Encapsulated Mouse Follicle Culture

Mouse follicle isolation, encapsulation, and culture were carried out as previously described (33 (link), 34 (link), 70 (link), 71 (link)). In short, morphologically normal multilayered secondary follicles (150 to 180 μm in diameter) were mechanically isolated and selected from 16-d-old CD-1 female mice in L15 media (Invitrogen) containing 1% FBS (Invitrogen). Follicles were then incubated in the maintenance media (50% minimal essential medium [αMEM GlutaMAX; Invitrogen] and 50% nutrient mixture [F-12 with GlutaMAX]) with 1% FBS at 37 °C, 5% CO₂ in air for 2 h. Afterward, individual follicles were encapsulated in 5 μL 0.5% (weight/volume) alginate hydrogel (Sigma-Aldrich), immediately immersed in 50 mM CaCl₂ and 140 mM NaCl for 2 min to allow cross-linking, and incubated in the maintenance media to recover for 2 h. Individual follicles were then cultured in 96-well plates for 7 d in the follicle-culture media (maintenance media + 3 mg/mL bovine serum albumin + 10 mIU/mL human recombinant follicle-stimulating hormone [FSH; from A. F. Parlow, National Hormone and Peptide Program, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD] + 1 mg/mL bovine fetuin [Sigma-Aldrich] + ITS [5 μg/mL insulin, 5 μg/mL transferrin, and 5 ng/mL selenium; Sigma-Aldrich]). Half of the follicle-culture media was replaced every other day.