Isolation and culture of human UREC

Following informed and written consent, human URECs (hURECs) were isolated from urine collected from two JBTS CEP290 patients and two unrelated wild-type controls. One JBTS patient carried in homozygosis the common CEP290 variant NM_025114.4; c.5668G>T; p.G1890X, whereas the second JBTS patient was compound het for the CEP290 variants c.5668G>T; p.G1890X and c.2495_2512delInATCT; p.T832Nfs*12. hURECs were cultured as described previously (Molinari et al., 2020 (link)). Briefly, cells were isolated from urine samples through repeated centrifugation passages and kept for the first 96 h in Dulbecco's medium Eagle medium/high glucose and Ham's F12 nutrient mix (1:1), supplemented with 10% (v/v) fetal bovine serum (FBS), 140 U/ml penicillin, 140 μg/ml streptomycin, 3.5 μg/ml amphotericin B and REGM SingleQuot kit supplements (Lonza), at 37°C in a humidified atmosphere of 5% (v/v) CO₂. After isolation for 96 h, medium was changed to Renal Epithelial Cell Growth Basal Medium (REBM, Lonza) supplemented with 0.5% FBS, 140 U/ml penicillin, 140 μg/ml streptomycin, 3.5 μg/ml amphotericin B and REGM SingleQuot kit supplements (Lonza). Before collection, cells were cultured in the same medium devoid of FBS for 48 h to induce ciliogenesis.