Cd 1 mouse

Chimeric organoids were constructed and cultured as previously described (Xinaris et al., 2015 ). Briefly, embryonic day (E) 12.5 CD1 mouse (Charles River Italia SpA) kidneys were dissected in MEM (M5650; Sigma-Aldrich, St. Louis, MO), placed in 1 × trypsin/EDTA (Biochrom AG) for 5 min at 37 °C, and dissociated into single cell suspensions by trituration and then filtration through a 40 μm cell strainer (BD Falcon). A total of 1.2 × 10⁵ mouse cells were mixed with 1.2 × 10⁴ human iPSC-derived podocytes or undifferentiated iPSC (10:1, mouse:human) and centrifuged at 900 × g for 5 min. The resulting pellets were placed on top of the 5 μm filter (Merck Millipore Ltd) supported by a metal grid in a humidified atmosphere with 5% CO₂ at 37 °C. Chimeric organoids were cultured in Advanced DMEM (12494; Gibco, Invitrogen Corporation) supplemented with 2% Embryonic Stem cells Fetal Bovine Serum (ES-FBS, Gibco), 1% l-glutamine (Invitrogen Corporation) and 1% penicillin/streptomycin (Invitrogen). For the first 24 hour organoids were cultured in the presence of 1.25 μM Glycyl-H1152 dihydrochloride (Tocris), a ROCK inhibitor.

CLBQ14 (purity ≥ 98%) was purchased from TCI Chemicals (Tokyo, Japan). LC-MS grade water and acetonitrile, clioquinol, formic acid, trifluoroacetic acid (TFA), ethanol, Tween 20, Tween 80, soybean oil, paraffin oil, olive oil, dimethyl sulfoxide (DMSO), N’N dimethyl acetamide (DMA), polyethylene glycol 400 (PEG 400), glycerol, 1-octanol, 0.85% sodium chloride solution, phosphate buffered saline tablets, and CD-1 mouse, Sprague Dawley (SD) rat, cynomolgus monkey and human microsomes were purchased from Sigma Aldrich (St. Louis, MO). Transcutol High Purity (HP)^®, Labrasol^® and Capyrol 90^® were gifts from Gattefosse (Lyon, France). Recombinant human cytochrome P450 enzymes (rhCYP) were purchased from BD Biosciences (San Jose, CA). Heparin (1000 units/mL) and pharmaceutical grade normal saline were purchased from Hospira (Lake Forest, IL). Human plasma was purchased from Gulf Coast Blood Center (Houston, TX) and fresh rat plasma was collected from male SD rats (Envigo RMS Inc., Indianapolis, IN) and stored at −80°C until use. CD-1 mouse and cynomolgus monkey plasma were purchased from BioIVT (Westbury, NY). All chemicals and reagents were used as received.