Cell culture

Immortalized mouse inner medullary collecting duct (IMCD) cells [isolated from αv^flox/flox mice, as described previously (Zhang et al., 2009; Husted et al., 1988) were maintained in Dulbecco's modified Eagle's medium (DMEM)/Nutrient Mixture F-12 Ham (DMEM/F-12 50/50; D8437, Sigma-Aldrich)] supplemented with 10% fetal bovine serum (Life Technologies) and 1% antibiotic-antimycotic solution (MT-30-004-CI, MediaTech). Renal proximal tubule cells (RPTCs; isolated from β1^flox/flox mice, as described previously; Elias et al., 2014) were maintained in DMEM/F-12 (50/50 D8437, Sigma-Aldrich) supplemented with 2.5% fetal bovine serum (Life Technologies), 20 ng/ml hydrocortisone (H0135, Sigma-Aldrich), ITS medium supplement (5 µg/ml insulin-5 µg/ml transferrin-5 ng/ml selenite; I1884, Sigma-Aldrich), 6.7 mg/ml 3,3′,5-triiodo-L-thyronine (T5516, Sigma-Aldrich), 0.096 mg/ml D-valine (V1255, Sigma-Aldrich) and 100 µg/ml Normocin (ant-nr-2, InvivoGen). RPTCs were maintained at 33°C with 0.1% interferon γ (14777, Sigma-Aldrich) in culture medium during growth and expansion. At least 10 days before 3D encapsulation, RPTCs were cultured in absence of interferon γ at 37°C to induce differentiation.