Isolation and culture of chicken growing follicles in 2- and 3-dimensional models.

The isolation and culture of ovarian follicles is essential for the studies of follicular development and function. In contrast to the relative ease of culture for mammalian follicles, developing in vitro cultures of high viability for the much larger avian follicles has always proven to be more challenging. In this study, the growing follicles from domestic hens (Gallus domesticus) were isolated using enzymatic and mechanical methods and then investigated for the optimized conditions for culture. Assessments of viability and hormonal responsiveness were also considered. A larger percentage of healthy follicles was achieved by mechanical separation than enzymatic dissociation (83% vs. 55% by collagenase I or 63% by trypsin), despite a lower recovery yield for the former (126 vs. 275 by collagenase I or 261 by trypsin) from each ovary. All of the mechanically isolated follicles (800 μm) survived when cultured in the 3-dimensional (3D) system for 7 days whereas only 93% of the follicles survived in the 2-dimensional (2D) group. Follicles cultured in the 3D system also had a higher cell proliferation rates but lower apoptotic rates as assessed by BrdU incorporation and TUNEL assays. Ultrastructural examination showed that the granulosa cells in the 3D group were organized tightly with adjacent layers in contrast to the loose attachment in the 2D system group. After treatment with follicle-stimulating hormone in the 3D culture for 3 days, the mechanically isolated follicles (800 μm) displayed elevated mRNA expression of steroidogenic enzymes, cytokines and cell cycle-regulating proteins. The 3D culture model established in this study thus provides a useful tool for in vitro culture using growing follicles in a large diameter to study the mechanisms of growing follicle development in the avian species.