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2 protocols using hepatocyte culture medium

1

In Vitro Transporter Assay Protocol

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Rifampin, diphenhydramide hydrochloride (98%), dimethyl sulfoxide (DMSO), ammonium acetate, acetonitrile Chromasolv (ACN), formic acid, dexamethasone, diclofenac, thiazolyl blue tetrazolium bromide (MTT), and methanol Chromasolv were purchased from Sigma-Aldrich (St. Louis, MO, USA). Bosentan was purchased from Actelion Pharmaceuticals (Allschwil, Switzerland). Cryopreserved human hepatocytes, HEK293 cells overexpressing OATP1B1, OATP1B3, and OATP2B1 transporter, HEK293 mock cells, phosphate-buffered saline (PBS), Gentest High Viability CryoHepatocyte Recovery kit, hepatocyte culture medium, ITS+ culture supplement, Dulbecco’s Modification Eagle’s Medium (DMEM), Minimal Essential Medium (MEM) non-essential amino acid solution, epidermal growth factor (EGF), and Hank’s balanced salt solution (HBSS) were purchased from Corning (Corning, NY, USA). Penicillin/streptomycin, William’s E medium (without phenol red), fetal bovine serum (FBS), l-glutamine, gentamycin sulfate, SYBR Green ER qPCR Supermix, and Superscript First-strand Synthesis kit were purchased from Life Technologies (Burlington, ON, Canada). Sodium butyrate solution and testosterone were purchased from EMD-Millipore (Darmstadt, Germany) and Acros (Geel, Belgium), respectively. Easy Spin Total RNA extraction kit was purchased from iNtRON (Seongnam, Korea).
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2

Establishing Human Stem Cell Lines

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hESCs (BG01 hESCs, WiCell, WI, USA) were stably maintained using feeder-free cell culture systems. In brief, hESCs were cultured on the top of Matrigel (Becton Dickinson, NJ, USA)-coated cell culture dishes and fed with fresh mTESR1 (Stemcell Technology, Vancouver, BC, Canada). hESCs were regularly passaged every three or four days using TryPLE (Thermo Fisher Scientific, Waltham, MA, USA) reagent. Wilson’s disease induced pluripotent stem cells (iPSCs) generated from patient fibroblast were kindly provided by Dr. Yong-Mahn Han from Korea Advanced Institute of Science and Technology (KAIST). Primary human hepatocytes (PHHs, Lot No.303-Caucasian, BD Biosciences, Franklin Lakes, NJ, USA) were cultured on the top of collagen-I coated dishes and maintained using Hepatocyte Culture Medium (Corning Inc., Corning, NY, USA).
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