T5516

Pkd1-depleted mouse kidney tubule cell lines (Pkd1^+/- and Pkd1^-/- cells) (a kind gift from Dr. Changlin Mei of Changzheng Hospital, Second Military Medical University, Shanghai, China) were cultured in Dulbecco’s modified Eagle’s medium/Ham’s F-12 medium (DMEM/F12) (Gibco) supplemented with 2% fetal bovine serum (Gibco), Insulin-Transferrin-Selenium (41400045, Gibco), triiodothyronine (2 x 10^-9 M, T5516, Sigma) and recombinant γ-interferon (10 units/ml, Sigma) at 33°C. For cilia formation, cells were transferred to 37°C and grown in γ-interferon-free medium for 7 days.

At DD12, the same volume of retinal maturation medium 2 supplemented with CHIR99021 and SU5402 was added to dilute Y27632. If cells did not completely cover the surface of the culture well, this procedure was skipped. At DD13, medium was changed to retinal maturation medium 2 supplemented with CHIR99021 and SU5402 without Y27632. At DD16, medium was changed to MEM / N1 / FBS medium (MEM-alpha (M-4526, Sigma) / 1% FBS / 1% N1 supplement (N-6530, Sigma) / 2 mM L-Glutamine (G7513, Sigma) / 0.1 mM NEAA / 250 mg/L L-taurine (T8691, Sigma) / 20 μg/L hydrocortisone (H-0396, Sigma) / 0.013 μg/L triiodo-thyronine (T-5516, Sigma)) which was used for human fetal RPE culture [43 (link)]. The medium was changed three times per week.

Human primary fetal retinal pigmented epithelium (hfRPE) cells were purchased from Sciencell (6540). Cells were seeded in transwells (Costar, 3450\3460) previously coated with Laminin 521 (BioLamina, LN521–03). During the first week EpiCM 2% FBS (Sciencell, 0010F,) media was employed to allow for attachment and proliferation (Sciencell, 4101). At day 4, FBS concentration was decreased to 1%. At day 7, cells were transferred to a maturation media; a modification of the formulation (MEM Alpha media (Sigma, M-4526) supplemented with N1 supplement at 1% concentration (Sigma, N-6530), Glutamine-Penicillin-Streptomycin 1 × (Sigma, G-1146), Non-essential Amino Acid (NEAA, M-7145), Taurine 0.25 mg/mL (Sigma, T-0625), Hydrocortisone 20 μg/L (Sigma, H-03966) and Triiodo-thyronin 13 ng/L (Sigma, T-5516) previously reported (Maminishkis et al., 2006 (link), Sonoda et al., 2009 (link)). For the apical side of the transwells we used supplemented media 0.1% Bovine Serum Albumin (BSA) (Sigma, A-9647). For the basolateral side media with 1% FBS was employed. Barrier function was evaluated measuring the trans-epithelial electrical resistance (TER) from 2 wells in 3 independent experiments after 5 weeks of culture (CellZscope, NanoAnalytics). A mean value of 6 wells and SD was calculated.