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Using ATCC ES C57BL/6 as an example, it is shown here how to culture mouse embryonic stem (ES) cell line. The clonal embryonic stem cell line #693 ES C57BL/6 was derived from a strain C57BL/6J (B6) mouse blastocyst [PubMed: 11730008]. The ES cells were shown to populate the germ line of two host blastocyst donors, FVB/NJ (FVB) and the coisogenic strain C57BL/6-Tyrc-2J (c2J). Coat-color chimera production was high using c2J blastocysts while FVB blastocysts produced a low number of chimeras [PubMed: 11730008].

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[Bio101] Culturing of C57BL/6 Mouse Embryonic Stem (ES) Cell Line
[Bio101] C57BL/6老鼠胚胎干细胞系的体外培养

干细胞 > 胚胎干细胞 > 维持和分化
作者: Yuqiong Pan
10/5/2011, 8277 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.142

[Abstract] Using ATCC ES C57BL/6 as an example, it is shown here how to culture mouse embryonic stem (ES) cell line. The clonal embryonic stem cell line #693 ES C57BL/6 was derived from a strain C57BL/6J (B6) mouse blastocyst [PubMed: 11730008]. The ES cells were shown to populate the germ line of two host blastocyst donors, FVB/NJ (FVB) and the coisogenic strain C57BL/6-Tyrc-2J (c2J). Coat-color chimera production was high using c2J blastocysts while FVB blastocysts produced a low number of chimeras [PubMed: 11730008].

[Abstract] 这里以ATCC ES C57BL/6为例介绍如何培养小鼠胚胎干细胞(ES)株系。 编号 #693 ES C57BL/6的干细胞系是从C57BL/6J (B6)小鼠囊胚取得[PubMed: 11730008]。 这些 ES细胞是由两个囊胚供体生殖系移植而来。这两个供体株系分别为FVB/NJ (FVB)和同源株系 C57BL/6-Tyrc-2J (c2J)。使用c2J囊胚株系产生的花色嵌合产物多于使用FVB囊胚株系[PubMed: 11730008]。

Materials and Reagents

  1. Cells and cell line:
    1. C57BL/6J (B6) mouse blastocyst (PubMed: 11730008 )
    2. #693 C57BL/6 mouse ES cell line (ATCC, catalog number: SCRC-1002 ™)
    3. CF1 mouse embryonic fibroblast (MEF) feeder cells (self-made from day 12.5 CF1 strain mouse embryos) or other mouse feeder cells such as DR4 (Applied StemCell, catalog number: 1013 )

  2. Medium and growth factors:
    1. DMEM (Life Technologies, Invitrogen™, catalog number: 11995-073 )
    2. Fetal bovine serum (FBS), Qualified (US) (Life Technologies, Invitrogen™, catalog number: 26140-079 )
    3. 100x MEM non-essential amino acids (NEAA) (Life Technologies, Invitrogen™, catalog number: 11140-050 )
    4. 1,000x 2-Mercaptoethanol, liquid (Life Technologies, Invitrogen™, catalog number: 21985-023 )
    5. 100x L-Glutamine (20 mM), liquid (Life Technologies, Invitrogen™, catalog number: 25030-081 )
    6. Penicillin/streptomycin (Pen/Strep), liquid (Life Technologies, Invitrogen™, catalog number: 15140-122 )
    7. 1,000 U/ml ESGRO® mouse leukemia inhibitory factor (LIF) (Merck KGaA, catalog number: ESG1107 )
    8. TrypLE™ express stable trypsin-like enzyme with phenol red (Life Technologies, Invitrogen™, catalog number: 12605-028 )
    9. Gelatin from porcine skin-BioReagent, Type A, powder (Sigma-Aldrich, catalog number: G1890-100G )
    10. 1x PBS (pH 7.4), liquid (Life Technologies, Invitrogen™, catalog number: 10010-049 )
    11. Ethanol
    12. 0.25% gelatin (see Recipes)
    13. Mouse ES cell medium (see Recipes)

Equipment

  1. Incubator: 5% CO2 in humidified air, 37 °C (Thermo Fisher Scientific)
  2. Centrifuges and rotor (Thermo IEC) 
  3. BD Primaria* Tissue Culture Dishes, 100 x 20 mm, 08-772-4F (Thermo Fisher Scientific, catalog number: 353803)
  4. 10 cm tissue culture dish
  5. Water bath
  6. 0.22 µm filter

Procedure

  1. Day 0
    1. Coat the 10 cm tissue culture dish with 10 ml 0.25% gelatin in a laminar flow hood at room temperature (RT) for 20-30 min.
    2. Remove gelatin, do not wash, plate 2 x 106 mitotically arrested MEF (CF-1 or DR4) as a feeder layer in 10 ml MEF medium (everything is the same as MES cell medium with the exception of LIF). MEF can stay for 4 days before plating mESC.

  2. Day 1
    1. One hour before thawing the vial of ES cells, perform PBS wash twice, then a 100% medium change using 9 ml mESC medium.
    2. Thaw the vial of C57BL/6 mouse ES cell line by gentle agitation in a 37 °C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water. Thawing should be rapid (approximately 90 sec).
    3. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.
    4. Transfer the cells from the vial to a 50 ml centrifuge tube. Use an additional 1 ml of media to rinse the vial and transfer the liquid to the tube. Add 8 ml of mESC medium dropwise to bring the total volume to 10 ml.
    5. Spin the cells at 270 x g for 3 min. Aspirate the supernatant and resuspend the pellet in 1 ml of mESC medium.
    6. Add the 1 ml of cell suspension to the previously prepared 10 cm dish containing feeder cells. Shake to move the cells to distribute evenly.
    7. Incubate the culture at 37 °C in a humidified 5% CO2/95% air incubator.

  3. Perform medium change every day, and passage cells every 1 to 3 days. Also, the subcultivation ratio is 1:4 to 1:7 as recommended.
    1. Subculturing:
      1. Aspirate the medium from the 10 cm culture dish containing the C57BL/6 mouse ES cells and rinse with 10 ml of PBS twice.
      2. Aspirate the PBS and add pre-warmed 5 ml of TrypLE™ Express, place the dish in a incubator for 5 min or until the ES cells are dissociated.
      3. Add 5 ml of ES cell medium and gently neutralize the contents of the dish.
      4. Transfer the cell suspension to the 50 ml centrifuge tube, spin the cells at 270 x g for 3 min. Aspirate the supernatant and resuspend the pellet in 4 ml or 7 ml of mESC medium depending on the subcultivation ratio.
      5. Add the 1 ml of cell suspension to the previous prepared 10 cm dish containing irradiated feeder cells. Shake to move the cells to distribute evenly.
      6. Place the dish in the 37 °C incubator overnight.

  4. When freeze the cells, use the following freezing medium:
    1. 10% DMSO
    2. 90% FBS

Notes

  1. Place vials in liquid nitrogen immediately upon receipt until it is convenient to proceed to culture.
  2. To insure the highest level of viability, be sure to warm media to 37 °C before using it on the cells.
  3. C57BL/6 mouse ES cells grow as small, tight colonies with phase bright borders. It’s optimal to passage them timely before they grow to over confluence.

Recipes

  1. 0.25% gelatin (800 ml)
    1.75 g gelatin
    Add Mill Q H2O to final volume 800 ml, autoclave before use.
  2. Mouse ES cell medium (500 ml) 
    DMEM                                            409 ml
    FBS                                                75 ml
    NEAA (100x)                                   5 ml
    Pen/Strep (100x)                             5 ml
    L-Glutamine (100x)                         5 ml
    2-mercaptoethanol (1,000x)            1 ml
    1,000 U/ml LIF                               50 µl
    Filtered by 0.22 µm filter unit, kept at 4 °C

References

  1. Brook, F. A. and Gardner, R. L. (1997). The origin and efficient derivation of embryonic stem cells in the mouse. Proc Natl Acad Sci U S A 94(11): 5709-5712.
  2. Brook, F. A., Evans, E. P., Lord, C. J., Lyons, P. A., Rainbow, D. B., Howlett, S. K., Wicker, L. S., Todd, J. A. and Gardner, R. L. (2003). The derivation of highly germline-competent embryonic stem cells containing NOD-derived genome. Diabetes 52(1): 205-208.
  3. C57BL/6 mouse ES cell line product datasheet (ATCC).
  4. Evans, M. J. and Kaufman, M. H. (1981). Establishment in culture of pluripotential cells from mouse embryos. Nature 292(5819): 154-156.

材料与试剂

 

1.        细胞和细胞系:

1)    #693 C57BL/6小鼠细胞系(ATCC SCRC-1002)

2)    CF1小鼠胚胎成纤维细胞(MEF) 饲养层细胞 (12.5天的 CF1 株系小鼠胚胎自行产生)或其他饲养层细胞如DR4 (应用了 StemCell ASF 1013).

2.        培养基和生长因子:

1)    DMEM (Invitrogen 11995-073)

2)    胎牛血清(FBS),合格 (US) (Invirogen 26140-079)

3)    MEM 非必须氨基酸(NEAA) (100X) (Invitrogen 11140-050)

4)    2-巯基乙醇(1,000X),液体(Invitrogen 21985-023)

5)    L-谷氨酰胺 (20mM) (100X),液体(Invitrogen 25030-081)

6)    青霉素/链霉素(Pen/Strep),液体 (Invitrogen 15140-122)

7)    1000 U/ml ESGRO? 小鼠白血病抑制因子(LIF) (Chemicon / Millipore ESG1107)

8)    TrypLE? 稳定表达胰蛋白酶样酶(带酚红)(Invitrogen 12605-028)

9)    猪皮明胶- BioReagentA型粉末 (Sigma, G1890-100G)

10)  PBS 7.4 (1X),液体(Invitrogen 10010-049)

 

仪器

 

1.        恒温箱: 5% CO2 存在于加湿空气中, 37°C (Thermo Fisher)

2.        离心机和转子(Thermo IEC)

3.        BD Primaria* 组织培养皿, 100 x 20 mm, 08-772-4F (Fisher 353803)

 

步骤

 

1.        0

1)   10cm组织化培养皿倒入10 ml 0.25%明胶,室温晾20-30 min

2)   移除明胶,不洗,将2x 106 有丝分裂期停止的小鼠胚胎因子MEF (CF-1 or DR4) 作为饲养层铺于10 ml MEF培养基中(一切与小鼠ES细胞培养基相同,加入额外的LIF)MEF可以在加入mESC前保持4天。

2.        1

3)   提前1小时解冻小瓶中的ES细胞,用PBS清洗2次,然后100%更换培养基,使用9 ml mESC培养基。

4)   37°C水浴中轻摇装有C57BL/6小鼠 ES细胞的小瓶以解冻。为最大可能减少污染,保持环形垫圈远离水面。应快速解冻(90s左右)。

5)   内容物一解冻立即将小瓶移出水浴锅,浸泡或喷洒70%乙醇以减少污染。这一步之后的所有步骤都要严格控制在无菌条件。

6)   将细胞从小瓶中移到一个50 ml离心管。使用额外1 ml培养基清洗小瓶并将液体转移到试管。将8 ml mESC培养基定容到终体积10 ml

7)   将细胞离心,270 x g3 min。吸走上层清液弃去,用1 ml mESC培养基重悬细胞颗粒。

8)   将那1 ml细胞悬浮液加入之前准备好的含有饲养层细胞的10 cm培养皿中。振摇以使细胞均匀分布。

9)   将培养物置于37°C,湿度 5% CO2/95% 的空气恒温箱中。

3.        每天更换培养基,1-3天继代一次细胞。推荐的继代比例是1:41:7

1)    继代培养:

a)      从含有C57BL/6 小鼠ES细胞的10 cm培养皿中吸出培养基并用10 ml PBS清洗2次。

b)      吸出PBS并加入5 ml预热过的 TrypLE? Express,将培养皿置于恒温箱中5 min或等到ES细胞离解。

c)      加入 5 ml ES细胞培养基并温和得中和培养皿内容物。

d)      将细胞悬浮液转移到50 ml离心管中,离心270 x g3 min。吸出上层清液并用4 ml 7ml mESC(取决于继代比例)培养基重悬细胞颗粒。

e)      1 ml细胞悬浮液加入之前准备好的含有经过紫外辐射灭菌的饲养层细胞的10 cm培养皿中。振摇以使细胞均匀分布。

f)       将培养皿置于37°C过夜孵育。

4.        冷冻细胞时,使用如下培养基:

2)    10% DMSO

3)    90% FBS

 

注:

 

1.        收集后立即将小瓶置于液氮中知道方便进行培养。

2.        确保最高的可行性,给细胞添加培养基前确保将培养基加温到37oC

3.        C57BL/6 小鼠 ES细胞生长体积小,克隆紧密,边缘明亮。最好在它们长到边缘相遇之前及时继代。

 

配方

 

1.        0.25% 明胶 (800 ml)

1.75 g 明胶

Mill Q H2O定容到终体积800,用前高压灭菌。

2.        小鼠 ES细胞培养基(500 ml) 

DMEM                                         409 ml

FBS                                               75 ml

NEAA (100X)                                   5 ml

Pen/Strep (100X)                             5 ml

L-谷氨酰胺 (100X)                           5 ml

2-巯基乙醇(1000X)                          1 ml

1000 U/ml LIF                                 50 μl

0.22 μm滤器过滤,4°C保存。

 

参考文献

 

1.         ATCC C57BL/6 mouse ES cell line product datasheet.

2.         Evans M.J., Kaufman M.H. (1981). Establishment in culture of pluripotential cells from mouse embryos. Nature 292(5819): 154-6. 

3.         Brook F.A., Gardner R.L. (1997). The origin and efficient derivation of embryonic stem cells in the mouse. Proceedings of the National Academy of Sciences of the United States of America 94(11): 5709-12. 

4.         Brook F.A., Evans E.P., Lord C.J., Lyons P.A., Rainbow D.B., Howlett S.K., Wicker L.S., Todd J.A., Gardner R.L. (2003). The derivation of highly germline-competent embryonic stem cells containing NOD-derived genome. Diabetes 52(1): 205-8.

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How to cite this protocol: Pan, Y. (2011). Culturing of C57BL/6 Mouse Embryonic Stem (ES) Cell Line. Bio-protocol Bio101: e142. DOI: 10.21769/BioProtoc.142; Full Text



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