Can Roscovitine and Trichostatin A be Alternatives to Standard Protocols for Cell Cycle Synchronization of Ovine Adult and Fetal Fibroblast Cells?

Abstract

Synchronization of donor cells is an important step for the success of somatic cell nuclear transfer (SCNT) application and facilitates the development of embryos. Contact inhibition, serum starvation and different chemical agents are used in synchronizing different types of somatic cells. In this study, to synchronize the primary ovine adult (POF) and fetal (POFF) fibroblast cells to G0/G1 phases, the contact inhibition, the serum starvation, Roscovitine and Trichostatin A (TSA) methods were used. In the first part of the study, Roscovitine (10, 15, 20 and 30 μM) and TSA (25, 50, 75 and 100 nM) were applied for 24 hours to determine the optimal concentration for POF and POFF cells. In the second part, optimal concentrations of Roscovitine and TSA for these cells were compared with contact inhibition and serum starvation methods. Cell cycle distribution and apoptotic activity were performed by flow cytometry to compare this synchronization methods. Serum starvation method resulted in higher cell synchronization rate in both cells compared to other groups. Although contact inhibition and TSA also achieved high success rates of synchronized cell value, it was observed that the difference between serum starvation and these groups was significant (p<0.05). When the apoptosis rates of the two cell types were examined, it was observed that the early apoptotic cells in contact inhibition and late apoptotic cells in the serum starvation were higher than the other groups (p<0.05). Although the 10 μM and 15 μM concentrations of Roscovitine gave the lowest apoptosis rates, it was observed that it failed to synchronize both of the ovine fibroblast cells to G0/G1 phase. As a result, it was concluded that while Roscovitine was not successful to synchronize both of the POFF and POF cell lines, TSA (50 nM for POF cells and 100 nM for POFF cells) can be used efficiently as an alternative to the contact inhibition and the serum starvation methods.