Development of a placental cell culture system for studying the control of mouse placental lactogen II secretion

Fetal placental tissue from 11 days pregnant mice was dissociated in collagenase and DNase solution and then separated on a 40 per cent Percoll gradient. Trophoblast cells banded at a density of 1.05 g/ml. When cultured on rat tail collagen, these cells formed colonies of mono- and binucleate cells varying in size from 40 to 70 microns. At the time of plating, about 13 per cent of the trophoblast cells secreted mouse placental lactogen II (mPL-II) as determined by reverse haemolytic plaque assay. The ratio of mPL-II-producing cells increased significantly in culture and reached 63 per cent after 48 h. The secretion of mPL-II increased continuously during six days of culture, whereas the total protein release was highest after the first day, declined the second day and then remained relatively constant for the last four days of culture. The DNA content of the cells did not change significantly during the six-day period. When the trophoblast cells were incubated with insulin (1 ng/ml to 5 micrograms/ml), a modest but significant reduction in mPL-II secretion was observed. No change in the mPL-II secretion was seen when epidermal growth factor was administered to the culture in concentrations from 1 ng/ml to 10 micrograms/ml. It is concluded that this in vitro culture system is suitable for studying both mPL-II secretion and the differentiation of mPL-II-producing cells.