学 | A kind of method that slow-virus transfection blastaea trophocyte studies its gene function |
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| CN102286532B (en) * | 2011-09-05 | 2012-10-17 | 浙江大学 | Method for obtaining inductive pluripotent stem cell |
| CN102286532A (en) * | 2011-09-05 | 2011-12-21 | 浙江大学 | Method for obtaining inductive pluripotent stem cell |
| CN102965393B (en) * | 2012-11-06 | 2015-08-26 | 上海交通大学 | The preparation method and its usage of people's induced multi-potent stem cells |
| CN102965393A (en) * | 2012-11-06 | 2013-03-13 | 上海交通大学 | Method for preparing human induced pluripotent stem cells and use of human induced pluripotent stem cells |
| CN103589686B (en) * | 2013-11-06 | 2015-11-18 | 四川新生命干细胞科技股份有限公司 | A kind of human umbilical cord mesenchymal stem cells that uses is as the method for feeder layer cultivator induced multi-potent stem cells |
| CN103589686A (en) * | 2013-11-06 | 2014-02-19 | 四川新生命干细胞科技股份有限公司 | Method of utilizing human umbilical cord mesenchymal stem cells as feed layer to culture human induced pluripotent stem cells |
| CN104140951A (en) * | 2014-08-06 | 2014-11-12 | 深圳市科晖瑞生物医药有限公司 | Method for developing and cultivating human induced pluripotent stem cells |
| CN104140951B (en) * | 2014-08-06 | 2016-08-24 | 深圳市科晖瑞生物医药有限公司 | A kind of method set up and cultivate people induced multi-potent stem cell |
| CN106381282A (en) * | 2016-10-12 | 2017-02-08 | 广东艾时代生物科技有限责任公司 | Induced pluripotent stem cell subculture method |
| CN106381282B (en) * | 2016-10-12 | 2020-08-07 | 广东艾时代生物科技有限责任公司 | Passage method for induced pluripotent stem cells |
| CN107236703A (en) * | 2017-05-25 | 2017-10-10 | 佛山科学技术学院 | A kind of feeder cells, cultural method and application |
| CN107236703B (en) * | 2017-05-25 | 2021-06-01 | 佛山科学技术学院 | Feeder layer cell, culture method and application |
| CN109975264A (en) * | 2019-04-18 | 2019-07-05 | 安徽农业大学 | A kind of method that slow-virus transfection blastaea trophocyte studies its gene function |
| CN109975264B (en) * | 2019-04-18 | 2022-04-08 | 安徽农业大学 | Method for researching gene function of lentivirus transfected blastocyst trophoblast cells |
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