Theca cells (TCs) are one of the most important cell types in follicles and play an essential role during follicular development. However, the suitable isolation method for buffalo TCs remains unclear. In the present study, we compared three separation methods (collagenase II digestion, trypsin digestion, and tissue cultivation) to seek the best isolation method for buffalo TCs. The results showed that, compared with the method of trypsin digestion, the isolation methods of collagenase II digestion and tissue cultivation exerted a significantly higher cell viability of buffalo TCs. Furthermore, compared to the methods of trypsin digestion and tissue cultivation, the isolation method of collagenase II manifested a significantly higher cell purity of buffalo TCs. This study indicated that, with regards to cell viability and purity, the method of collagenase II digestion was the optimal choice to isolate buffalo TCs. Our study provides a basis for the procurement of high-quality buffalo TCs to further explore buffalo somatic cell nuclear transfer.
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