Culture pH and osmolality influence proliferation and embryoid body yields of murine embryonic stem cells

The realization of many stem cell based regenerative therapies will depend on culture production. Murine embryonic stem cells (mESC) provide a practical model for stem cell process research as they can be readily obtained at relatively high numbers and purities. However, an understanding of their environmental tolerance ranges is still lacking. These tolerance ranges were explored using an embryoid body (EB) formation assay as a functional measure of mESC maintenance. The mESC were exposed to wide ranges of initial medium glucose, glutamine, ammonium, lactate, pH and osmolality conditions. Within the common ranges of conventional maintenance cultures, the only environmental variables that significantly influenced EB yields were pH and osmolality. Dose–response experiments with these two variables revealed that, within 48 h, the EB yield was, for example, ∼3-fold decreased (p < 0.05) when mESC were cultured either at an initial pH of 7.0 or an osmolality of 400 mOsm/kg compared to a medium at pH 7.3 and 300 mOsm/kg. This decline was due to decreases in both the growth rate and in the fraction of EB-forming cells. More extreme pH (6.7 or 7.75) as well as osmolality (200 or 500 mOsm/kg) conditions reduced the EB formation potential to even lower levels. The decreased growth rates and EB forming potential of mESC cultured at pH 6.7 or 7.75 for 24 or 48 h, when returned to pH 7.3 medium, recovered to control levels within 96–144 h. These studies provide guidance in developing optimal environmental tolerance ranges within which both the mESC output from maintenance cultures as well as the subsequent EB yields can be maximized.