| Abstract |
Cell lines from the early embryonic stages of fish have potential uses in
the development of embryonic stem (ES) cell technology, either as ES cell
lines or embryonic fibroblasts (EF) to act as feeders for ES lines. As
well, they would be useful in other biological disciplines, such as
toxicology. Therefore, we attempted to initiate cell cultures from
individual rainbow trout embryos at approximately stage 6 or blastula
stage of development. Embryos were treated in various proteolytic
solutions, and then with the chorions still intact, single embryos were
placed into individual wells of 24-well plates in Leibovitz's medium with
10% fetal bovine serum. At various times afterwards, the chorions were
ruptured with a Pasteur pipette, releasing cells into the medium. The
proportion of the approximately 100 embryos that gave rise to primary
cultures is unknown, as most wells were overrun by bacterial contamination.
Of the uncontaminated wells, a few embryos generated some adherent cells
without further proliferation. One embryo, however, gave rise to an
attached ball of cells, from which grew an adherent monolayer. These cells
were subcultured, giving rise to mixed colonies of epithelial-like and
fibroblast-like cells. These cells have now been passaged over 25 times,
cryopreserved, and designated RTeeb. RTeeb express CYP1A in response to
dioxin (TCDD), which is a useful property in toxicological studies. They
are alkaline-phosphatase-positive, express CD9 mRNA, and sporadically form
compact circular colonies in monolayer cultures, properties reminiscent of
mammalian ES cells. Yet, the cells are aneuploid, and are thus more
similar to mammalian embryonal carcinoma cell lines.
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