NWRs and SWR fibroblast cell lines were obtained from the San Diego Zoo Institute for Conservation Research Frozen Zoo biobank and expanded. No IACUC approval was required for the transfer of previously established cell lines. Cells were cultured at 37°C with 5% CO2 in either Dulbecco's modified Eagle's medium (DMEM) 1X+GlutaMAX™ (Gibco; supplemented with 10% fetal bovine serum, and 1% MEM nonessential amino acids; NEAA, Gibco) or a 1:1 mix of the supplemented DMEM and Fibroblast Growth Medium (FGM™-2; Lonza). The additional proprietary growth factors and supplements (including 0.5% recombinant human fibroblast growth factor-b (rhFGF-b) and 0.05% recombinant human insulin) in the FGM-2 formulation have been shown to improve the growth of rhinoceros fibroblast cells [25,26] and have been key to the growth and biobanking of many rhinoceros species in the Frozen Zoo. We refer to this medium as 50:50 throughout.

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