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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, 8494 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.

材料和试剂

  1. 细胞和细胞系:
    1. C57BL/6J(B6)小鼠胚泡(PubMed:11730008)
    2. #693 C57BL/6小鼠ES细胞系(ATCC,目录号:SCRC-1002 TM)
    3. CF1小鼠胚胎成纤维细胞(MEF)饲养细胞(从12.5天CF1菌株小鼠胚胎自制)或其他小鼠饲养细胞如DR4(Applied StemCell,目录号:1013)
  2. 中和生长因子:
    1. DMEM(Life Technologies,Invitrogen TM,目录号:11995-073)
    2. 胎牛血清(FBS),Qualified(US)(Life Technologies,Invitrogen TM,目录号:26140-079)
    3. 100x MEM非必需氨基酸(NEAA)(Life Technologies,Invitrogen TM,目录号:11140-050)
    4. 1,000x 2-巯基乙醇,液体(Life Technologies,Invitrogen TM,目录号:21985-023)
    5. 100x L-谷氨酰胺(20mM),液体(Life Technologies,Invitrogen TM,目录号:25030-081)
    6. 青霉素/链霉素(Pen/Strep),液体(Life Technologies,Invitrogen TM,目录号:15140-122)
    7. 1000U/ml ESGRO小鼠白血病抑制因子(LIF)(Merck KGaA,目录号:ESG1107)
    8. TrypLE TM用酚红表达稳定的胰蛋白酶样酶(Life Technologies,Invitrogen TM,目录号:12605-028)
    9. 来自猪皮肤的明胶BioReagent,A型粉末(Sigma-Aldrich,目录号:G1890-100G)
    10. 1x PBS(pH 7.4),液体(Life Technologies,Invitrogen TM,目录号:10010-049)
    11. 乙醇
    12. 0.25%明胶(见配方)
    13. 小鼠ES细胞培养基(参见配方)

设备

  1. 培养箱:在37℃的潮湿空气中5%CO 2(Thermo Fisher Scientific)
  2. 离心机和转子(Thermo IEC)
  3. BD Primaria * Tissue Culture Dishes,100 x 20mm,08-772-4F(Thermo Fisher Scientific,目录号:353803)
  4. 10厘米组织培养皿
  5. 水浴
  6. 0.22μm过滤器

程序

  1. 第0天
    1. 在层流罩中在室温(RT)下用10ml 0.25%明胶涂覆10cm组织培养皿20-30分钟。
    2. 去除明胶,不洗,平板2×10 6有丝分裂逮捕的MEF(CF-1或DR4)作为饲养层在10ml MEF培养基中(一切都与MES细胞培养基相同,除了 的LIF)。 MEF可以在铺板mESC前停留4天。

  2. 第1天
    1. 在解冻小瓶ES细胞前1小时,进行PBS洗涤两次,然后使用9ml mESC培养基更换100%培养基。
    2. 通过在37℃水浴中温和搅拌解冻小瓶C57BL/6小鼠ES细胞系。 为了减少污染的可能性,将O型圈和盖子从水中取出。 解冻应该快速(大约90秒)。
    3. 一旦内容物解冻,从水浴中取出小瓶,并通过浸入或用70%乙醇喷雾净化。 从这一点开始的所有操作都应在严格的无菌条件下进行。
    4. 将细胞从小瓶转移到50ml离心管。 使用额外的1 ml培养基冲洗小瓶,将液体转移到试管中。 滴加8毫升mESC培养基,使总体积为10毫升。
    5. 旋转细胞在270×g 3分钟。 吸出上清液并将沉淀重悬在1ml mESC培养基中。
    6. 将1ml细胞悬浮液加入到预先制备的含有饲养细胞的10cm培养皿中。 摇动以移动单元格均匀分布。
    7. 在37℃,在潮湿的5%CO 2/95%空气培养箱中培养培养物。

  3. 每天进行培养基更换,并每1至3天传代细胞。 此外,推荐的传代培养比为1:4至1:7。
    1. 亚文化:
      1. 从包含C57BL/6小鼠ES细胞的10cm培养皿中吸出培养基,并用10ml PBS冲洗两次。
      2. 吸出PBS,加入预热的5ml TrypLE™Express,将培养皿放置在孵化器中5分钟或直到ES细胞解离。
      3. 加入5毫升ES细胞培养基,轻轻中和盘中的内容物。
      4. 转移细胞悬浮液到50ml离心管,细胞在270×g离心3分钟。 吸出上清液,并将沉淀重悬在4ml或7ml的mESC培养基中 取决于次培养比率。
      5. 将1ml细胞悬浮液加入到先前制备的包含经辐照的饲养细胞的10cm培养皿中。 摇动以移动单元格均匀分布。
      6. 将培养皿置于37℃培养箱中过夜。

  4. 当冷冻细胞时,使用以下冷冻培养基:
    1. 10%DMSO
    2. 90%FBS

笔记

  1. 一旦收到,立即将小瓶置于液氮中,直到方便进行培养。
  2. 为了确保最高水平的生存能力,确保将培养基温热至37°C,然后将其用于细胞。
  3. C57BL/6小鼠ES细胞生长为具有相位明亮边界的小的紧密集落。 最好在他们成长到汇合之前及时通过

食谱

  1. 0.25%明胶(800ml) 1.75克明胶
    加入Mill Q H 2 O至终体积800ml,在使用前高压灭菌
  2. 小鼠ES细胞培养基(500ml)
    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(1,000x)             1 ml
    1,000 U/ml LIF                             50微升
    用0.22μm过滤器过滤,保持在4℃下

参考文献

  1. Brook,F.A。和Gardner,R.L。(1997)。 小鼠胚胎干细胞的起源和有效衍生。 Natl Acad Sci USA 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。和Gardner, 含有NOD衍生基因组的高度生殖细胞能力的胚胎干细胞的衍生。 em> Diabetes 52(1):205-208。
  3. C57BL/6小鼠ES细胞系产品数据表(ATCC)。
  4. Evans,M.J.and Kaufman,M.H。(1981)。 在小鼠胚胎中培养多能性细胞的建立 自然 292(5819):154-156
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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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