Cryopreservation of chicken feather follicle-derived cells using chicken serum-based media: broadening cellular diversity in avian biobanking

Feather follicles are dynamic and heterogeneous mini-organs containing epithelial, mesenchymal, pigmentary, and progenitor cell populations. However, cryopreservation studies have focused mainly on fibroblasts, and other follicular populations remain poorly explored. This study aimed to establish favorable cryopreservation conditions for cells isolated from growing feathers of Gallus gallus domesticus using chicken serum. Primary cultures were assessed by morphology, differentiation-associated staining, and reverse transcription quantitative polymerase chain reaction (RT-qPCR). Cells were cryopreserved for 60 days in media containing 10% or 80% chicken serum combined with 5%, 7.5%, or 10% dimethyl sulfoxide (Me 2 SO). Post-thaw cell status was evaluated by Annexin V/propidium iodide flow cytometry immediately after thawing and after 72 h of recovery, and metabolic activity was assessed using a colorimetric tetrazolium assay. Cultures progressively became dominated by small, rounded cells arranged in compact colonies. Morphological and molecular findings were consistent with preferential maintenance of a population displaying epithelial-associated characteristics after culture and cryopreservation. Media containing 80% chicken serum produced more favorable post-thaw outcomes than media containing 10%, with lower late apoptosis immediately after thawing, greater metabolic recovery, and higher retention of adherent cells. Serum concentration accounted for 77.87% of the variation in metabolic activity and had a stronger effect than Me 2 SO concentration. Overall, the 80% chicken serum formulations containing 5% or 7.5% Me 2 SO showed the most consistent recovery across the evaluated endpoints. These findings support the use of chicken serum-based media for cryopreservation of feather follicle-derived cells and broaden the range of cellular resources available for avian biobanking and conservation-oriented biotechnologies.