COVR Development In Vitro

Inguinal fat from male rabbits was digested in 0.1% type I collagenase, as previously described.^14,15 After centrifugation, infranatant was washed free of lipid and red blood cells. The resulting stromovascular fraction was plated in culture. Adherent adipose-derived stem cells (ASC) were harvested at third passage. Cell multipotency was confirmed by differentiation to osteogenic and adipogenic phenotypes.^14,15

Cell-based constructs were formed within 12 mm Corning Transwell (Corning, Corning, NY) culture inserts.¹² Rabbit fibrinogen at 5 mg/mL was mixed in a 4:1:1 ratio with bovine thrombin (2 units/mL in HEPES-buffered saline with CaCl₂) and cell suspension (6 × 10⁶ cells/mL) to form fibrin gels with embedded ASC. After gelation, additional ASC were pipetted onto the surface to replicate an epithelial layer. Culture medium within the annulus supplied the tissue constructs only through the insert base. Medium contained 10% fetal bovine serum and 10 ng/mL epidermal growth factor and was changed every 2 to 3 days for 2 weeks.