The resulting pellet of granulosa
cells was washed twice by centrifugation at 180 g for
5 min with 20 mL of calcium and magnesium free PBS
(WAKO, Tokyo, Japan) containing 100 IU/mL of penicillin
and 100 g/mL of streptomycin. The pellet was
then suspended in 10 mL of 0.1% hyaluronidase solution
for 10 to 20 min at 38.5 °C.