Data

Preliminary work has already resulted in valuable data sets. Australian native amphibian cells, amenable to culture, passaging, cryopreservation and recovery have been prospectively validated and karyotyped. Below are the results of this early work on two individual genera and species.

Results

Limnodynastes tasmaniensis: Up to P3 pre- and P3 post-cryopreservation

(Total = 7 passages). Cryopreservation = 3 months; 2N = 24 (n = 27, i = 78%)

Conclusions

• Cells from Australian native frogs are amenable to culture, repeat passaging, cryopreservation, recovery and continued passaging.

• Combined with our recent publications, the culture, cryopreservation and recovery techniques developed here are applicable to both adult frogs and tadpoles.

• These techniques will now be employed to cryobank prospectively validated cell lines from critically endangered frogs.

• Cell viability will be further validated by iPSC generation and cloning.

Materials and Methods

All experimental tissue was sourced according to appropriate governmental and ethical permissions. All animals were either sacrificed for alternative experimental procedures or euthanized due to injury or sickness. All experimental procedures were undertaken following appropriate permissions from relevant government and ethical authorities. Tissue was grown and passaged in Amphicell media^TM. Imaging for karyotyping was performed on colchicine (GIBCO) treated cells stained with DAPI (Sigma). Imaging was performed at 1000 x using an Olympus BX60 microscope, colour CCD Leica DFC425C camera, and EL-6000 light source. Images were captured using Leica LAS-AF software.

Contributions

Dr Richard Mollard: Experimental design and execution; data interpretation and preparation.

Prof Michael Mahony: Supply of deceased frogs.

 Abbreviations

D = day; HP = high power; LP = low power; M = months of cryopreservation; P = passage