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 (Me₂SO). 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 Me₂SO concentration. Overall, the 80% chicken serum formulations containing 5% or 7.5% Me₂SO 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.

Follicular cells represent a valuable resource for genetic research, biotechnology and cryopreservation in biobanks, particularly for the conservation of endangered species. They offer a more practical alternative to gametes, embryos and fibroblasts. Collection of these cells can be achieved through feather plucking. Feather samples were opened with a scalpel and the feather pulp was washed with PBS, cut into cubes and digested in collagenase type IV. Cultivation was carried out in DMEM culture medium with 15% fetal bovine serum, 1% penicillin/streptomycin and 0.5% amphotericin, under incubation conditions of 39.5 °C and 5% CO₂. Passages were carried out with 5% EDTA for 5 min. The culture was successful, with great cell proliferation, adherence to plastic and aggregation into cell colonies. This method was effective in obtaining feather follicle cells from wild birds, especially when collected up to 6 h after their death, and can serve as a base protocol for research with feather follicle cells aiming to create biobanks.