The Modified Cultured Method for Siamese Crocodile (Crocodylus siamensis) Primary Cell Culture derived from Eyelid and Blood Vessel

Abstract

Siamese crocodile (Crocodylus siamensis) is a freshwater crocodile. Its wildly distributed habitat was Southeast Asia. It was commonly found in wild nature of Thailand in the past and presently declared as critically endangered by International Union for Conservation of Nature (IUCN). In Thailand, crocodile farming is allowed in order to conserve pure bred and facilitate rational purpose. However, the purity of Siamese crocodiles' genetic has been decreased due to cross-breeding with saltwater crocodile in many farms. The objectives of this research were to study cell culture method by modifying the cell culturing methods and maintain Siamese crocodile genetic material as primary cell culture for further study on crocodiles’ chromosome, host specific disease and providing resource for other diagnosis tools. Eyelid and blood vessel were obtained from the newly dead Siamese crocodile suffering from fighting. The specimens were then excising into small pieces and cultured with two supplement formulas including i) Iscove's Dulbecco’s Modified Eagle Medium (IMDM) supplemented with 10% Fetal Bovine Serum (FBS) and ii) IMDM supplemented with 10% crocodile plasma. The antibiotic and antifungal were added in both types of media for preventing contamination. Thereafter the cultured flasks were incubated at 28 °C and 37 °C with 5% CO₂ supplied. The fibroblast-like cells were observed under the inverted microscope at 2 weeks later. The cells were subcultured until 10 passages and preserved at every single passage under -196 °C in liquid nitrogen. The result indicated that both supplement formulas were successfully applied for primary cell culture at the temperature of 28°C and 37°C. The supplementation by FBS is more applicable than crocodile plasma due to commercially available.