CN102329771A - Method and special culture medium for subculturing chicken embryonic stem cells for long time - Google Patents

Abstract

A method for long-term subculture of chicken embryonic stem cells and a special medium thereof, which is characterized in that The cells in the bright area of the blastodisc are dispersed into single cells or small cell clusters by mechanical blowing, then inoculated on the STO feeder layer, cultured with the above-mentioned chicken embryonic stem cells in a special medium and at 37-38°C, and passaged for 3-5 days Once, the special medium includes 600-900mL of conditioned medium; 50-150mL of fetal bovine serum; 5-20mL of chicken serum; 5-25mL of one or several mixtures of non-essential amino acids; 0.146-0.292g of L-glutamine ; β-mercaptoethanol 6-14 μL; mouse leukemia inhibitory factor 1—2×10 ⁶ IU; basic fibroblast growth factor 10-50 μg; stem cell growth factor 5-20 μg, and the above components were established in DMEM high glucose medium To 1000mL, adjust the pH value to 7.2-7.5, the conditioned medium is obtained in this way, after culturing BRL-3A cells until the cells are confluent, collect the supernatant of the cultured cells, centrifuge and then filter through a filter membrane to obtain the conditioned medium culture medium. Compared with the prior art, the invention has the advantages of realizing long-term subculture of chicken embryonic stem cells, fast cell proliferation and high clone acquisition rate.

Description

Method for long-term subculture of chicken embryonic stem cells and its special medium

technical field

The invention relates to the field of animal cell engineering, in particular to a method for cultivating chicken embryonic stem cells and a special culture medium thereof.

Background technique

Embryonic stem cells are highly undifferentiated cells with developmental pluripotency isolated from the inner cell mass or primordial germ cells of early animal embryos. It has broad application prospects in tissue and organ engineering, cell therapy, and animal genetic modification and manipulation.

Embryonic stem cells have a tendency to differentiate spontaneously. The key technology for culturing embryonic stem cells is to select a suitable culture system so that the cells can proliferate rapidly and indefinitely while maintaining an undifferentiated state. Commonly used methods include: (1) feeder layer cells are used to inhibit the differentiation of embryonic stem cells through a mechanism that is not yet fully understood; (2) conditioned medium: conditioned medium contains factors that inhibit cell molecules; (3) Cytokines that inhibit cell differentiation or promote cell proliferation are used, including leukemia inhibitory factor, stem cell growth factor, basic fibroblast growth factor, etc. Since Evans et al. successfully established embryonic stem cell lines from delayed implantation mouse early embryos in 1981, based on the combination of one or more of the above methods, it has been used in humans, pigs, cattle, sheep, goats, mink, and Ganges. Species such as monkeys, horses, and rabbits have achieved long-term subculture of embryonic stem cells and successfully established embryonic stem cell lines or embryonic stem cell lines. But so far, although there are reports on the culture of chicken embryonic stem cells, they can only maintain their undifferentiated state in a short period of time, and the long-term subculture of chicken embryonic stem cells in vitro and the establishment of cell lines have not yet been realized. An important reason why chicken embryonic stem cells cannot be subcultured in vitro for a long time is that the current embryonic stem cell culture system, including the medium formula, is designed based on mammals such as mice or humans, and there are great differences between avian cells and mammalian cells , some culture conditions or medium formulations suitable for mammalian embryonic stem cells may not fully meet the needs of chicken embryonic stem cells in vitro. Therefore, although these culture conditions or medium formulations can maintain chicken embryonic stem cells in an undifferentiated state for a period of time, they will not be able to maintain this state during long-term subculture, resulting in rapid cell differentiation.

Contents of the invention

The object of the present invention is to provide a culture system suitable for chicken embryonic stem cells to realize the long-term subculture of chicken embryonic stem cells in vitro, the method for long-term subculture of chicken embryonic stem cells and its special medium.

期胚盘明区的细胞，经机械吹打分散成单个细胞或小细胞团后，接种于STO饲养层，用上述鸡胚胎干细胞在专用培养基及37~38℃环境下培养，3~5天传代一次，专用培养基包括条件培养液600—900mL；胎牛血清50—150 mL；鸡血清5—20 mL；非必需氨基酸中的一种或几种混合物5—25 mL；L-谷氨酰胺0.146—0.292 g；β-巯基乙醇6—14 μL；小鼠白血病抑制因子1—2×10⁶ IU；碱性成纤维细胞生长因子10—50 μg；干细胞生长因子5—20 μg，将上述成分用DMEM高糖培养基定容至1000 mL，调pH值为7.2—7.5，条件培养液是这样获得的，培养BRL-3A细胞至细胞汇合后（通常为3天），收集培养细胞的上清液，离心分离后再经滤膜过滤所得即为条件培养液。这里，滤膜是0.22um微孔得滤膜，离心分离的转速为1000rpm，离心分离时间不少于5min。 The method for long-term subculture of chicken embryonic stem cells of the present invention is achieved in this way, separating

The cells in the bright area of the blastodisc are dispersed into single cells or small cell clusters by mechanical blowing, then inoculated on the STO feeder layer, cultured with the above-mentioned chicken embryonic stem cells in a special medium and at 37-38°C, and passaged for 3-5 days Once, the special medium includes 600-900 mL of conditioned medium ; 50-150 mL of fetal bovine serum; 5-20 mL of chicken serum; 5-25 mL of one or several mixtures of non-essential amino acids ; 0.146 mL of L-glutamine —0.292 g; β-mercaptoethanol 6—14 μL; mouse leukemia inhibitory factor 1—2×10 ⁶ IU; basic fibroblast growth factor 10—50 μg; stem cell growth factor 5—20 μg. The DMEM high-glucose medium was adjusted to 1000 mL, and the pH value was adjusted to 7.2-7.5. The conditioned medium was obtained in this way. After culturing BRL-3A cells until the cells became confluent (usually 3 days), the supernatant of the cultured cells was collected , centrifuged and then filtered through a filter membrane to obtain the conditioned medium. Here, the filter membrane is a 0.22um microporous filter membrane, the rotational speed of centrifugation is 1000rpm, and the centrifugation time is not less than 5min.

The special medium of the present invention includes 600-900 mL of conditioned medium ; 50-150 mL of fetal bovine serum; 5-20 mL of chicken serum; 5-25 mL of one or more mixtures of non-essential amino acids ; L-glutamine 0.146—0.292 g; β-mercaptoethanol 6—14 μL; mouse leukemia inhibitory factor 1—2×10 ⁶ IU; basic fibroblast growth factor 10—50 μg; stem cell growth factor 5—20 μg, the above ingredients Use DMEM high-glucose medium to adjust the volume to 1000 mL, and adjust the pH value to 7.2-7.5. The conditioned medium is obtained in this way. After culturing BRL-3A cells until the cells are confluent (usually 3 days), collect the supernatant of the cultured cells After centrifugation and filtration through a filter membrane, the conditioned medium was obtained. Here, the filter membrane is a 0.22um microporous filter membrane, the rotational speed of centrifugation is 1000rpm, and the centrifugation time is not less than 5min.

Compared with the prior art, the present invention can realize the long-term subculture of chicken embryonic stem cells, and has the advantages of fast cell proliferation and high clone acquisition rate.

Description of drawings

Fig. 1 (A) is the chicken embryonic stem cells passed to the second generation cultured in the culture system of the present invention;

Fig. 1 (B) is the chicken embryonic stem cells passed to the 5th generation cultured in the culture system of the present invention;

Fig. 1 (C) is the chicken embryonic stem cells passed to the 10th generation cultured in the culture system of the present invention;

Fig. 1 (D) is the chicken embryonic stem cells cultured in the culture system of the present invention and passed to the 15th generation;

Fig. 1 (E) is the chicken embryonic stem cells cultured in the culture system of the present invention and passed to the 19th generation;

Figure 1 (F) is the chicken embryonic stem cells that have been cultured in the culture system of the present invention and passed to the 25th generation

Fig. 2 (A) is the identification of the chick embryo stem cell that alkaline phosphatase staining is blue-purple;

Fig. 2 (B) is the identification of chicken embryonic stem cells positive for SSEA-1 identified by immunocytofluorescence;

Figure 2 (C) is the identification of chicken embryonic stem cells that form embryoid bodies and highly express mesoderm in suspension culture in differentiation medium;

Figure 2(D) is the identification of chicken embryonic stem cells with the marker gene CH-T of endoderm and ectoderm;

Fig. 2 (E) is the identification of chicken embryonic stem cells of endoderm and ectoderm marker gene TTR;

Fig. 2 (F) is the identification of chicken embryonic stem cells of endoderm and ectoderm marker gene β-activin;

Figure 3 is the primer sequences for RT-PCR detection of three germ layer-specific genes in embryoid bodies.

Detailed ways:

The present invention is described in further detail in conjunction with embodiment now:

(1) Experimental animals and cell lines

Fertilized eggs were purchased from Guangdong Provincial Food Enterprise Group Foshan Nanhai Breeding Poultry Co., Ltd.; clean-grade pregnant 12.5-day Kunming mice were purchased from the Experimental Animal Center of Southern Medical University; STO cell lines were purchased from the Wuhan University Collection Center (China Type Culture Collection Center ); the rat liver cell line (buffalo rat liver cell, BRL-3A cell) was purchased from the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

(2) Solution or reagent preparation

1. Preparation of PBS: Weigh 8.0g NaCl, 3.63g Na ₂ HPO ₄ , 0.2g KCl 0.2g KH ₂ PO ₄ 0.24g, dissolve in an appropriate amount of ultrapure water, adjust the pH value to 7.4, and set the volume to 1000mL; 100ml/bottle package, autoclave at 15 pounds for 30 minutes, and store at 4°C for later use.

2. The preparation of DMEM: get a bag of DMEM (glucose content 13.5 grams, Gibco company) and dissolve with appropriate ultrapure water, take by weighing _NaHCO3 3.7g, be dissolved in ultrapure water and mix, add penicillin streptomycin 1 mL (100,000 IU/mL), dilute to 1000 mL with ultrapure water, adjust the pH to about 7.2, filter and sterilize with a 0.22 μm microporous membrane, aliquot 100 mL, and store at 4°C for later use.

3. Preparation of Hanks solution: Take a bag of Hanks (Gibco company) and dissolve it in an appropriate amount of ultrapure water, weigh 0.35g of NaHCO ₃ , dissolve in ultrapure water and mix well, add penicillin streptomycin 1 mL (100,000 IU/ mL), dilute to 1000 mL with ultrapure water, adjust the pH to about 7.2, filter and sterilize with a 0.22 μm microporous membrane, aliquot 100 mL, and store at 4°C for later use.

4. Preparation of trypsin: Weigh 0.25 g of trypsin and dissolve it in 100 mL of PBS, filter and sterilize with a 0.22 μm microporous membrane, aliquot 1 mL, and store at 4°C for later use.

5. Dispensing of stem cell growth factor: Take a packaged stem cell growth factor (Invitrogen Company) and dissolve it in 100 μL ultrapure water, add 9.9 mL of DMEM culture solution containing 10% (volume percentage) fetal bovine serum, mix well, 0.22 After sterilizing with a μm microporous membrane filter, aliquot 100 μL and store in a sealed seal at -20°C. When used, the embryonic stem cell culture medium is diluted to the desired concentration.

6. Packing of basic fibroblast growth factor: Take a packaged basic fibroblast growth factor (Invitrogen) and dissolve it in 100 μL Tris (PH7.6), add 10% (volume percentage) fetal bovine serum 4.9 mL of DMEM culture solution, mixed thoroughly, sterilized by 0.22 μm microporous membrane filter, aliquoted in 100 μL/tube, and sealed at -20°C. Dilute to the desired concentration with embryonic stem cell culture medium when used.

7. Subpackaging of leukemia inhibitory factor: Take a packaged leukemia inhibitory factor (Millipore Company), add 9.5 mL of PBS containing 10% (volume percentage) fetal bovine serum, fully dissolve and filter through a 0.22 μm microporous membrane to sterilize. Aliquot 200 μL/tube and store in a sealed container at 2-8°C. When used, the embryonic stem cell culture medium was diluted to the desired concentration.

8. Preparation of mitomycin-C: Dissolve 2 mg mitomycin-C in 20 mL PBS to make it completely dissolved, make a stock solution with a concentration of 100 μg/mL, filter and sterilize with a 0.22 μm microporous membrane Then aliquot 1 mL/tube, store at -20°C, and dilute to 10 μg/mL in DMEM when used.

9. Preparation of 0.1% gelatin: Weigh 0.1g of gelatin and fully dissolve it in 100 mL of three-distilled water, autoclave at 15 pounds for 30 minutes, and store at 4°C for later use.

10. Preparation of EDTA: Weigh 0.2g of EDTA, 0.8g of NaCl, 0.02g of KCl, 0.289g of Na ₂ HPO ₄ 12H ₂ O, 0.02g of KH ₂ PO ₄ and dissolve them in 100 mL of ultrapure water, autoclave at 15 pounds 30 min, stored at 4°C for later use.

11. Preparation of 0.25% trypsin-EDTA digestion solution: 0.25% trypsin and EDTA were prepared at a ratio of 1:1.

12. BRL-3A cell culture medium: Add the following substances to the high-glucose DMEM medium: 5% (volume percentage) fetal bovine serum, 2 mM L-glutamine, 1% (volume percentage) double antibody.

Embodiment 1 The cultivation of chicken embryonic stem cells

1. Preparation of feeder layer

(1) Mouse embryonic fibroblasts, chicken embryo fibroblasts and STO cell lines were cultured in high-glucose DMEM medium containing 10% (volume percentage) fetal bovine serum and 1% (volume percentage) double antibody according to conventional methods;

(2) When 80%-90% of the cells are confluent, discard the culture medium and wash with PBS 2-3 times;

(3) Add mitomycin-C at a final concentration of 10 μg/mL, and incubate at 37°C for 2-2.5 h;

(4) Discard mitomycin-C and wash the cells with PBS for 6-7 times to ensure complete removal of mitomycin-C;

(5) Add an appropriate amount of 0.25% trypsin-EDTA digestion solution to digest for about 30 seconds, add DMEM solution to stop the digestion, and fully pipette to prepare a single-cell suspension;

(6) Centrifuge at 1000 rpm for 5-8 min, resuspend the cells in high-glucose DMEM medium containing 10% (volume percentage) fetal bovine serum and 1% (volume percentage) double antibody, and adjust the cell density to 5× ¹⁰⁵ /mL, inoculated on a culture plate coated with 0.1% gelatin, and cultured in an incubator at 37°C and 5% CO ₂ ;

(7) Observe the growth of the cells after 24 h. If the cells fail to cover the monolayer, add mitomycin C-treated cells to ensure that the cells can be connected into one piece without gaps; use within d.

2. Preparation of conditioned medium

(1) BRL-3A cells were seeded in culture flasks and cultured with BRL-3A cell culture medium;

(2) When the cells are 80%-90% confluent, discard the culture medium, and add 10 mL of fresh BRL-3A cell culture medium for every 55 ^cm2 growth area of cells;

(3) After 3 days, collect the BRL-3A cell culture medium, put it in a sterile bottle, and store it at -20°C. Before use, it should be sterilized by filtration with a 0.22 μm microporous membrane, and the pH value should be adjusted to 7.2.

3. Preparation of special medium for chicken embryonic stem cells

Take 600, 800, and 900 mL of the above BRL-3A cell conditioned medium, respectively, and add the following ingredients: 50 or 100 or 150 mL of fetal bovine serum, 5 or 10 or 20 mL of chicken serum, one or more of non-essential amino acids A total of 5 or 10 or 25 mL, L-glutamine 0.146 or 0.200 or 0.292 g, β-mercaptoethanol 6 or 8 or 14 μL, mouse leukemia inhibitory factor 1×10 ⁶ or 1.5×10 ⁶ or 2×10 ⁶ IU, basic fibroblast growth factor 10 or 30 or 50 μg, stem cell growth factor 5 or 10 or 20 μg, use DMEM high-glucose medium to make up to 1000 mL, prepare chicken embryonic stem cell special medium, store at 4°C for later use .

4. Isolation and culture of chicken embryonic stem cells

(1) Take fresh fertilized eggs, soak them with bromogeramine, then sterilize them with 75% alcohol, and let them stand for 5 minutes with the air cell facing upwards;

(2) Open the egg under aseptic conditions, place the egg in a sterile petri dish, separate the egg white and egg yolk, and make the blastodisc face up, and remove the egg white on the membrane around the yolk;

(3) Place a small piece of filter paper with a central perforation (about 5 mm in diameter) on the egg yolk, so that the blastodisc is exposed in the center of the hole, quickly cut the vitelline membrane along the filter paper with ophthalmic scissors, and gently lift the filter paper to separate it. germinal disc;

(4) Immerse the blastoderm attached to the filter paper in PBS, and carefully wash off the attached egg yolk;

(5) Use a hair ring to remove the dark area of the blastoderm under the microscope, pipette the cells in the bright area, add chicken embryonic stem cell medium, blow gently into discrete single cells or cell clusters, and inoculate the above mouse embryos respectively The feeder layer prepared by fibroblasts, chicken embryo fibroblasts or STO cells was cultured in an incubator at 37°C and 5% CO ₂ , half of the liquid was changed every 24 h, and the growth status of the cells was observed;

(6) After 7-8 days, colony-like growth of chicken embryonic stem cells was observed on the feeder layer;

(7) Determine the optimized chicken embryonic stem cell culture system based on conditions such as the growth state of chicken embryonic stem cells, the number and size of colonies formed, and the differentiation state of colonies: among them, the best feeder layer cells are STO cells, and the optimal medium formula For: 800 mL of conditioned medium, 100 mL of fetal bovine serum, 10 mL of chicken serum, 10 mL of non-essential amino acids, 0.146 g of L-glutamine, 8 μL of β-mercaptoethanol, and 1×10 ⁶ IU of mouse leukemia inhibitory factor , basic fibroblast growth factor 20 μg, stem cell growth factor 10 μg, and DMEM high glucose medium to 1000 mL, pH 7.4.

5. Subculture of chicken embryonic stem cells

(1) Select cell colonies that grow well, have obvious bulges, clear edges, and do not show differentiation in shape, and wash them with PBS once or twice;

(2) Add new PBS, incubate at 37°C for 8 or 9 or 10 min, discard the PBS when the cell colony is slightly separated from the feeder layer under a microscope, and add chicken embryonic stem cell special medium;

(3) Aspirate the cells with a pipette, blow them moderately to obtain small cell clusters, inoculate the cells into 2 new culture wells with feeder layers, and culture them in an incubator at 37°C and 5% CO ₂ , half the amount every 24 h change fluid;

(4) After the cells grow into obvious clusters, repeat the operations of (1)-(3) for the next generation of cell passage. In this culture system, the undifferentiated state and high proliferation of chicken embryonic stem cells can still be maintained after 25 passages ability.

Characterization of chicken embryonic stem cells:

The chicken embryonic stem cells passed to 2-25 generations obtained in Example 1 are identified by the following method:

1. Morphological Identification

The morphology of chicken embryonic stem cells is similar to that of embryonic stem cells of other species, and they grow in a typical colony shape. Its shape can be preliminarily identified.

2. Alkaline phosphatase staining identification

Take chicken embryonic stem cells in good growth state, discard the culture medium, and wash with PBS to remove residual medium; add 4% paraformaldehyde to fix at 4°C for 15 or 20 or 30 min, discard the fixative, and wash with PBS for 3 times; add BCIP/NBT The staining solution was incubated at 37°C for 1.5 or 1.8 or 2 h, washed 3 times with PBS, and observed under an inverted microscope. The results showed that the chicken embryonic stem cells were blue-purple, while the feeder cells and differentiated cells were not stained (see Figure 2(A)).

3. Immunocytofluorescence staining identification of embryonic stem cell surface specific antigen (SSEA-1)

Take chicken embryonic stem cells in good growth state, discard the culture medium, wash with PBS, add pre-cooled fixative solution (acetone: absolute ethanol 3:2) and fix for more than 30 min; rinse with PBS for 3 times, add blocking solution (containing 10% fetal Bovine serum in PBS) for 2 h; Rinse with PBS, add primary antibody (SSEA-1), incubate in 37°C water bath in the dark for 1 h, wash with PBST for 5 min × 3 times, add secondary antibody (IgG-FITC), 37°C water bath Incubate in the dark for 45 min, wash with PBST for 5 min x 3 times, add a drop of 60% glycerol, and observe under a fluorescent microscope. The results showed that the chicken embryonic stem cells were positive for SSEA-1, and the cells showed yellow-green fluorescence, while the feeder cells and differentiated cells were negative (see Figure 2(B)).

4. In Vitro Differentiation Assay

(1) Hanging drop culture of chicken embryonic stem cells: Pick out chicken embryonic stem cell clones with a mouth pipette and a stripping needle, digest them into dispersed chicken embryonic stem cells with 0.05% trypsin digestion solution, centrifuge, discard the supernatant; add 10 mL Differentiation medium, pipetting repeatedly to form a cell suspension, use a multi-hole pipette to drop 5 rows of 30 μL/drop (10 ⁴ cells/mL) cell suspension under the lid of a 100 mm tissue culture dish, carefully flip the lid to make the liquid The drop was suspended on the lid of the petri dish, and placed on a petri dish containing 10 mL of PBS, and cultured in a 5% CO ₂ incubator at 37.5°C for 2 days;

(2) Suspension culture of differentiated embryonic stem cells: After embryonic stem cells were cultured in hanging drops for 2 days, the lid of the culture dish was carefully turned over to collect the droplets, and transferred to a 35 mm culture dish. The culture medium contained 20% (volume percentage) fetal bovine Serum, without adding any differentiation-inhibiting factor components, was blown with a pipette every day to prevent the formed embryoid bodies from adhering to the wall, and continued to suspend culture in the incubator for 5 days, and half the amount of medium was changed every other day. 2(c));

(3) Natural differentiation of ES cells: transfer the embryoid bodies obtained above to a 24-well culture plate containing 2 mL of differentiation medium and treated with 0.1% gelatin, inoculate one embryoid body in each well, and allow chicken embryonic stem cells to adhere to the wall natural differentiation;

(4) RT-PCR method to detect the expression of three germ layer-specific genes in embryoid bodies: CH-T, TTR and β-activin are the marker genes of mesoderm, endoderm and ectoderm, respectively, and conventional RT-PCR detection The expression of these three marker genes in embryoid bodies. The results showed that embryoid bodies could highly express the marker genes of the above three germ layers after natural differentiation, suggesting that chicken embryonic stem cells could form cells of the three germ layers after spontaneous differentiation (see D, E, F of Figure 2 and Figure 3).

Claims (8)

1. the method for the culturing chicken embryonic stem cells that goes down to posterity for a long time is characterized in that separating

The cell in phase blastodisc area pellucida after machinery piping and druming is dispersed into individual cells or small cell cluster, is inoculated in the STO feeder layer, cultivates under special culture media and 37 ~ 38 ℃ of environment with above-mentioned chicken embryonic stem cells, goes down to posterity once in 3 ~ 5 days, and special culture media comprises Conditioned medium600-900mL; Foetal calf serum 50-150 mL; Chicken serum 5-20 mL; Non-essential amino acidIn one or more mixtures 5-25 mL; L-glutaminate 0.146-0.292 g; Beta-mercaptoethanol 6-14 μ L; Mouse leukemia supressor 1-2 * 10 ⁶IU; Prostatropin 10-50 μ g; Stem cell factor 5-20 μ g are settled to 1000 mL with mentioned component with the DMEM high glucose medium, and adjust pH is 7.2-7.5, Conditioned medium is to obtain like this,After cultivation BRL-3A cell converges to cell, collect the supernatant of culturing cell, be conditioned medium through the membrane filtration gained again after the spinning.

2. the method for the culturing chicken embryonic stem cells that goes down to posterity for a long time according to claim 1; It is characterized in that separating the cell in phase blastodisc area pellucida; After machinery piping and druming is dispersed into individual cells or small cell cluster; Being inoculated in the STO feeder layer is achieved in that and comprises

(1) MEC, CEF and STO cell strain are incubated at by ordinary method that to contain volume percent be that 10% foetal calf serum, volume percent are defended two anti-high sugared DMEM nutrient solutions of 1%;

When (2) the 80%-90% cell converges, abandon nutrient solution, PBS washes 2-3 times;

(3) adding final concentration is the Mitomycin-C of 10 μ g/mL, hatches 2-2.5 h for 37 ℃;

(4) abandon Mitomycin-C, PBS thorough washing cell 6-7 times guarantees to remove fully Mitomycin-C;

(5) add about an amount of 0.25% trypsinase-EDTA Digestive system digestion 30s, add DMEM liquid and stop digestion, fully piping and druming, preparation single cell suspension;

Centrifugal 5-8 min of (6) 1000 rpm, it is that 10% foetal calf serum, volume percent are two anti-high sugared DMEM nutrient solution re-suspended cells of 1% that use contains volume percent, the adjustment cell density is 5 * 10 ⁵Individual/mL, be inoculated in the culture plate that encapsulates through 0.1% gelatin, put 37 ℃, 5%CO ₂, cultivate in the incubator;

Observation of cell growing state behind (7) 24 h is failed to be paved with individual layer as if cell and then added the cell that ametycin was handled, and is very close to each other to guarantee cell to join together.

3. the method for the culturing chicken embryonic stem cells that goes down to posterity for a long time according to claim 1 and 2 is characterized in that cultivating the BRL-3A cell and is the BRL-3A cell inoculation in culturing bottle, cultivates with the BRL-3A cell culture fluid.

4. the method for the culturing chicken embryonic stem cells that goes down to posterity for a long time according to claim 3 when treating when it is characterized in that cultivating the BRL-3A cell that cell 80%-90% converges, is abandoned nutrient solution, every 55cm ²The cell of growth area adds the fresh BRL-3A cell culture fluid of 10 mL.

5. the method for the culturing chicken embryonic stem cells that goes down to posterity for a long time according to claim 4; It is characterized in that adding fresh BRL-3A cell culture fluid and collect BRL-3A cell culture fluid-be supernatant after 3 days; Put aseptic bottle;-20 ℃ frozen, uses preceding spinning and through 0.22 μ m filtering with microporous membrane degerming, adjust pH value to 7.2 then.

6. the special culture media of the culturing chicken embryonic stem cells that goes down to posterity for a long time is characterized in that comprising Conditioned medium600-900mL; Foetal calf serum 50-150 mL; Chicken serum 5-20 mL; Non-essential amino acidIn one or more mixtures 5-25 mL; L-glutaminate 0.146-0.292 g; Beta-mercaptoethanol 6-14 μ L; Mouse leukemia supressor 1-2 * 10 ⁶IU; Prostatropin 10-50 μ g; Stem cell factor 5-20 μ g are settled to 1000 mL with mentioned component with the DMEM high glucose medium, and adjust pH is 7.2-7.5, Conditioned medium is to obtain like this,After cultivation BRL-3A cell converges to cell, collect the supernatant of culturing cell, be conditioned medium through the membrane filtration gained again after the spinning.

7. the special culture media of the culturing chicken embryonic stem cells that goes down to posterity for a long time according to claim 6 when treating when it is characterized in that cultivating the BRL-3A cell that cell 80%-90% converges, is abandoned nutrient solution, every 55cm ²The cell of growth area adds the fresh BRL-3A cell culture fluid of 10 mL.

8. the special culture media of the culturing chicken embryonic stem cells that goes down to posterity for a long time according to claim 7; It is characterized in that adding fresh BRL-3A cell culture fluid and collect BRL-3A cell culture fluid-be supernatant after 3 days; Put aseptic bottle;-20 ℃ frozen, uses preceding spinning and through 0.22 μ m filtering with microporous membrane degerming, adjust pH value to 7.2 then.

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