Generation of healthy bovine ovarian organoids: a proof-of-concept derivation technique.

BACKGROUND: Organoids have emerged as powerful tools in reproductive medicine and bioengineering, offering three-dimensional (3D) models that closely mimic native tissues. However, the development of protocols for generating healthy epithelial ovarian organoids (OvaOs) remains significantly underexplored, as most studies focus on ovarian cancer models. This work presents an effective protocol for generating healthy bovine OvaOs as a physiological and translational model for ovarian research, mimicking the anatomical and functional similarities between bovine and human ovarian surface epithelium (OSE). RESULTS: Healthy bovine OvaOs were successfully derived using a mechanical-enzymatic method with a predominant mechanical approach, which proved superior to exclusively enzymatic techniques that failed to yield an adequate number of OSE cells. The biological potential of the resulting OvaOs to establish long-term organoid lines was demonstrated by their exponential growth over a 21-day culture period, extensive passaging capacity, and high viability after freeze-thaw cycles. Histological analyses confirmed that healthy bovine OvaOs recapitulated OSE tissue characteristics, including the expression of Cytokeratin 18, Vimentin, and CD44, while the absence of Paired box gene-8 (PAX8) expression excluded contamination by fimbrial cells. CONCLUSIONS: This study describes an effective mechanical protocol for deriving healthy OvaOs from bovine ovaries. These 3D models faithfully replicate the biological features of bovine OSE, with sustained viability across long-term cultures, passaging, and freeze-thaw cycles. These findings underscore their potential as translational models for advancing ovarian physiology research and adapting protocols to human ovarian tissue.