Culturing of C57BL/6 Mouse Embryonic Stem (ES) Cell Line   

Abstract

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.