William s e medium

Primary human hepatocytes were isolated from samples taken from the specimen of patients undergoing major hepatectomy at the Department of General, Visceral, and Transplantation Surgery of Heidelberg University Hospital. All patients were screened for eligibility irrespective of the indication for major hepatectomy and included provided they have signed the valid informed consent form. Informed consent of the patients for the use of tissue for research purposes was obtained corresponding to the ethical guidelines of University Hospital Heidelberg (reference number S-557/2017). A non-tumor tissue sample with intact Glisson’s capsule, weighing approximately 20 g was collected and immediately transported in William’s E medium (PAN Biotech) to the laboratory for further processing.

Primary hepatocytes were isolated from C57BL/6NHsd male mice via collagenase perfusion as described previously (61 (link), 62 (link), 63 (link), 64 (link)). Cells were plated onto collagen (0.9 mg/ml) coated 24-well plates at 200,000 cells/well in Williams E medium (PAN Biotech), substituted with 10% FBS, 100 nM dexamethasone, and penicillin/streptomycin and maintained at 37°C in an atmosphere with 5% CO₂. After 4 h of attachment, cells were washed with PBS and allowed to rest in dexamethasone-free medium overnight before treatment.

Human cell lines A549, Caco2, HCT116, HEK 293, Hela, HepaRG, Hs 578T, Huh7, MDA-MB-231, MCF7, SK-BR-3, T84 and Chinese hamster ovary cells CHO-CAR, CHO-CD46, and CHO control cells were cultured in high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM, PAN-Biotech, Aidenbach, Germany). Huh7 cells and all three CHO cells were supplemented with non-essential amino acids (PAN-Biotech, Aidenbach, Germany). CHO-CAR and CHO-CD46 cells were selected with 50 µg/mL G418. HepG2 cells were cultured in RPMI 1640 (PAN-Biotech, Aidenbach, Germany). HepaRG cells were cultured in William’s E Medium (PAN-Biotech, Aidenbach, Germany). Breast cancer cell line SK-BR-3 was cultured in McCoy’s 5A Medium (PAN-Biotech, Aidenbach, Germany). All the above media were supplemented with 10% (except 20% for CaCo2) FBS, (GE Healthcare, Solingen, Germany), 100 units per mL (U/mL) penicillin, and 100 µg/mL streptomycin (PAN-Biotech, Aidenbach, Germany). All cells were maintained in a humidified atmosphere at 37 °C and 5% CO₂.

Human hepatocellular carcinoma (HepG2) (ATCC, HB-8065) were maintained in DMEM (4.5 g/l glucose, pyruvate) (Gibco), supplemented with 10% fetal bovine serum (PAA Laboratories) and penicillin/streptomycin (25 μg/ml each, Gibco) at 37 °C, 5% CO₂. Cells were seeded at a density of 60,000 cells/cm² and allowed to proliferate for 3 days. Following medium change, cells were treated as indicated. Cells were routinely (6 times per year) tested for mycoplasma contamination.
Primary human hepatocytes (PHH) were obtained from BioIVT (donors IAN, IPH, GID) and from Lonza (donors HUM4108, HUM4055B, HUM4229, HUM181501B). Cells were seeded on collagen-coated plates (250 μg/ml rat collagen, Roche) at a density of 150,000 cells/cm² and cultivated for 3 h in William’s E medium (PAN Biotech) containing penicillin (100 U/ml, Gibco), streptomycin (0.1 mg/ml, Gibco), gentamycin (10 μM, PAN Biotech), dexamethasone (100 nM, Sigma), stable l-glutamine (2 mM, Sigma), insulin supplement (2 ng/ml, Sigma), and 10% Sera Plus (PAN Biotech). After 3 h for cell attachment, medium was exchanged to Sera Plus-free medium and cells were allowed to adjust for 16 h before treatment.

Primary hepatocytes were isolated from 3- to 4-month-old C57BL/6 mice as described previously [25 (link)]. Primary hepatocytes were kept in culture medium (DMEM; PAN Biotech, Aidenbach, Germany) supplemented with 10% FBS, 1% penicillin-streptomycin, and l-glutamine (PAN Biotech). After overnight incubation, the medium was replaced by William’s E medium (PAN Biotech), and cells were treated for 6 h with IL-1β (20 ng/ml; Peprotech, Hamburg, Germany), IL-6 (40 ng/ml; Sigma, St. Louis, MO, USA), TNFα (30 ng/ml; Peprotech), and the HNF4α antagonist BI6015 (50 μM; Millipore, Burlington, MA, USA) or for 24 h with TGFβ1 (10 ng/ml; R&D Systems, Minneapolis, MI, USA). Alternatively, the cells were transfected with the siRNA against HNF4α (60 pmol; Thermo Fisher Scientific ID: 158081 and 158082, Germany) according to the manufacturer’s protocol (Lipofectamine® RNAiMAX™ Transfection Reagent, Thermo Fisher Scientific, Germany) and cultured for 48 h. RNA was isolated via RNeasy tissue mini isolation kit (Qiagen, Hilden, Germany). The RNA samples were translated to cDNA with the M-MLV reverse transcriptase kit (Promega, Madison, WI, USA) and random hexamers (Thermo Scientific, Waltham, MA, USA). The relative expression of genes of interest was determined using specific primers (Additional file 1: Table S1). The mouse ribosomal gene L7 was used as an internal loading control.

Primary hepatocytes were isolated from male 8–12-wk-old mice according to the well-established collagenase perfusion protocol (Klingmüller et al., 2006 (link)). They were lysed immediately for RNA isolation or cultured in a collagen sandwich in 24-well plates (200,000 cells/well) in William’s E medium (Pan Biotech; cat. no. P04-29150) supplemented with 10% FBS, 100 nM dexamethasone (Sigma-Aldrich; cat. no D1756-25MG), and penicillin/streptomycin until they polarized (Zeigerer et al., 2017 (link)). The polarized hepatocytes were fixed with 4% PFA for 30 min.

Hepatocytes were isolated from male LRep1−/− and LRep1+/+ mice at an age of 6 weeks as described previously^{55 (link)}. Cells were plated on collagen A (200 µg/ml in PBS; Biochrom, Berlin, Germany) coated cell culture dishes with Williams E medium (PAN Biotech, Aidenbach, Germany) containing L-glutamine, 10% (v/v) FCS (for the first 4 hours, subsequently FCS free) and 100 nM dexamethasone (Sigma Aldrich) and incubated in a 5% CO₂ humidified atmosphere at 37 °C.
The human hepatocellular carcinoma cell line HepG2 was cultured in Dulbecco’s modified Eagle’s medium (DMEM, high glucose) supplemented with 10% (v/v) FCS at 37 °C in 5% CO₂.