Successful cryopreservation of PCLS would allow the building of a tissue bank and reduce the use of laboratory animals. During vitrification, tissue may be damaged by toxicity of the cryoprotective agent (CPA), chilling injury (injury due to temperature reduction per se) and injury from ice crystal formation. The mechanism of chilling injury is unknown. We aim to identify critical genes and signaling pathways responding to chilling injury by microarray analysis to enhance insight into its mechanism and to facilitate development of vitrification methods for PCLS. Rat PCLS impregnated with CPA were cooled to -15 o C and held for 10 min to induce chilling injury. Changes in mRNA expression profile in the slices after cooling were compared to those exposed to the CPAs only using Affymetrix arrays. Chemical toxicity of the CPAs was determined by ATP content. Differential Scanning Calorimetry (DSC) indicated that ice crystal formation was prevented in slices loaded with CPA during cooling. CPA toxicity appeared negligible (ATP content >95% of control slices). By cooling at -15o C, the ATP content was decreased by 20-30% compared to that of slices exposed to CPA, indicating moderate chilling injury. Microarray analysis showed that chilling-induced gene expression changes were clearly distinguishable from those induced by the CPAs. 1800 genes were changed (FDR<0.05) with 30 genes up- and 6 genes down-regulated> 1.5 fold. IL-1 and MAPK pathways were among the most interesting regulated pathways. In conclusion: we were able to separate chilling injury from other damaging events that occur during PCLS vitrification, and identified changes in gene expression and signaling pathways due to chilling injury by microarray analysis. This is the first effort to investigate the mechanism of chilling injury in integrated tissue by microarray analysis under conditions in which other sources of injury are absent.
|Tijdschrift||The Toxicologist. Supplement to Toxicological Sciences|
|Nummer van het tijdschrift||1|
|Status||Published - mrt.-2012|