Viable pluripotent chick blastodermal cells can be maintained long term in an alkaline defined medium.

Most chicken embryonic cell culture methods call for neutral pH media of different natures, with disregard of the peculiar electrochemical environment in which avian embryos develop, with a 4 pH unit gradient across the thin blastoderm and the vitelline membrane. We report results of a culture system in alkaline media (pH >9) with atmospheric conditions. Blastodermal and blood cells, maintained for 8 wk with minor differentiation in the absence of the standard growth factors, developed a thick, mucoid-like matrix in which a large proportion of the cell mass grew embedded, with no direct contact to cultureware. After up to 8 wk, blastoderm explants and dissociated blastodermal cells, cultured in either M199 or Dulbecco's modified Eagle's medium (DMEM) in the absence of supplemental CO2, expressed several pluripotency markers (SSEA1, VASA) and embryoid bodies were formed. The assayed conditions impose an undoubted electrolyte stress on the cells which, notwithstanding, maintained their viability and remained undifferentiated. We hypothesize that a rise in pH and the activation of active cation exchanger like Na(+)/H(+) antiporter could mediate the observed differentiation arrest.