Chemically defined, serum-free and xeno-free generation of mature type 2 alveolar epithelial cells from hiPSCs
Chemically defined, serum-free and xeno-free generation of mature type 2 alveolar epithelial cells from hiPSCs
For dissociation, spheroids were collected and centrifuged at 200g for 4 minutes and resuspended in a dissociation mix of  0.125 mg/mL Collagenase (Merck, C9891), 1 U/mL Dispase II (Thermo Fisher Scientific, #17105041) and 0.1 U/μL DNase (Merck, D4527) mixed in DMEM/F12 (Dulbecco's Modified Eagle's Media: Nutrient Mixture F-12; Thermo Fisher Scientific, #11330-057) for 20 minutes at 37°C on a DLAB MX-T6-Pro Roller at 10 RPM. Dissociation mix was neutralised using 1:3 dilution with basal media and centrifuged at 200 RCF for 5 minutes. Spheroids were resuspended in dissociation mix consisting of 0.05% trypsin-EDTA (Gibco, #25300062) and 0.1 U/μL DNase and placed at 37°C for 10-15 minutes, resuspending manually every 3 minutes. Dissociation mix was neutralised using 10% foetal bovine serum solution in DMEM/F12 at 1:3 dilution. Cells were centrifuged at 300 RCF for 5 minutes, and then counted. RNA extraction was done following RNeasy Mini kit (Qiagen, #74106) instructions. Samples were outsourced to GENEWIZ for nucleic acid library construction, and processed for ribosomal RNA depletion, and library preparation using standard NEB kit from New England Biolabs.