Establishment and cryopreservation of liver, heart and muscle cell lines derived from the Chinese alligator （Alligator sinensis）

The Chinese alligator, Alligator sinensis, is a critically endangered species. A conservation project of gene resources for an endangered species first involves the preservation of organs, tissues, gametes, genomic DNA libraries and cell lines. The present study is the first to establish and cryopreserve cell lines of liver, heart and muscle tissues from the Chinese alligator. The study revealed that there was a large discrepancy in cell migration time in primary cultures among liver (11-12 d), heart (13-14 d) and muscle (17-18 d) tissue pieces. The differences in time in primary cell culture suggested that it was relatively easy to build visceral-derived cell lines for reptiles. Biological analysis showed that the population doubling time for thawed cells was approxi- mately 36 h. Karyotyping revealed that the frequency of Chinese alligator cells showing chromosome number as 2n=32 was 88.6%-93.4%. Chinese alligator cell lines established here provide a vital resource for research and are likely to be useful for protection of this rare and critically endangered species. Furthermore, the establishment of these methods may supply technical and theoretical support for preserving genetic resources at the cellular level for other reptile species.