Culturing of C57BL/6 Mouse Embryonic Stem (ES) Cell Line
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].
Materials and Reagents
- Cells and cell line:
- C57BL/6J (B6) mouse blastocyst (PubMed: 11730008 )
- #693 C57BL/6 mouse ES cell line (ATCC, catalog number: SCRC-1002 ™)
- 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 )Medium and growth factors:
- DMEM (Life Technologies, Invitrogen™, catalog number: 11995-073 )
- Fetal bovine serum (FBS), Qualified (US) (Life Technologies, Invitrogen™, catalog number: 26140-079)
- 100x MEM non-essential amino acids (NEAA) (Life Technologies, Invitrogen™, catalog number:11140-050 )
- 1,000x 2-Mercaptoethanol, liquid (Life Technologies, Invitrogen™, catalog number: 21985-023 )
- 100x L-Glutamine (20 mM), liquid (Life Technologies, Invitrogen™, catalog number: 25030-081 )
- Penicillin/streptomycin (Pen/Strep), liquid (Life Technologies, Invitrogen™, catalog number: 15140-122 )
- 1,000 U/ml ESGRO® mouse leukemia inhibitory factor (LIF) (Merck KGaA, catalog number: ESG1107 )
- TrypLE™ express stable trypsin-like enzyme with phenol red (Life Technologies, Invitrogen™, catalog number: 12605-028 )
- Gelatin from porcine skin-BioReagent, Type A, powder (Sigma-Aldrich, catalog number: G1890-100G)
- 1x PBS (pH 7.4), liquid (Life Technologies, Invitrogen™, catalog number: 10010-049 )
- 0.25% gelatin (see Recipes)
- Mouse ES cell medium (see Recipes)
- Incubator: 5% CO2 in humidified air, 37 °C (Thermo Fisher Scientific)
- Centrifuges and rotor (Thermo IEC)
- BD Primaria* Tissue Culture Dishes, 100 x 20 mm, 08-772-4F (Thermo Fisher Scientific, catalog number: 353803)
- 10 cm tissue culture dish
- Water bath
- 0.22 µm filter
- Day 0
- 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.
- 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.
- Day 1
- One hour before thawing the vial of ES cells, perform PBS wash twice, then a 100% medium change using 9 ml mESC medium.
- 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).
- 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.
- 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.
- Spin the cells at 270 x g for 3 min. Aspirate the supernatant and resuspend the pellet in 1 ml of mESC medium.
- 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.
- Incubate the culture at 37 °C in a humidified 5% CO2/95% air incubator.
- 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.
- Aspirate the medium from the 10 cm culture dish containing the C57BL/6 mouse ES cells and rinse with 10 ml of PBS twice.
- 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.
- Add 5 ml of ES cell medium and gently neutralize the contents of the dish.
- 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.
- 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.
- Place the dish in the 37 °C incubator overnight.
- When freeze the cells, use the following freezing medium:
- 10% DMSO
- 90% FBS
- Place vials in liquid nitrogen immediately upon receipt until it is convenient to proceed to culture.
- To insure the highest level of viability, be sure to warm media to 37 °C before using it on the cells.
- 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.
- 0.25% gelatin (800 ml)
1.75 g gelatin
Add Mill Q H2O to final volume 800 ml, autoclave before use.
- Mouse ES cell medium (500 ml)
- DMEM 409ml
- FBS 75 ml
- NEAA(100x) 5 ml
- Pen/Strep(100x) 5 ml
- L Glutamine(100x) 5 ml
- 2-mercaptoethanol(1,000x) 1ml
- 1,000 U/ml LIF 50 µl
- Filtered by 0.22 µm filter unit, kept at 4 °C
- 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.
- 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.
- C57BL/6 mouse ES cell line product datasheet (ATCC).
- Evans, M. J. and Kaufman, M. H. (1981). Establishment in culture of pluripotential cells from mouse embryos. Nature 292(5819): 154-156.
Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Pan, Y. (2011). Culturing of C57BL/6 Mouse Embryonic Stem (ES) Cell Line. Bio-101: e142. DOI:10.21769/BioProtoc.142.