C 12360

The primary HPF cells were purchased from PromoCell GmbH (C-12360, Heidelberg, Germany). As per the catalog information provided by PromoCell GmbH, these primary HPF cells are isolated from human lung tissue, and the passage number is two after thawing. The cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich Co., St. Louis, MO, USA) and 1% penicillin–streptomycin (GIBCO BRL, Grand Island, NY, USA). Cultures were maintained in a humidified incubator containing 5% CO₂ at 37 °C. For experiments, HPF cells within the passage range of four to eight were utilized.

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.

Biocompatibility of the various inks was assessed through incorporation of human pulmonary lung fibroblasts (HPFs) (cryopreserved, C12360, LOT #446Z031), isolated from healthy human respiratory tissue, purchased from PromoCell (Heidelberg, Germany) into the bulk materials. Each material was loaded with 2 × 10⁶ primary human pulmonary fibroblasts per mL, and 100 µL of a specified material was pipetted into each well of a 96-well plate with one row (n = 12) for each material of interest. Crosslinker (50 mM CaCl₂) was added to each well and left for 20 min before it was removed and replaced with cell culture media (Fibroblast Growth Media 2, PromoCell). The plates were cultured in an incubator (37 °C, 5% CO₂), with a plate removed after 1, 3, 5, 7, 10, and 14 days. The cell-containing material was then dissolved with EDTA. The plates were centrifuged and the EDTA was removed and replaced with PBS. WST-1 was then added and the plates were incubated for 1 h before absorbance measurements were taken using a BioRad xMark microplate spectrophotometer (Bio-Rad, Hercules, CA, USA) at a wavelength of 440 nm.

Human NSCLC A549 adenocarcinoma cells, SCLC Calu-6 cells, and normal lung WI-38 VA-13 subclone 2RA cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Primary normal HPF cells were purchased from PromoCell GmbH (C-12360, Heidelberg, Germany) and used between passages five and ten. Lung cells were kept in a standard humidified incubator at 37 °C with 5% CO₂ and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich Co., St. Louis, MO, USA) and 1% penicillin–streptomycin (GIBCO BRL, Grand Island, NY, USA). Cells were grown in 100 mm plastic cell culture dishes (BD Falcon, Franklin Lakes, NJ, USA) and harvested with trypsin–EDTA (Gibco BRL).