Culture, cryobanking and passaging of karyotypically validated native Australian amphibian cells

Abstract

This study describes the culture, cryobanking, thawing and passaging of karyotypically validated cells from two life history stages of the “least concern” Australian native amphibian species Litoria infrafrenata. Adult frog toe and tadpole macerates were generated from animals euthanized due to ill health following injury. Cultured cells proliferated and formed colonies after one to two weeks of culture. Cultures were cryopreserved in liquid nitrogen for a minimum of one month, thawed, passaged for expansion and karyotyped. Post-thaw karyotypes revealed the expected 2N = 24 diploid chromosome number in approximately 90% of all metaphase spreads. Further, metacentric, submetacentric and subtelocentric configurations were the same as previously described karyotype configurations obtained from living frogs of this same species. Using cryobanked and prospectively validated cell lines, conservation programs including assisted reproduction technologies and genomic, mitochondrial and proteomic mining initiatives may therefore be complemented with minimal or no disturbance to living and healthy animals.