Developmental potential of cumulus cell-derived culture frozen in a quiescent state after nucleus transfer

An efficient method for freezing donor cells is necessary when using nucleus transfer of somatic cells for large-scale cloning. In the present study, we developed a method for freezing and thawing bovine cumulus cell-derived cultured cells to be used as nucleus donors. Cumulus cells were obtained from ovaries of living and slaughtered bovine and cultured in vitro. Cumulus cell-derived cultured cells were serum-starved for several days to induce a quiescent state and then frozen at -70 degrees C for at least 2 d. Immediately thereafter or 2 h after thawing, the cells were used as donor cells for nuclear transfer without additional in vitro culture. The fusion rate with recipient cytoplasts was not affected by the cumulus cell source (slaughtered or living) or time after thawing (0 and 2 h). The cleavage rate of frozen-thawed cumulus cell-derived cultured cells from slaughtered cows immediately after thawing (0 h) was highest (97%) and was significantly higher than that of controls (85%) or cells transferred 2 h after thawing (85%). There were no significant differences among any of the groups in the potential of the nuclear transfer embryos to develop into blastocysts (34 vs 44 and 44%, 39 vs 45 and 46%). Thus, storage of bovine cumulus cell-derived cultured cells in the quiescent state at -70 degrees C is effective and might be useful and convenient for large-scale cloning. The maximum storage periods and developmental potential of embryos after such nucleus transfers requires further examination.