Long-term culture and cryopreservation of interstitial cells of Cajal

Objective. Interstitial cells of Cajal (ICCs) in the gastrointestinal tract generate and propagate slow waves and mediate neuromuscular neurotransmission. Damage to ICCs has been described in several gastrointestinal motor disorders, and although many studies have examined ICCs in culture, they have been largely limited to freshly dissociated cells or short-term cultures. An efficient and reliable method to establish a source of ICCs is much needed. The aim of this study was to investigate methods for culturing, subculturing, cryopreservation, and recovery of ICCs. Methods. ICCs were derived from intestinal segments of domestic rabbits, and immunohistochemistry for c-Kit was used to identify ICCs in culture and after recovery. Recovered ICCs were also examined for motilin receptor expression. Results. Optimal conditions for ICC culture and cryopreservation were based on cell growth curves and MTT assay. On the basis of these findings, recovered cells were cultured for 7 days and then sorted via flow cytometry based on c-Kit immunoreactivity. The percent of c-Kit positive cells was 64.3%, and the number of ICCs sorted was 6.7 x 10(5). Reverse-transcription polymerase chain reaction and western blotting verified motilin receptor expression in c-Kit-positive ICCs. Conclusions. This is the first study to describe the culture, passage, and recovery of ICCs and to show motilin receptor expression. Our results suggest that ICCs play an important role, at least in some species, in initiating the migrating myoelectric complex induced by motilin.