Primary hepatocyte maintenance supplements

This study evaluated mRNA and miRNA expression data collected in a previously published human hepatocyte experiment.5, 6 In brief, freshly isolated human hepatocytes from 7 different donors were obtained from CellzDirect (Durham, NC) and were plated on 12‐well collagen‐coated plates cultured in Williams’ E medium without phenol red containing Primary Hepatocyte Maintenance Supplements (Life Technologies Corporation, Carlsbad, CA). Cultures from each donor were considered biological replicates (n = 7). All studies were performed within 72 and 120 hours following the time of hepatocyte isolation. Hepatocytes were treated with rifampin (10 μmol/L) or corresponding vehicle control (0.1% methanol) for 24 hours. The commercially obtained human hepatocytes were deidentified and specific demographic and/or clinical information were not available from the supplier.

CD235a⁻ liver cells were plated in William's E Medium (Thermo Fisher Scientific, Grand Island, NY) with 5% FBS and Primary Hepatocyte Maintenance Supplements (#CM4000, Thermo Fisher Scientific) providing 0.1 µM dexamethasone, 6.25 µg ml⁻¹ human recombinant insulin, 6.25 µg ml⁻¹ human transferrin, 6.25 ng ml⁻¹ selenous acid, 1.25 mg ml⁻¹ bovine serum albumin, 5.35 µg ml⁻¹ linoleic acid, 2 mM GlutaMAX and 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. Cultures were also supplemented with 10 ng ml⁻¹ oncostatin M (OSM) and 10 ng ml⁻¹ epidermal growth factor (EGF). Cells were grown on BioCoat Collagen I coated tissue culture plates (BD, Franklin Lakes, NJ) for 6 days at 37°C. Confluent cultures were harvested using TrypLE Enzyme solution (Life Technologies) and then stained for flow cytometric analysis or transplantation into mice. Some plates were fixed with 10% formalin in PBS and stained for immunofluorescence microscopy analysis.

iHepSCs were generated as previously described [13 (link)]. Briefly, mouse embryonic fibroblasts (MEFs) were isolated from C57BL/6J mice E13.5 (Hyochang Science, Daegu, Korea). We plated 1.0 × 10⁴ MEFs (passage 2–3) on a 0.1% gelatin-coated 12-well plate and cultured them at 37 °C with 5% CO₂ in an MEF medium consisting of Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Thermo Fisher Scientific, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Grand Island, NY, USA), 1% penicillin/streptomycin, L-glutamine, 2-mercaptoethanol and MEM non-essential amino acids (Thermo Fisher Scientific, Massachusetts, USA). The next day, the cells were cultured in an MEF medium containing 6 μg/mL of protamine sulfate. At 30 min after protamine sulfate treatment, MEF cells were transduced with lentivirus-encoding Oct4 and Hnf4α in an MEF medium. After three days, the MEF medium containing the lentivirus was switched to the hepatic culture medium (HEP medium) that consisted of William’s E Medium (Gibco, Thermo Fisher Scientific, Grand Island, NY, USA) supplemented with Primary Hepatocyte Maintenance Supplements (Gibco, Thermo Fisher Scientific, Grand Island, NY, USA). The culture medium was changed every 2 days.

Cell samples were harvested and prepared from two healthy donors and two NASH patients. The cell samples were provided by Viscient Biosciences Inc. Viscient Biosciences Inc. provides assurances that the cells come from tissues collected in compliance with applicable laws and provided based on informed consent by the donors. Frozen vials of HSC, LEC cells, and hepatocytes were thawed in a 37 degree Celsius water bath. HSC and LEC cells were then transferred to 5 ml of their respective media—HSC in DMEM+10% FBS, LEC cells in EGM2 media from Lonza (Catalog# CC-3162). HSC and LEC cells were then spun at 200g for 5 minutes. The supernatant was aspirated, and the pellet was resuspended in 1ml of media. Hepatocytes were thawed in Gibco hepatocyte thaw media (Catalog number: CM7500) per the manufacturer's instructions. They were then transferred to 2 ml of Williams E maintenance medium with no phenol red (Catalog number: A1217601, Thermo Fisher). Gibco Williams E basal media was supplemented with primary hepatocyte maintenance supplements (Catalog number# CM4000) to make complete maintenance media used for tissue maintenance.

Each liver was perfused with 10 ml of HBSS (without Ca²⁺ or Mg⁺) containing 0.5 mM EDTA, 4 ml of HBSS (without Ca²⁺ or Mg⁺) and 10 ml of collagenase solution [2 mg/ml collagenase H (11074059001; Roche), 0.1 mg/ml DNaseI (10104159001; Roche), 5 mM CaCl₂ and 0.9 mM MgCl in HBSS] sequentially. All the solutions were pre-warmed to 37 °C before use. Livers were dissected and further incubated in 7 ml of collagenase solution at 37 °C for 5 min and dissociated. Dissociated cells were passed through a cell strainer and centrifuged at 50 g for 5 min to precipitate hepatocytes. For qRT-PCR, the pellet was immediately dissolved in Trizol for the hepatocyte fraction. The supernatant containing NPCs was washed with PBS at 50 g for 5 min three times to eliminate hepatocytes, and precipitated at 400 g for 5 min, and dissolved in Trizol, and used as NPC fraction. For hepatocyte culture, the hepatocyte pellet after the first centrifugation was resuspended and washed with PBS at 50 g for 5 min three times to eliminate NPCs, resuspended in the culture media [Williams’ Media E supplemented with Primary Hepatocyte Maintenance Supplements (CM4000; Gibco) as instructed] and seeded on collagen-coated plates.

Each liver was perfused with 10 ml of HBSS (without Ca²⁺ or Mg⁺) containing 0.5 mM EDTA, 4 ml of HBSS (without Ca²⁺ or Mg⁺) and 10 ml of collagenase solution [2 mg/ml collagenase H (11074059001; Roche), 0.1 mg/ml DNaseI (10104159001; Roche), 5 mM CaCl₂ and 0.9 mM MgCl in HBSS] sequentially. All the solutions were pre-warmed to 37 °C before use. Livers were dissected and further incubated in 7 ml of collagenase solution at 37 °C for 5 min and dissociated. Dissociated cells were passed through a cell strainer and centrifuged at 50 g for 5 min to precipitate hepatocytes. For qRT-PCR, the pellet was immediately dissolved in Trizol for the hepatocyte fraction. The supernatant containing NPCs was washed with PBS at 50 g for 5 min three times to eliminate hepatocytes, and precipitated at 400 g for 5 min, and dissolved in Trizol, and used as NPC fraction. For hepatocyte culture, the hepatocyte pellet after the first centrifugation was resuspended and washed with PBS at 50 g for 5 min three times to eliminate NPCs, resuspended in the culture media [Williams’ Media E supplemented with Primary Hepatocyte Maintenance Supplements (CM4000; Gibco) as instructed] and seeded on collagen-coated plates.