40 EARLY DEVELOPMENT OF RECONSTRUCTED MOUSE EMBRYOS USING BONE MARROW CELLS FROZEN WITHOUT CRYOPROTECTANT

Animal cells frozen with suitable cryoprotectants have been successfully cryopreserved for long periods of time, maintaining viability upon thawing. Animal cells frozen without cryoprotectant, however, may suffer serious damage and not be useful as donors in somatic cell nuclear transfer (SCNT). However, in some cases, old animal samples were frozen only as a whole body or a piece of tissue without cryoprotectant. If the cells from such old samples could be useful for SCNT, then there are potentially many candidates where individual animals could be reproduced. In this study, we examined the possibility of using mouse bone marrow cells frozen without cryoprotectant as nuclear donors in SCNT. Thigh bones were collected from B6C3F1 mice and frozen in either a –25�C or a –80�C freezer for more than one month. Thawing of frozen bones was performed by placing them in an incubator at 37�C. Bone marrow cells were collected by washing the bone cavity with saline. Recipient oocytes for SCNT were collected from B6D2F1 female mice. The enucleation of recipient oocytes and the injection of nuclei were performed as previously reported (Wakayama et al. 1998 Nature 394, 369–374) with a piezo-actuated micromanipulator system. In this study, 4 groups of mouse cells (fresh bone marrow cells, bone marrow cells frozen at –25�C, bone marrow cells frozen at –80�C, and fresh cumulus cells) were used as the nuclear donors in SCNT. After nuclear injection, embryos were kept in mCZB medium for 1 h at 37�C. Subsequently, embryos were cultured for 3 h with 5 µg mL–1 cytochalasin B and 10 mm SrCl2 for activation and cultured for an additional 20 h in mKSOM medium. The nuclear dynamics of SCNT embryos in each donor cell group was observed using 42,6-diamidino-2-phenylindole (DAPI) staining and a fluorescent microscope at 0, 1, 7, and 24 h after nuclear injection. Data were analyzed by Student's t-test. The cell viability after thawing by trypan blue vital staining was about 20% regardless of freezing temperature. At 7 h after nuclear injection, the SCNT embryos injected with frozen bone marrow cells, regardless of freezing temperature, had more single pronuclei (67%, 54/81; P < 0.05) than SCNT embryos injected with either fresh bone marrow cells (36%, 26/73) or cumulus cells (28%, 67/236). At 24 h after nuclear injection, fewer SCNT embryos injected with bone marrow cells, either fresh or frozen, developed to the 2-cell stage (fresh: 11%, 6/56; frozen at –25�C: 21%, 5/24; frozen at –80�C: 20%, 10/49) than SCNT embryos injected with cumulus cells (58%, 185/319; P < 0.05). There was no difference in the embryonic development to the 2-cell stage among SCNT embryos injected with either fresh or frozen bone marrow cells. Further studies are required to determine whether cells frozen without cryoprotectant are capable of resulting in viable clones.