I. Principle
HepG2 is a human hepatocellular carcinoma cell line with hepatocyte-like features. Under excess free fatty acid (FFA) exposure, intracellular triglycerides (TG) markedly accumulate and abundant lipid droplets form, phenocopying hepatocyte steatosis in morphology and lipid metabolism. Thus, HepG2 is widely used as an in-vitro model for NAFLD and related studies. Induction typically uses a combination of oleic acid (OA) and palmitic acid (PA): OA more readily promotes TG synthesis and lipid-droplet formation with relatively lower toxicity, whereas PA is more lipotoxic and better mimics cellular injury and inflammatory responses under lipid overload. Using both at defined ratios (e.g., 0.5 mM OA + 0.25 mM PA) yields robust droplet formation while maintaining viability and lipotoxic effects, better reflecting in-vivo high-fat exposure.
II. Materials
1.Cells
HepG2 human hepatocellular carcinoma cell line.
2.Reagents
DMEM + 10% FBS for routine culture;0.25% trypsin–EDTA for dissociation/passaging;FFAs (e.g., sodium palmitate, sodium oleate);PBS for washes;4% paraformaldehyde for fixation;Nile Red solution, Oil Red O solution for lipid staining;DAPI for nuclear staining;ALT/AST assay kits and TG assay kit for biochemical readouts.
3.Equipment
CO₂ incubator (37℃, 5% CO₂); biosafety cabinet; centrifuge; microscope; microplate reader; culture plates and disposables.
III. Procedures
1.Cell culture
Seed HepG2 in DMEM + 10% FBS and culture at 37℃, 5% CO₂. Passage every 2–3 days using 0.25% trypsin–EDTA. Use cells in logarithmic growth for experiments.
2.Preparing FFA solutions
Follow the IFU to add the supplement to complete medium (10% FBS) to the desired FFA working concentration. A common combo is 0.5 mM sodium oleate + 0.25 mM sodium palmitate .
3.Inducing steatosis
When HepG2 reach ~80%–100% confluence, replace medium with fresh complete medium (10% FBS, 1% pen/strep). Add the high-fat supplement at the preset concentration; add equal volumes of vehicle to controls. Add gently and mix by gentle rocking to avoid local high concentrations. Incubate in the CO₂ incubator; 24 h is a typical starting condition. Before formal runs, perform pilot gradients of concentration and time to determine optimal FFA dosing and exposure for your HepG2 batch. Common induction times: 24/48/72 h, selecting conditions based on droplet formation, morphology, and viability.
4.Readouts
(1) Biochemical assays
Measure ALT/AST in supernatants or lysates per kit IFUs; measure TG per kit to assess lipid accumulation and metabolic changes.
(2) Staining and imaging
Rinse with PBS, fix with 4% paraformaldehyde at RT ~10 min, rinse with PBS, stain with Nile Red protected from light ~10 min, then counterstain nuclei with DAPI; alternatively use Oil Red O per standard protocol with appropriate washes and nuclear counterstain. Observe lipid droplets and distribution under a microscope and compare control vs high-fat groups to evaluate model success and steatosis severity.
IV. FAQ
Q1: Must I use serum-containing medium during induction?
A: Literature and experience support adding OA/PA in complete medium with 10% FBS to balance viability with robust droplet accumulation—this is a common, stable condition. Serum-free induction can alter lipid uptake and stress responses and may enhance lipotoxicity; it requires re-optimization and safety assessment. For most routine pharmacology/mechanism studies, serum-containing medium yields more reproducible results.
Q2: Massive HepG2 death or detachment after FFA addition—why?
A: Common causes: FFA too high, improper addition causing local spikes, overlong exposure, or poor initial cell status. Troubleshoot by lowering total FFA or adjusting OA/PA ratio (reducing PA often mitigates lipotoxicity); premix FFA thoroughly with complete medium before gently adding to plates; shorten induction (start at 24 h, extend as needed); ensure cells are healthy pre-induction (avoid over-confluence or over-aged cultures).
Aladdin: https://www.aladdinsci.com/