Isolation and characterization of permanent cell lines from inner cell mass cells of bovine blastocysts

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Abstract
Inner cell masses (ICM) from in vitro produced day 8 or 9 bovine blastocysts were isolated by immunosurgery and cultured under different conditions in order to establish which of two feeder cell types and culture media were most efficient in supporting attachment and outgrowth of the bovine ICM cells. The efficiency of attachment and outgrowth of the ICM cells could be markedly improved when STO feeder cells were used instead of bovine uterus epithelial cells, and by using charcoal‐stripped serum instead of normal serum to supplement the culture medium. More than 20 stable cell lines were obtained. Some of these lines were examined by immunofluorescence for developmentally regulated markers. From these results we conclude that the cell lines resemble epithelial cells, rather than pluripotent ICM cells. The developmental potential of cells of one of the lines was tested in the nuclear transfer assay. The cell line could support the initial development of enucleated oocytes, but none of the reconstructed embryos passed the eight‐cell block.