Establishment, Characterization, and Cryopreservation of Feather Follicle Fibroblast Lines From Hyacinth Macaw (Anodorhynchus hyacinthinus).

Abstract

Cryopreservation, biobanking, and in vitro propagation of cells from endangered species represent key strategies for advancing biodiversity conservation. The Hyacinth Macaw (Anodorhynchus hyacinthinus), a flagship avian species of the Brazilian Pantanal, is among the critically vulnerable taxa that stand to benefit significantly from such cutting-edge biotechnological interventions. This study developed and validated minimally invasive methods for isolating, culturing, and cryopreserving fibroblasts derived from feather follicles (FFFs) of Hyacinth Macaws. Cells isolated from nestlings under 100 days of age demonstrated superior yields and viability compared to older birds. Two cryopreservation media were tested-Cryomedium 1 (55% DMEM-F12, 35% FBS, 10% DMSO) and Cryomedium 2 (90% FBS, 10% DMSO)-with Cryomedium 1 proving more effective in maintaining FFFs viability immediately post-thaw. Several culture conditions were evaluated, including conventional plating, drop plating, gelatin coating, and supplementation with RevitaCell and bFGF. The optimal method involved conventional plating on gelatin-coated plates with RevitaCell supplementation during the first 24 h post-thaw. Additionally, two different culture media were tested, with KAV-1 emerging as the best option for the long-term propagation of Hyacinth Macaw fibroblasts. After some passages, FFFs maintained a stable karyotype of 2n = 70, expressed classical fibroblast markers such as Vim, Fap, Acta2, Col1a1, Col1a2, synthesized vimentin and Type I collagen in the cytoplasm, and were confirmed to be free of mycoplasma contamination. We successfully established the first primary fibroblast cell lines derived from the Hyacinth Macaw and demonstrated their efficient responsiveness to lipid nanoparticle-mediated transfection and exogenous gene expression, representing a significant advancement toward the development of somatic reprogramming strategies. These experiments enabled the optimization of protocols for cell collection, cryopreservation, in vitro propagation, and inducing sustained heterologous expression, thereby laying a valuable foundation for the future generation of induced pluripotent stem cells (iPSCs) to support ex situ conservation efforts for this endangered species.