Estimation of viability of bovine granulosa cells

Granulosa cells harvested from non-atretic, antral follicles of cow (and pig and sheep) ovaries were incubated over glass cover slips in medium containing 20% (v/v) donor calf serum. Cell attachment to the cover slips was rapid, being in most cases complete within 3 h at 37 degrees C. There was little further change over the next 20 h. The number of bovine granulosa cells which attached to a cover slip was proportional to the volume of cell suspension added to the medium; and the amount of oestradiol secretion by attached cells in a testosterone-enriched medium increased in parallel with their number. Granulosa cells which did not attach within 3 h produced little oestradiol. There was no clear relationship between the number of nigrosin-impermeable cells in suspensions prepared from different follicles and plating efficiency. It is concluded that the 3-h attachment of granulosa cells in culture is a useful measure of the number of viable cells in a suspension and is to be preferred to less direct techniques based on dye exclusion.