Effects of Trypsinization on Viability of Equine
Chondrocytes in Cell Culture
B. C. Sutradhar, J. Park, G. Hong, S. H. Choi and
G. Kim*
Laboratory of Veterinary Surgery, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk 361-673, Korea
*Corresponding author:
ghkim@cbu.ac.kr
Abstract
Trypsin is an essential reagent for routine cell
culture work. In the cultivation of mammalian cells, it has been extensively
used for cell isolation from tissues or cell dislodging in subculturing. It may
damage the cell membrane in contact of cells during long trypsinization.
However, there is no specific report on time-dependent effect of trypsinization
on cells. In the present study, we investigated the time dependent effects of
trypsinization on equine chondrocytes. Cell viability after trypsinization with
0.25% trypsin-EDTA for 5 to 60 minutes was quantified by trypan blue exclusion
assay, propidium iodide-Hoechst double staining, flow cytometry analysis and
XTT assay. The results showed
that trypsin-EDTA decreased the proliferation of equine chondrocytes depending
on the exposure time of trypsinization. After 20 and 60 minutes of
trypsinization, the cell membranes were strongly affected and the percentages of
viable cells reduced to 91% and 85% respectively detected by trypan blue
exclusion assay. Similar results were
observed both in flow cytometric evaluation and propidium iodide-Hoechst double staining. The XTT assay result also showed decreased cell
viability with the extended time of trypsinization. In order to minimize
the time dependant cytotoxicity of trypsinization, as minimum as short time
exposure is suggestive that maximizes live cell isolation from tissue as well as
subculture of equine chondrocytes or other cells.