J. Zachary Bruneel and Dr. Jeralyn J. Franson, Animal Science
Introduction
Embryonic stem (ES) cells are obtained through the culture of undifferentiated cells of embryos in early devel-opment. Because embryonic cells will undergo differentiation and development when placed in standard culture conditions, a media which contains differentiation inhibitors must be used in order for successful development of ES cells. ES cells are capable of becoming any cell type in the body when removed from the influence of differentiation inhibitors, making ES cells a potential agent for gene transfer and livestock cloning. ES cells are therefore an invaluable research tool, providing both a better understanding of genetics as well as providing genetically superior animals. The successful culture of embryonic stem cells depends upon accurate and reliable isolation techniques. The purpose of this research was to determine the optimum method for isolation and culture of ES cells and also to determine which differentiation inhibitor works most effectively. Embryos were collected and cultured for the purpose of both perfecting current techniques and studying which differentiation inhibiting media supported maximum growth.
Procedure
Seven to eleven days prior to slaughter, sows and gilts from the BYU swine unit were induced to ovulate using either injectable or physical means and subsequently bred. Immediately following slaughter, uteri from each animal were collected, labeled and placed in a isotonic saline solution for transport. Uteri were then physically examined for signs of ovulation, one or more corpora lutea. If ovulation had occurred, the uteri were then flushed internally using Dulbecco’s modified eagle media and any embryos were collected into a sterile dish. Embryos were then evaluated for developmental stage and health, as assessed by light microscopy. Embryos which appeared healthy were then put through a series of phosphate buffered saline rinses and cultured overnight in an oxygen rich incubator. Evaluation and rinsing of embryos occurred under a sterile hood.
Discussion
No embryos survived the preliminary culture stage. This was due to a myriad of factors, many of which were overcome with time and practice. It was discovered that the later stage embryos (day 11) had a lower mortality rate than their earlier stage counterparts (day 7). The fragility of early stage embryos would account for this finding. There were also problems associated with media preparation and aseptic technique. The pH of the collecting media was not correct and resulted in embryonic death. Improper aseptic technique resulted in bacterial contamination. Steps were taken to correct both problems. The collecting media (Dulbecco’s modified eagle media) was prepared and adjusted to the proper pH and careful attention was paid to aseptic technique including sterilization of all instruments and trays.
Temperature was also a contributing factor to embryonic mortality. If the uterine tracts were allowed to cool at all during collection, embryonic mortality increased. The temperature of the collecting and rinsing media were also critical. Care was taken to keep all media warm. To keep tracts warm a heated tray proved useful as well as a slide warmer to keep the tray at the optimum temperature, and mortality subsequently improved. The biggest problem to overcome was that often a pig would ovulate only three or four embryos, giving us fewer specimens to use in this research. We did not find a solution to this problem, nor did we find a way to predict this phenomenon.
Conclusion
We were unable to test our hypothesis due to problems associated with temperature, pH, embryonic shock, and contamination. These problems were identified and when possible corrected, and embryonic mortality subsequently decreased. However, we were still unable to maintain cell life for a period of more than 24 hours. With time and more practice, we feel confident that more conclusive results would have been achieved.