Effects of confluent, roscovitine treatment and serum starvation on the cell-cycle synchronization of bovine foetal fibroblasts.
The present study was designed to examine the effects of cell-cycle synchronization protocols, such as confluent, roscovitine treatment and serum starvation, in bovine foetal fibroblasts on synchronization accuracy at G0/G1, viability, apoptosis, necrosis and ploidy for use as a nuclei donor. The cells in 5-10 passages were randomly allocated into three treated groups. Cells were cultured either in Dulbecco's modified Eagle's medium (DMEM) + 10% foetal bovine serum (FBS) until 90% confluent (group 1, confluent), in DMEM + 10% FBS + 30 microM roscovitine for 12 h (group 2, roscovitine), or in DMEM + 0.5% FBS for 5 days (group 3, serum starvation). Most of the cells (>80%) in all groups were arrested at the G0/G1 stage. Although the rates did not differ, cells in group 1 showed an increased cell population arrested at the G0/G1 phase. Significantly (p < 0.05) higher rates of apoptosis occurred in group 3 than in group 1 and 2 (10% vs 6% and 6%, respectively). No differences in chromosomal abnormality were observed among groups. However, by increasing the number of cell culture passages up to 15, significantly (p < 0.05) higher chromosomal abnormality was observed than in 5 and 10 passages (39% vs 28% and 23%, respectively) in group 1. The results clearly indicated that bovine foetal fibroblasts could be effectively synchronized at G0/G1 stages by all the three different treatments, confluent, roscovitine and serum starvation. However, cells in confluent showed reduced apoptosis and necrosis when they underwent 5-10 passages, exhibiting increased percentage of cells with stable chromosome diversity. Hence, cells in confluent merit further studies before they could be used as nuclear donors.
Cho
S R
SR
College of Veterinary Medicine, Gyeongsang National University, Chinju, Korea.
Ock
S A
SA
Yoo
J G
JG
Mohana Kumar
B
B
Choe
S Y
SY
Rho
G J
GJ
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002417:Cattle / Q000196:embryology*; D018929:Cell Culture Techniques / Q000662:veterinary; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D005260:Female; D005347:Fibroblasts / Q000166:cytology* / Q000502:physiology; D006131:Growth Inhibitors / Q000494:pharmacology*; D011687:Purines / Q000494:pharmacology*; D000077546:Roscovitine
D016895:Culture Media, Serum-Free; D006131:Growth Inhibitors; D011687:Purines; D000077546:Roscovitine