Immunofluorescence analysis was performed on the Hepa-RG cell line, seeded into sterile chamber slides (Thermo-Fisher) at a density of 1 × 104 cells/well and treated as reported in the Supporting Information using exosomes derived from 20 patients affected by NAFLD and 10 healthy subjects.
Heparg
Manufactured by Thermo Fisher Scientific
Sourced in United States
The HepaRG is a human-derived liver cell line that can differentiate into hepatocyte-like and biliary-like cells. It is used as an in vitro model system for various applications, including drug metabolism and toxicology studies.
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Lab products found in correlation
Penicillin, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Heparg, by Merck Group (3 mentions)
Streptomycin, by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Merck Group (3 mentions)
Nk 92, by American Type Culture Collection (2 mentions)
Mirneasy mini kit, by Qiagen (2 mentions)
Sk hep1, by American Type Culture Collection (2 mentions)
Penicillin, by Merck Group (2 mentions)
F7524, by Merck Group (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Hydrocortisone hemisuccinate, by Merck Group (2 mentions)
Caco 2, by Merck Group (2 mentions)
Mcf 7, by Merck Group (2 mentions)
Penicillin g, by Merck Group (2 mentions)
Amphotericin b, by Merck Group (2 mentions)
Insulin, by Merck Group (2 mentions)
Mcf 7, by American Type Culture Collection (2 mentions)
Lncap, by American Type Culture Collection (2 mentions)
22 protocols using heparg
1
Exosome-mediated Immunofluorescence in NAFLD
2
Cultivation of Hepatic Cell Lines and Primary Hepatocytes
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Human HCC HepG2 (cat #HB-8065), SK-Hep1(cat #HTB-52), and Hep3B (Cat #HB-8064) cells were obtained from American Type Culture Collection (ATCC). HCC cells were grown in DMEM medium (Invitrogen, Carlsbad, CA, USA) supplemented with 5% Fetal Bovine Serum (FBS, Access Biologicals, Vista, CA, USA) and 50 U/mL penicillin/streptomycin. HepaRG, a human bipotent progenitor cell line capable of differentiating into 2 different cell phenotypes (i.e., biliary-like and hepatocyte-like cells) [38 (link)], was obtained from ThermoFisher Scientific (Waltham, MA, USA). The terminally differentiated HepaRG cells (Cat #HPRGC10) and media ingredients were obtained from ThermoFisher Scientific (Waltham, MA, USA). As per the manufacturer’s instructions, specific thawing and plating media (Cat #HPRG770) were used, and cells were expanded in William’s E Medium (Cat #12551032) supplemented with 1% GlutaMax (Cat #35050061). Human cryopreserved hepatocytes (HPCH05+) and hepatocytes thawing and plating medium were obtained from Xenotech (Kansas City, KS, USA). Cells were incubated at 37 °C in a cell culture incubator supplied with 5% CO2 and used in experiments when they reached 70–80% of the confluence level.
3
Immortalized Cell Lines: Myocardial and Hepatic
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Myoblast (heart, myocardium) immortalized cell line (H9C2(2-1); code CRL-1446™) was provided as a frozen vial by ATCC. The cells were cultured in their specific media (DMEM). Hepatic immortalized cell line (HepaRG; code HPRGC10) was purchased from ThermoFisher Scientific (Gibco Waltham, MA, USA) and cultured in William’s E Medium enriched with insulin and hydrocortisone 21-hemisuccinate sodium salt at final concentrations of 4 µg/mL and 50 µM, respectively. Both media contained a 10% FCS and 1% penicillin (100 U/mL)/streptomycin (100 µg/mL) mixture. The cells were kept in an incubator at 37 °C and 5% CO2 during the experiments.
4
Differentiation and Senescence of HepaRG Cells
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The human cell line HepaRG was purchased by Merck Millipore (MMHPR116; Merck KGaA, Darmstadt, Germany). Undifferentiated HepaRG cells exhibit a fibroblast-like morphology, and the differentiation process induces both hepatocyte- and biliary-like epithelial phenotypes at confluence, indicating bipotent progenitor features[11 (link),18 (link)]. HepaRG cells were seeded at 27000 cell/cm2 confluence in a base medium composed by William’s E Medium + GlutaMAX (3255-020; Thermo Fisher Scientific, Waltham, MA, United States) supplemented with 10% fetal bovine serum (F7524; Merck KGaA), 100 U/mL penicillin (13752; Merck KGaA), and 100 μg/mL streptomycin (P4333; Merck KGaA). Medium was changed twice a week and cells were passaged once every 7 d. Cells in passage 10 (P10, young cells) and P20 (senescent cells) were used for assays and compared. To obtain HepaRG differentiation, a two-step procedure was used as previously described. Cells were cultured in the medium for 2 wk and then in the presence of 2% DMSO for an additional 2 wk[11 (link)].
5
Differentiation of HepaRG Cells for Hepatotoxicity Studies
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Human hepatic stem cell line HepaRG is the most suitable cell line for studying drug-induced hepatitis because of the richer CYP450 proteins in HepaRG than L-02, HepG2 and hiHeps cell lines 46 (link), 47 (link). HepaRG was obtained from ThermoFisher Scientific (#HPRGC10). The cells were seeded at 1 × 105 undifferentiated cells/cm2 in Hepatocyte Wash Medium (ThermoFisher Scientific, #17704024) containing additives for growth (Gibco). Then the cells were cultured at 37 °C with 21% O2 and 5% CO2 for 14 days before differentiation. The medium was renewed every 3 days. Cell differentiation was induced as described 48 (link). Briefly, the growth medium was composed of William's E medium supplemented with 10% FBS, 100 units/mL penicillin, 100 μg/mL streptomycin, 5 μg/mL insulin, and 50 μM hydrocortisone hemisuccinate. For the routine differentiation process, a two-step procedure was used. Cells were first maintained in the growth medium for 2 weeks, then maintained in the differentiation medium (supplemented with 2% DMSO) for 2 more weeks. The cells were maintained up to 4 weeks after differentiation for use. As for the mouse macrophage RAW264.7 cells, they were cultured in DMEM-based complete medium.
6
Hepatocyte Differentiation and Viability Assay
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All chemicals were purchased from Sigma-Aldrich (St. Louis, Missouri, USA) and were of the highest-purity grade available. The chemicals were dissolved in dimethyl sulfoxide (DMSO) at appropriate concentrations before use. The human hepatoma cell line, HepaRG™ was purchased from Thermo Fisher Scientific (Waltham, Massachusetts, USA). The frozen cells were thawed, seeded directly, and were maintained in Williams' E medium (Sigma-Aldrich, St. Louis, Missouri, USA) supplemented with 10% FetalClone™ II serum (HycloneTM, GE Healthcare, Chicago, Illinois, USA), 1 × L-glutamine, 5 μg/mL human insulin, and 50 μM hydrocortisone hemisuccinate without antibiotics for 2 weeks. Next, the medium was replaced with the aforementioned medium containing 2% DMSO for 2 more weeks to induce differentiation into hepatocytes. The cells were then cultured in a humidified atmosphere of 5% CO2 at 37°C. The cell viability was assessed using para-nitrophenylphosphate (PNPP) as previously reported to measure their cellular acid phosphatase (ACP) activity (19 (link)).
7
Immortalized Hepatocyte-like Cell Lines for HBV Studies
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An immortalized hepatocyte-like cell line (imHC) [39 (link),40 (link)], HepG2 cell line (ATCC, Manassas, VA, USA) and HepaRG (Thermo Fisher Scientific, Waltham, MA, USA) were used. All cell lines were cultured in DMEM/F12 (Hyclone), 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin at 37 °C, 5% CO2. Prior to hepatitis B virus (HBV) infection, 1 × 106 cells were seeded onto each well of a 6-well plate overnight. The quiescent stage was achieved in differentiated medium [42 (link)] (Williams’ E medium, 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 5 µg/mL insulin, 50 µM hydrocotisone, 2 mM L-glutamine (GlutaMax, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and 2% DMSO (Sigma Corporation of American, Ronkonkoma, NY, USA) for 2 weeks.
8
Western Blot Analysis of Hepatocyte Proteins
9
Hepatocyte Isolation and EGR1 Promoter Analysis
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Human hepatoma cells (HepaRG, Thermo Fisher, San Jose, CA, USA) were maintained in DMEM supplemented with 10% FBS (Hyclone, Logan, UT, USA). Primary murine hepatocytes were isolated as previously described24 ,25 and seeded at 2 × 105 cells/well for 12-well culture dishes, or 4 × 105 cells/well for 6-well culture dishes, or 4 × 106 cells/well for p100 culture dishes. Cell viability was examined at the time of seeding by trypan blue staining; typical isolation yielded >95% viability. EGR1 promoter-luciferase construct was generated by amplifying genomic DNA spanning the proximal promoter and the first exon of EGR1 gene (-900/+50) and ligating into a pGL3-basic vector (Promega, Madison, WI, USA). Truncation mutants were made using a QuikChange kit (Thermo Fisher Scientific, Waltham, MA, USA) and verified by direct sequencing. Small interfering RNAs (siRNAs) were purchased from Dharmacon (Lafayette, CO, USA). Transient transfections were performed with Lipofectamine 2000 (Thermo Fisher Scientific). Luciferase activities were assayed 24–48 h after transfection using a luciferase reporter assay system (Promega) as previously described.26
10
Hepatocyte Cell Culture for HBV Research
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imHC was maintained in 1:1 DMEM/F12 (Hyclone). imHC could harbor the entire HBV life cycle22 (link). HepaRG (Thermo Fisher Scientific, Waltham, MA, USA), Huh7 and HepG2 (ATCC, Manassas, VA, USA) were maintained in DMEM/F12 (Hyclone). HeLa cell was maintained in DMEM (Hyclone). All media were supplemented with 10% FBS (Hyclone), 100 U/mL penicillin and 100 µg/mL streptomycin (Invitrogen, USA) at 37 °C with 5% CO2. Prior to HBV infection, imHCs and HepaRG were differentiated with 2% DMSO (Sigma, NY) in Williams’ E medium, 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 5 μg/mL insulin, 50 μM hydrocortisone, 2 mM l -glutamine (GlutaMax, Gibco, Thermo Fisher Scientific, MA) for 2 weeks.
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