Placental alkaline phosphatase (PLAP) staining and human chorionic gonadotropin (hCG) production in cultures of fresh and cryopreserved cytotrophoblasts isolated by CD9/MHC class I/MHC class II immunoelimination.

We have further characterized villous trophoblasts isolated by trypsinization and purified by elimination of CD9/MHC class I/MHC class II expressing cells. The cells isolated were >99.99% cytotrophoblasts by criteria of cytokeratin (positive) and vimentin (negative) expression. Purified cells directly after isolation (fresh) were compared with cryopreserved and thawed cells (frozen) for production of human chorionic gonadotropin (hCG) and expression of placental alkaline phosphate (PLAP) after 4 h of culture. We found that fresh cells may adhere slightly more strongly than frozen cells, contained approximately 8-fold more PLAP-positive cells (indicating syncytial fragments) after adherence but neither preparation would secrete hCG until day 4 of culture. We conclude that the cells isolated by cell elimination were cytotrophoblasts with only a small number of PLAP (<0.2% of the fresh population plated) positive cells and that both populations shared the property of very low hCG production until cultured past day 4. We speculate that cells isolated by other methods (accompanying paper) may be contaminated by even more syncytial fragments, detectable by PLAP staining and by production of hCG in the first 48 h of culture.