CN102250830B - In-vitro separating and culturing method for germline stem cell - Google Patents
In-vitro separating and culturing method for germline stem cell Download PDFInfo
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Abstract
The invention discloses an in-vitro separating and culturing method for a germline stem cell. The method comprises the following steps of: obtaining a cell from germinal epithelium of an ovary of a female mammal, differentiating and separating a non-adherent germline stem cell from other adherent cultured cells by adopting gelatin with the concentration 0.2 percent and a Matrigel differential adherence combination cloning method; and culturing the germline stem cell obtained by separating on an MEF (Mouse Embryonic Fibroblast) feeder layer. The separated germline stem cell has ES (Embryonic Stem) characteristic and the potential of differentiating into a muscle cell, an adipose cell and oocyte-like cell and various cells with three internal germinal layers.
Description
Technical field
The invention belongs to biological technical field, relate to in-vitro multiplication, differentiation, the separation of stem cell animal, particularly the in-vitro separation of a kind of female reproduction stem cell and cultural method.
Background technology
The research of the generation of sexual cell, propagation, differentiation is one of important topic of life science.In the animal reproduction breeding, the genetic resources of high yield, the high-quality sexual cell that places one's entire reliance upon is finished heredity; The mankind, because the Infertility that the generation of ovum, dysmaturity etc. cause perplexs numerous patients' a realistic problem especially; In fundamental research, ovum is one of the most important research object of developmental biology research especially; Therefore the generation of ovocyte, the research of differentiation have great importance in livestock industry production and biomedical research.Yet the generation of higher mammal ovocyte, transfer, differentiation are finished fully in vivo, therefore be difficult to the dynamic molecular events (Hua etc. that detect in real time and study wherein, Recent advances in the derivation of germ cells from the embryonic stem cells.StemCells and Development, 2008,17:399-411).
Stem cell is that a class has self, highly propagation and the cell colony of multi-lineage potential.Embryonic stem cell (ES cell or ESCs) is to be separated and next multipotential stem cell by body early embryo inner cell mass (ICM) or archeocyte (PGCs).Because unique biological characteristics of embryonic stem cell, make embryonic stem cell at cell replacement therapy, histoorgan reparation and reconstruction, gene therapy and developmental biology model, new drug development and toxicological experiment etc., nearly all life science and biomedicine field all have great importance.
Before more than 40 year, scientist just finds to have in the boar reproductive organ existence of male germ stem cells-stem spermatogonium, and has successfully carried out vitro culture.It is found that recently, the spermatogonium of adult animals testis is under external suitable condition, may be converted into the male germ stem cells (Matsui etc. of versatility, Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture.Cell, 1992,70:841-847; Dong Wuzi etc., cultivation and the detection of new born bovine male sex-cell source class ES, journal of animal science and veterinary medicine, 2007,38 (2): 144-148; Guan etc., Pluripotency of spermatogonial stem cells from adult mouse testis.Nature, 2006,440:1199-1203; Sesndel etc., Generation of functional multipotent adult stem cells from GPR125
+Germline progenitors.Nature, 2007,449:346-350; Kanatsu-Shinohara etc., Pluripotency of a single spermatogonial stem cell in mice.Biol Reprod., 2008,78 (4): 681-687; Kanatsu etc., Generation of pluripotent stem cells from neonatal mouse testis.Cell, 2004,119:1001-1012; Bukovsky etc., Generation of pluripotent stem cells from adult human testis.Nature, 456,344-349; Kanatsu-Shinohara etc., Pluripotency of a single spermatogonial stem cell in mice.Biol Reprod., 2008,78 (4): 681-687).So mGSCs can be as the ideal carrier cell of transgenic animal and cloned animal, and compared with normal ES cell, mGSCs cell derived quantity is many, cultivate relatively easy.But the research for sexual cell, what is more important mGSCs can effectively break up and produces sperm (Ko etc., Induction of pluripotency in adult unipotent germline stem cells.Cell Stem Cell, 2009,5:87-96).
But for female mammal, traditional view is thought: all sexual cell all enter reduction division in female fetus fetal development, no longer produce new ovocyte after the birth.Therefore, in the life cycle after it, only have limited female sex cell to be present in the sexual cell pond, with advancing age, its quantity will constantly reduce, and not exist a kind of stem cell it is replenished and to regenerate.In recent years, there is the investigator to point out the sexual cell of the rear female mice of birth to have the activity of regeneration.
Professor Wu Ji of Shanghai Communications University separates from the adult mice ovary first and has successfully set up female reproduction stem cell (FGSCs), and proves that resulting FGSCs has the ovocyte of being differentiated to form, and finishes the potentiality of transmitting from generation to generation.Therefore it is expected to provide new approach and platform as transgenic animal production, xenotransplant, the research of large animal disease model etc., also can be used as Mammalian Reproduction and the new way of growing research, with respect to the production method of traditional somatic cell clone and microinjection transgenic animal, it has advantages of simply, efficient is high.The mankind, may develop the numerous potential female sex cell on the ovary, be reproduction medical research and the new precious ovocyte source of clinical development.To help the mechanism such as the generation of postgraduate's cell colonization and ovum, apoptosis; Treatment to Infertilities such as animal species preservation, regenerative medicine and premature ovarian failures will produce far-reaching influence.
The female reproduction stem cell has made some progress nematode, fruit bat and mouse isotype animal, as culture system (Zou etc., the Production of offspring from a germline stem cell line derived from neonatal ovaries.Nat Cell Biol.2009May of mouse FGSCs have been set up; 11 (5): 631-6.; Zou etc., Improved efficiency of female germline stem cell purification using Fragilis-based magnetic bead sorting.Stem Cells Dev.2011May 26.[Epub ahead of print] .).But to the germline stem cell of domestic animal such as pig, ox, sheep etc., it is still not enough that people understand, and there is not yet the isolation cultivation method of female reproduction stem cell.
Summary of the invention
The problem that the present invention solves is to provide in-vitro separation and the cultural method of a kind of female reproduction stem cell, ovary in-vitro separation clone female reproduction stem cell from female mammal, and the female reproduction Stem cells cultured in vitro to being separated to, for the research female sex cell lays the foundation.
The present invention is achieved through the following technical solutions:
The extracorporeal separation method of a kind of female reproduction stem cell may further comprise the steps:
1) isolated cell
The ovarian germinal epithelium that will separate from the ovary of aseptic collection shreds to fragment, with resuspended 2～3 times of the DMEM/F12 nutrient solution that comprises volume fraction 10%FBS, obtains the tissue mass of lower floor after leaving standstill; Then with the mixed solution digestion process tissue mass that comprises Collagenase I, the 10 μ g/ml DNase of massfraction 0.1% and massfraction 0.1% Unidasa 2～3 times, each 20～30min, resuspended with the DMEM/F12 nutrient solution that comprises volume fraction 10%FBS again, separate obtaining ovary source sexual cell;
2) the former culture of female reproduction stem cell
With the ovary source sexual cell that obtains with 2 * 10
5The density of individual/mL is seeded in the plastic culture dish through 37 ℃ of coated 30min of gelatin of massfraction 0.2%, and nutrient solution is female reproduction stem cell separation and Culture liquid;
Described female reproduction stem cell separation and Culture liquid also comprises following component take the DMEM/F12 substratum as basic medium: Basic Fibroblast Growth Factor, 2.5 μ mol/L BIO (6-bromoindirubin-30-oxime), 100IU/ml penicillin and the 100mg/mL Streptomycin sulphate of a kind of, the 2mmol/L L-glutaminate in the middle of the foetal calf serum of volume fraction 20% and the serum replacement (KSR), 1mmol/L Sodium.alpha.-ketopropionate, 0.1mmol/L beta-mercaptoethanol, 0.1mmol/L non-essential amino acid, 5～10ng/mL;
Cultivate behind the 12h plastic culture dish cultivation that will not adherent cell forwards to through 4 ℃ of coated 1h of Matrigel matrix membrane, nutrient solution is female reproduction stem cell separation and Culture liquid;
After not adherent cell shifts for the first time and cultivates 12h, with shift cultivate after again not adherent cell transfer cultivate through the plastic culture dish of 4 ℃ of coated 1h of Matrigel matrix membrane and cultivate, nutrient solution is female reproduction stem cell separation and Culture liquid;
After attached cell did not shift cultivation for the second time, 2d changed 1 nutrient solution; Behind the Growth of Cells 3～7 days, begin to occur fine and close embryonic stem cell sample colony;
3) cultivation of going down to posterity of female reproduction stem cell
When treating that fine and close embryonic stem cell sample colony grows to periphery and begins noble cells to occur, with the sucking-off of fine and close embryonic stem cell sample colony, washing is 2 times in PBS; Move on to again in the trypsinase of TrypLE cell dissociation buffer or massfraction 0.05% and digest 2～3min, and pressure-vaccum 5～20 times; The discrete unicellular or small cell cluster of opening is transferred to the plastic culture dish cultivation that is coated with 1～2h through 4 ℃ of Matrigel matrix membranes, and nutrient solution is female reproduction stem cell separation and Culture liquid; 37 ℃, 5%CO
2Cultivate under the saturated humidity condition;
After cultivating 3～7d, begin to occur fine and close embryonic stem cell sample colony, be the female reproduction stem cell.
The extracorporeal culturing method of above-mentioned female reproduction stem cell may further comprise the steps:
The female reproduction stem cell is inoculated on the MEF feeder layer, nutrient solution also comprises following component take the DMEM/F12 substratum as basic medium: the ITS of the non-essential amino acid of the serum replacement of volume fraction 20% or 20% foetal calf serum, 2mmol/L glutamine, 0.1mmol/L beta-mercaptoethanol, 100IU/mL penicillin, 100mg/mL Streptomycin sulphate, massfraction 1%, 2.5 μ mol/L BIO, 5～10ng/mL Basic Fibroblast Growth Factor and massfraction 1%; 2～3d changes a nutrient solution.
Compared with prior art, the present invention has following useful technique effect:
1, the extracorporeal separation method of female reproduction stem cell provided by the invention is applicable to the separation and Culture of mammiferous female reproduction stem cell.The female reproduction stem cell of institute's separating clone forms typical fine and close embryonic stem cell sample colony, has the embryonic stem cell characteristic, its alkaline phosphatase (AP) is positive or the weak positive, and SSEA1, SSEA4, CD49f, Oct4, C-myc, Sox2, Klf4, Nanog etc. also are positive; The female reproduction stem cell of separation and Culture is larger, and cell is bright, opacifying property is strong, nuclear is large, 3～7 days typical fine and close cell clones of formation after cultivating, similar ES cell clone.
2, pig is a kind of important economic animal, and simultaneously size, dissection and physiological structure and the mankind of pig are very approaching, is the optimal human diseases model except primate had an optimistic view of of people and the donor of organ transplantation.How to obtain, keep the high-quality indigenous blastogenesis resource of China, existing population is improved, become the pressing issues of pig cultivation and breeding, the research of pig germline stem cell is expected one of important means that becomes solution blastogenesis resource.
Specifically for the separation of pig female reproduction stem cell, it is positive that the pig female reproduction stem cell of the pig female reproduction stem cell separating clone of separating clone is Dazl, Vasa, and the part cell also is Filga, ZP3, Stra8, Scp3 etc. and also is positive; The pig female reproduction stem cell of separating clone has tridermic differentiation potential, can be differentiated to form embryoid, muscle like cell, fat-like cell and triploblastica mark positive cell, comprising PDX1, Desmin, A-ACTIN and NSE positive cell neurocyte, myocardial cell and ovocyte like cell; Especially the differentiation potential that has an ovocyte like cell has great importance for acquisition, maintenance, the improvement of pig high-quality germ plasm resource.
3, the extracorporeal separation method of female reproduction stem cell provided by the invention for the in-vitro separation of Mammals female reproduction stem cell provides new method, is convenient to follow-up using and studying.
Description of drawings
Fig. 1 is biological characteristics and the staining examine result of pig female reproduction stem cell; Wherein, A is that single pig female reproduction stem cell assembles the fine and close embryonic stem cell sample clone who forms, 100 *; B is that pig female reproduction stem cell is alkaline phosphatase staining and takes on a red color, the positive, 200 *.
Fig. 2 is the immunofluorescence detected result of pig female reproduction Stem cell surface marker; Wherein, the middle row in the middle of the A figure are respectively SSEA1, SSEA4 and CD49F is positive, and left column is respectively the nuclear colour developing of SSEA1, SSEA4 and CD49F dyeing, and the Merge of SSEA1, SSEA4 and CD49F dyeing and its nuclear is classified on the right side as; Middle row among the B figure are respectively Oct4, C-myc, Klf4, Sox2 are positive, and left column is respectively the nuclear colour developing of Oct4, C-myc, Klf4, Sox2 dyeing, and the Merge of Oct4, C-myc, Klf4, Sox2 dyeing and its nuclear is classified on the right side as.
Fig. 3 is that the differentiation potential of pig female reproduction stem cell detects; Middle row are respectively Dazl, Vasa, Filga, ZP3, Stra8 and the Scp3 positive, and left column is respectively nuclear colour developing of its corresponding dyeing, and the Merge of Dazl, Vasa, Filga, ZP3, Stra8 and Scp3 dyeing and its nuclear is classified on the right side as.Bar＝200μm。
Fig. 4 is that the differentiation potential of pig female reproduction stem cell detects; The embryoid (EB) that suspension culture 3d forms, A, bar=40 μ m; B, bar=100 μ m; C, bar=200 μ m; D, bar=400 μ m; E, the adherent muscle cell (mesoderm) that is differentiated to form similar spindle shape of embryoid, bar=200 μ m; F, the adherent fat-like cell (mesoderm) that is differentiated to form of embryoid is the oil red O stain positive, bar=200 μ m; The adherent 10d of embryoid is differentiated to form PDX1 (entoderm), Desmin (mesoderm), A-ACTIN (mesoderm) and NSE positive cell (ectoderm).
Fig. 5 is biological characteristics and the staining examine result of mouse female reproduction stem cell; Wherein, A, B are that single pig female reproduction stem cell assembles the fine and close embryonic stem cell sample clone who forms, 100 *; C is that pig female reproduction stem cell is alkaline phosphatase staining and takes on a red color, the positive, 200 *.
Embodiment
The invention provides the culture system of a kind of high efficiency separation cloning mammal female reproduction stem cell, comprise the separation of female reproduction stem cell, detection and the vitro culture of female reproduction stem cell.
Originate as starting materials with the Adult Mammals ovary; The separation purification method of female reproduction stem cell adopts 0.2% gelatin and Matrigel differential velocity adherent in conjunction with cloning, with other cell differentiation of not adherent female reproduction stem cell and adherent culture, separate.
Below specifically describe with the in-vitro separation of pig female reproduction stem cell:
The extracorporeal separation method of a, pig female reproduction stem cell specifically may further comprise the steps:
1) separates the pig ovary cell
With the aseptic physiological saline that contains two anti-(500IU/mL penicillin, 500mg/mL Streptomycin sulphates) with the pig ovary washing of aseptic collection 3 times, again with containing two anti-PBS (phosphate buffered saline buffer) washings 3 times, in sterile petri dish, peel off the pig ovary adventitia, separate germinal epithelium;
Germinal epithelium is shredded to 1mm
3Fine grained chippings, with the FBS nutrient solution of DMEM/F12+ volume fraction 10% resuspended (500 turn following centrifugal) germinal epithelium fragment, repeat 2～3 times, after leaving standstill 2～5 minutes, obtain lower floor's germinal epithelium cell mass;
Then be 0.1% Collagenase I (collagenase I with massfraction, Invitrogen)+10 the mixed solution digestion process 2～3 times of the Unidasa of μ g/mL DNase (DNA enzyme)+massfraction 0.1%, each 20～30min, use again the resuspended nutrient solution of DMEM/F12+10%FBS (500 turn following centrifugal), separate obtaining the germinal epithelium cell;
If cell mass is still larger, then be trypsin treatment 3～5min of 0.25% with massfraction, resuspended with DMEM/F12+10% (volume fraction) FBS, cell counting;
2) the former culture of female reproduction stem cell
With the germinal epithelium cell that obtains with 5 * 10
5It is the plastic culture dish of 37 ℃ of coated 30min of gelatin of 0.2% that the density of individual/mL is seeded in through massfraction, adds female reproduction stem cell separation and Culture liquid;
Described female reproduction stem cell separation and Culture liquid is take the DMEM/F12 substratum as basic medium, also comprise following component: the foetal calf serum of volume fraction 20% (or 20% serum replacement, KSR), 2mmol/L L-glutaminate, 1mmol/L Sodium.alpha.-ketopropionate, 0.1mmol/L beta-mercaptoethanol, 0.1mmol/L non-essential amino acid (NEAA, American I nvitrogen company product), the Basic Fibroblast Growth Factor (bFGF) of 5～10ng/mL, 2.5 μ mol/L BIO, 100IU/ml penicillin, 100mg/mL Streptomycin sulphate;
Cultivate behind the 12h plastic culture dish cultivation that will not adherent cell forwards to through 4 ℃ of coated 1h of Matrigel matrix membrane (BD company), nutrient solution is female reproduction stem cell separation and Culture liquid;
After not adherent cell shifts for the first time and cultivates 12h, with shift cultivate after again not adherent cell transfer cultivate through the culture dish of 4 ℃ of coated 1h of Matrigel matrix membrane and cultivate, nutrient solution is female reproduction stem cell separation and Culture liquid;
After attached cell did not shift cultivation for the second time, 2～3d changed 1 nutrient solution; Behind Growth of Cells 5～7d, begin to occur more typical fine and close embryonic stem cell sample colony;
3) cultivation of going down to posterity of female reproduction stem cell
Treat that fine and close embryonic stem cell sample colony grows to periphery and begins to occur noble cells, namely peripheral cell becomes large, and when boundary was obvious, with the sucking-off of fine and close embryonic stem cell sample colony, washing was 2 times in 100 μ L PBS; Move on to again 100 μ L TrypLE cell dissociation buffer (commercial cell dissociation buffer, the TrypLE of Invitrogen company
TMExpress) or in the trypsinase of massfraction 0.05% digest 2～3min, and with 5～20 discrete cells of 10 μ L liquid-transfering gun pressure-vaccums; Unicellular or the small cell cluster of opening dispersing moves on to through the plastic culture dish of 4 ℃ of coated 1h of Matrigel and cultivates, and nutrient solution is female reproduction stem cell separation and Culture liquid; 37 ℃, 5%CO
2Cultivate under the saturated humidity condition;
Behind Growth of Cells 3～7d, begin more typical fine and close embryonic stem cell sample colony to occur, be and separate the pig female reproduction stem cell that obtains.
The cell detection of b, the pig female reproduction stem cell that is separated to
1) alkaline phosphatase detects
With go down to posterity culture of isolated to the pig female reproduction stem cell of fine and close embryonic stem cell sample colony fix 3～5min with 4% Paraformaldehyde 96 after, wash twice with the PBS without calcium magnesium, with alkaline phosphatase dye liquor (in the firm red Tris-Cl solution that is dissolved in 10mL PH 8.2～8.4 of 2mg naphthyl alcohol phosphoric acid salt and 10mg) dyeing 8min, the sucking-off staining fluid, PBS washes 2 times, photographic recording;
The pig female reproduction stem cell of separating clone has the biological characteristics of ES cell characteristics and germline stem cell, forms typical colony, shown in Figure 1A; The alkaline phosphatase staining of pig female reproduction stem cell takes on a red color, and shown in figure B, for alkaline phosphatase (AP) positive, namely has the characteristic of multipotent stem cells.
2) immunohistochemical methods or immunofluorescence detect
The pig female reproduction stem cell cultivated with the fixing 15min of 4% Paraformaldehyde 96, is washed twice, 3%H with PBS
2O
2Incubated at room 10min, PBS washes twice, 10% foetal calf serum (PBS dilution) sealing, incubated at room 30min, serum deprivation inclines, drip respectively Oct4 (1: 500), SSEA1 (1: 200), SSEA4 (1: 200), Nanog (1: 200), CD49f (1: 200), Dazl (1: 500), Vasa (1: 1000), C-myc (1: 200), Sox2 (1: 200), Klf4 (1: 200), Filga (1: 100), ZP3 (1: 200), Stra8 (1: 500) and Scp3 (1: 300) primary antibodie working fluid, 4 ℃ are spent the night, PBS washes twice, drip two anti-(goat-anti rabbit FITC or sheep anti mouse FITC or Cy3,1: 500) working fluid, incubated at room 1h, PBS washes twice, each 5min, fluorescence microscopy Microscopic observation photographic recording result;
The primary antibodie sign of above-mentioned immunofluorescence is the mark of ES cell and germline stem cell, detected result is respectively such as Fig. 2, shown in Figure 3, wherein, SSEA1, SSEA4, CD49F (Fig. 2 A), Oct4, C-myc, Klf4, Sox2 (Fig. 2 B), the specific marks such as sexual cell, ovocyte and reduction division such as Dazl, Vasa, Filga, ZP3, Stra8 and Scp3 (Fig. 3) all are positive.
3) cells in vitro differentiation potential
Cultivate the making embryoid with the droplet hanging drop of 1000 cells/25 μ L, check the embryoid formational situation behind the 3d under the stereoscopic microscope, the embryoid (EB) that pig female reproduction stem cell suspension culture forms is shown in Fig. 4 A; Collect EB, (substratum is for removing the pig female reproduction stem cell parting liquid of bFGF and BIO on the 48 empty culture plates that 0.1% gelatin attaches in cultivation, namely take the DMEM/F12 high glucose medium as basic medium, add foetal calf serum, 2mmol/L L-glutaminate, 1mmol/L Sodium.alpha.-ketopropionate, 0.1mmol/L beta-mercaptoethanol, 0.1mmol/L non-essential amino acid, 100IU/mL penicillin and the 100mg/mL Streptomycin sulphate of volume fraction 20%), the cell type that 1～3 weekly check EB comes off and;
The pig female reproduction stem cell of separating clone has tridermic differentiation potential, can be differentiated to form EB (shown in Fig. 4 A～D), muscle cell (shown in Fig. 4 E), adipocyte (shown in Fig. 4 F, oil red O is positive) and ovocyte like cell (shown in Fig. 4 E); Adherent 10d EB is differentiated to form PDX1 (entoderm), Desmin (mesoderm), A-ACTIN (mesoderm) and NSE positive cell (ectoderm).
According to the isolation cultivation method of above-mentioned pig female reproduction stem cell, as the source, carry out the separation of female reproduction stem cell with the germinal epithelium of mouse ovarian, be separated to equally the mouse female reproduction stem cell of fine and close embryonic stem cell sample colony.Fig. 5 is biological characteristics and the staining examine result of mouse female reproduction stem cell, and wherein A, B are that single pig female reproduction stem cell assembles the fine and close embryonic stem cell sample clone who forms, 100 *; C is that pig female reproduction stem cell is alkaline phosphatase staining and takes on a red color, the positive, 200 *.
C, for the female reproduction stem cell that separation obtains, its external isolated culture method is:
The female reproduction stem cell is inoculated on the MEF feeder layer (is the mouse embryo fibroblasts feeder layer, processes 2-3h with the white mouse embryo fibroblasts of the elder brother of conceived 12-16d through 10 μ g/mL mitomycin, with 50000/cm
2Density spread be attached on the plastic culture plate that 0.1% gelatin is processed), nutrient solution is take the DMEM/F12 substratum as basic medium, also comprise following component: the serum replacement (KSR of volume fraction 20%, Invitrogen), the 2mmol/L glutamine, 0.1mmol/L beta-mercaptoethanol, 100IU/mL penicillin, the 100mg/mL Streptomycin sulphate, 0.1mmol/L non-essential amino acid (NEAA), 2.5 μ mol/L BIO, 5～10ng/mL Basic Fibroblast Growth Factor (bFGF), 1%ITS (Regular Insulin+Transferrins,iron complexes+Sodium Selenite, Sigma company).
Claims (2)
1. the extracorporeal separation method of a boar female reproduction stem cell is characterized in that, may further comprise the steps:
1) isolated cell
The ovarian germinal epithelium that will separate from the ovary of aseptic collection shreds to fragment, with resuspended 2～3 times of the DMEM/F12 nutrient solution that comprises volume fraction 10%FBS, obtains the tissue mass of lower floor after leaving standstill; Then with the mixed solution digestion process tissue mass 2～3 times of the Collagenase I, 10 μ g/ml DNase and massfraction 0.1% Unidasa that comprise massfraction 0.1%, each 20～30 minutes, resuspended with the DMEM/F12 nutrient solution that comprises volume fraction 10%FBS again, separate obtaining ovary source sexual cell;
2) the former culture of pig female reproduction stem cell
With the ovary source sexual cell that obtains with 2 * 10
5The density of individual/mL is seeded in the plastic culture dish through 37 ℃ of coated 30min of gelatin of massfraction 0.2%, and nutrient solution is female reproduction stem cell separation and Culture liquid;
Described female reproduction stem cell separation and Culture liquid also comprises following component take the DMEM/F12 substratum as basic medium: a kind of, the 2mmol/L L-glutaminate in the middle of the foetal calf serum of volume fraction 20% and the serum replacement (KSR), 1mmol/L Sodium.alpha.-ketopropionate, 0.1mmol/L beta-mercaptoethanol, 0.1mmol/L non-essential amino acid, the Basic Fibroblast Growth Factor of 5～10ng/mL, 2.5 μ mol/L BIO(6-bromoindirubin-30-oxime), 100IU/ml penicillin and 100mg/mL Streptomycin sulphate;
Cultivate behind the 12h plastic culture dish cultivation that will not adherent cell forwards to through 4 ℃ of coated 1h of Matrigel matrix membrane, nutrient solution is female reproduction stem cell separation and Culture liquid;
After not adherent cell shifts for the first time and cultivates 12h, with shift cultivate after again not adherent cell transfer cultivate through the plastic culture dish of 4 ℃ of coated 1h of Matrigel matrix membrane and cultivate, nutrient solution is female reproduction stem cell separation and Culture liquid;
After attached cell did not shift cultivation for the second time, 2d changed 1 nutrient solution; Behind Growth of Cells 3～7d, begin to occur fine and close embryonic stem cell sample colony;
3) cultivation of going down to posterity of pig female reproduction stem cell
When treating that fine and close embryonic stem cell sample colony grows to periphery and begins noble cells to occur, with the sucking-off of fine and close embryonic stem cell sample colony, washing is 2 times in PBS; Move on to again in the trypsinase of TrypLE cell dissociation buffer or massfraction 0.05% and digest 2～3min, and pressure-vaccum 5～20 times; The discrete unicellular or small cell cluster of opening is transferred to the plastic culture dish cultivation that is coated with 1～2h through 4 ℃ of Matrigel matrix membranes, and nutrient solution is female reproduction stem cell separation and Culture liquid; 37 ℃, 5%CO
2Cultivate under the saturated humidity condition;
After cultivating 3～7d, begin to occur fine and close embryonic stem cell sample colony, be pig female reproduction stem cell.
2. the extracorporeal culturing method of the pig female reproduction stem cell that separates of a claim 1, it is characterized in that, pig female reproduction stem cell is inoculated on the MEF feeder layer, nutrient solution also comprises following component take the DMEM/F12 substratum as basic medium: the ITS of the non-essential amino acid of the serum replacement of volume fraction 20%, 2mmol/L glutamine, 0.1mmol/L beta-mercaptoethanol, 100IU/mL penicillin, 100mg/mL Streptomycin sulphate, massfraction 1%, 2.5 μ mol/L BIO, 5～10ng/mL Basic Fibroblast Growth Factor and massfraction 1%; 2～3d changes a nutrient solution.
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| US20100196337A1 (en) * | 2007-04-23 | 2010-08-05 | Perry John M | Methods and compositions for stem cell self-renewal |
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| US20100196337A1 (en) * | 2007-04-23 | 2010-08-05 | Perry John M | Methods and compositions for stem cell self-renewal |
| WO2009021151A1 (en) * | 2007-08-07 | 2009-02-12 | Primegen Biotech Llc | Isolation, characterization and propagation of germline stem cells |
| CN101638633A (en) * | 2009-09-04 | 2010-02-03 | 西北农林科技大学 | Method for in-vitro separation and culture of goat male germ stem cells |
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