Preimplantation genetic diagnosis of an embryo generally requires cells obtained by biopsy, an invasive procedure. The objective of this study was to develop a procedure for efficient production of high quality blastocysts after biopsy and cryopreservation suitable for eventual implantation, while providing adequate biopsy material for genetic analysis. The zona pellucida of compact morulae cultured in either a serum-free or serum-containing medium were slit using a glass microblade without injuring the blastomeres, a partial zona pellucida dissection. Subsequent culture of the biopsied (slit) morulae in either the serum-free or serumcontaining medium resulted in proliferation of trophoblastic cells that herniated through the incision. The developmental rates of biopsied compact morulae into blastocysts and herniated blastocysts obtained from the serum-free medium were significantly (p <0.001) higher than those in the serum-containing medium. The survival rate (92.2%) of freshly biopsied blastocysts cultured in the serum-free medium was slightly higher than that (83.3%) of blastocysts derived in the serum-containing medium. The mean cell number (47.6) of herniated trophoblastic cells from serum-free-derived blastocysts was greater than that (33.8) of trophoblastic cells from serum-derived blastocysts. After the biopsied compact morulae obtained by the partial zona pellucida dissection were cultured in either the serum-free or serum-containing medium and then cryopreserved at the blastocyst stage, the post-thaw survival rate of the serum-free-derived blastocysts tended to be higher than that of serum-derived blastocysts. Cryopreserved blastocysts after biopsy by microdissection did not appear to recover as well as blastocysts after biopsy by partial zona pellucida dissection. Based on these results, partial zona pellucida dissection and the subsequent serum-free culture of bovine embryos could be useful for the efficient production of high quality biopsied blastocysts which can be cryopreserved, while at the same time providing adequate numbers of trophoblastic cells from the biopsied blastocysts for multiple genome analyses.

View full abstract

The new, three nontumorigenic epithelial cell lines, MSGα, M-SGβ1, M-SGβ2, has been established from mouse submandibular gland tissue. Cells were explanted and propagated in chemically defined serum-free medium including several supplements with fibroblast growth factor-1 (FGF-1) or epidermal growth factor (EGF). Either EGF or FGF-1 stimulated the growth of those MSG cells, and was essential factors for the cell growth. The mode of chromosome numbers of MSG/32 cells, which were serially cultured in the serum-free medium supplemented with FGF-1 exhibited diploid. But those of MSGα cells, which were serially cultured in the medium supplemented with EGF plus FGF-1, and MSGβ1, cells which were serially cultured in the medium supplemented with EGF were shifted from diploid to triploid with increasing passage level. These cell lines are nontumorigenic in athymic mice. These cell lines expressed some of the functional proteins which were characteristic to mouse submandibular gland ductal cells.
Taken together, these cell lines would be useful to study growth and differentiation of normal epithelial cells and malignant transformation studies with oncogenes and chemical carcinogenes.

View full abstract