3D Collagen Hydrogel Models with Primary Cells

The primary cells used to build the models were kept as low passage number frozen stocks in liquid nitrogen. The 3D models were grown on Celltreat (Celltreat Scientific Products, Pepperell, MA) 24-well hanging cell culture inserts with a permeable 8 μM PET membrane. The collagen hydrogel was developed using previously defined methods (Gappa-Fahlenkamp and Shukla, 2009 (link)) (Derakhshan et al., 2018 (link)), and consisted of 64.5 vol% 3.1 mg/mL type 1 bovine collagen (Advanced BioMatrix; Carlsbad, CA), 8.1 vol% 10x M199 (Gibco), 13.3 vol% 0.1 N NaOH, and 14 vol% phosphate-buffered saline (PBS). Primary HPFs (PromoCell, Catalog #C-12360 Heidelburg, Germany) were thawed and then directly seeded within the collagen gel solution at a concentration of 75,000 cells/mL, and 0.150 mL of collagen solution was aliquoted to the inner well of the hanging cell culture inserts. Following 45 min of incubation at 37°C 5% CO₂ concentration, complete SAEC medium (PromoCell, Catalog #C-21070 Heidelburg, Germany) was added to the bottom well chamber (1 mL), and 0.100 mL of medium was added to the top of the collagen gel. The hydrogels were incubated for 24 h before adding SAECs.