INITIAL CULTURE BEHAVIOR OF RAT BLASTOCYSTS ON SELECTED FEEDER CELL-LINES

To increase our understanding of rat embryos in culture and to attempt the isolation of blastocyst-derived cell lines, we examinated the ini tial growth behaviour of rat blastocysts from four strains of rat on f our different feeder cell layers. The feeders used were a continuous c ell line of murine embryonic fibroblasts (STO), primary mouse (MEF) or primary rat (REF) embryonic fibroblasts, and a continuous cell line o f rat uterine epithelial cells (RUCs). A medium that gave optimum plat ing efficiencies for murine ES cells was used in the rat embryo cultur e. Each culture system allowed hatching and attachment of the blastocy sts, that is, the behaviour was similar on each feeder and each strain for the first 2 days in culture. Subsequently, there was a rapid diff erentiation of the Inner Cell Mass (ICM) cells on fibroblastic feeder cell layers (STO > MEF > REF), and this was generally complete after 3 -6 days in primary culture. On RUCs, the ICM was found to increase in size without differentiation up to and including day 4 and in some cas es longer. Embryo-derived cells were obtained by disaggregating and pa ssaging ICMs on REF and RUC feeders. Rounded, refractile, and epitheli al-like cells were isolated on REF and colonies of ES-like cells on th e RUCs. The ES-like cells were positive for expression of alkaline pho sphatase and stage-specific embryonic-antigen 1. This is an important first step towards the derivation and culture of pluripotent ES cells from the rat. (C) 1995 Wiley-Liss, Inc.