In vitro primary hepatocyte assay
MB Biosciences offer in vitro platforms based on primary hepatocytes and hepatic cell models to define glucose and lipid metabolism profiles and to screen potential therapeutic agents for metabolic disorders such as diabetes and obesity. As a leading biotechnology company specializing in hepatic cells, MB Bioscience provides a wide range of assays to evaluate drug efficacy, toxicity, and overall liver physiology using primary human, rat, and mouse hepatocytes. In addition to conventional 2D culture systems, we also offer advanced 3D hepatocyte cultures and microtissue platforms.
Cytotoxicity and Viability Assays
Cytotoxicity and viability assays with primary hepatocytes are widely used to evaluate the potential liver toxicity of new compounds. These assays measure the overall health and survival of hepatocytes after exposure to test substances, providing early insights into safety risks. We offer reliable and sensitive readouts including ATP quantification for cellular energy levels and mitochondrial activity assays (MTT/MTS), or LDH release to determine compound-induced cell stress, damage, or death.
MTT / MTS assay – measures mitochondrial activity.
ATP assay – measures cellular energy levels.
LDH release assay – detects cell membrane damage.
Drug Metabolism: CYP450 enzyme induction assays
CYP induction assays evaluate whether a compound increases expression or activity of cytochrome P450 enzymes (CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A4), is a key for drug–drug interaction risk assessment. Induction can reduce drug exposure in vivo by accelerating metabolism
Primary human hepatocytes (gold standard, donor variability assessed with ≥3 donors).
Use cryopreserved human hepatocytes, plated and allowed to recover.
Expose cells to test compound across a concentration range.
Include positive controls (known inducers: rifampicin for CYP3A4, omeprazole for CYP1A2, phenobarbital for CYP2B6).
Include negative/vehicle controls.
Maintain exposure over several days to allow induction response (conceptually multiple dosing may be used).
Readout measurements: mRNA expression: quantify CYP transcripts by qRT-PCR.
Liver-Specific Functions Assays In Vitro
Primary hepatocytes retain many of the essential metabolic and synthetic functions of the liver, making them the most physiologically relevant in vitro model. These functions serve as critical markers of hepatocyte health and activity.
•Albumin secretion assay tests the hepatocyte’s ability to synthesize and secrete albumin, Readout: albumin levels measured in the culture media by ELISA.
•ALT and AST leakage assays test for liver-specific enzyme release due to cell damage, and the readout is enzymatic activity of ALT or AST in culture media.
•Ammonia metabolism assay measures the hepatocyte’s ability to clear ammonia via the urea cycle or glutamine synthesis, with the readout being ammonia depletion and urea or glutamine formation in media.
•Bile acid synthesis and secretion assay measures production and transport of bile acids into canaliculi, and the readout is bile acid concentration in the culture media detected by colorimetric kits.
•Cholesterol and lipid metabolism assays examine hepatic regulation of lipid synthesis and storage, with the readout being intracellular lipid staining such as Oil Red O or cholesterol quantification assays.
•Glucose metabolism assays evaluate gluconeogenesis and glycolysis in hepatocytes, and the readout is glucose or lactate levels in media or isotope tracing data.
•Glucose production assay (Gluconeogenesis) tests the ability of hepatocytes to perform gluconeogenesis, that is, generating glucose from non-carbohydrate substrates such as lactate, glycerol, or amino acids. The readout is usually the amount of glucose released into the culture media, measured with enzymatic glucose assays or sometimes isotope-tracing methods to confirm substrate utilization.
•Glycogen synthesis and storage assay (Glycogenolysis) tests the ability of hepatocytes to take up glucose and store it in the form of glycogen. The readout is usually detection of intracellular glycogen, either by PAS staining under microscopy for visualization, or by biochemical quantification kits that measure glycogen levels enzymatically.
•Indocyanine Green (ICG) clearance assay models overall liver clearance function, and the readout is uptake and excretion of ICG dye measured spectrophotometrically.
•Lactate dehydrogenase (LDH) release assay evaluates hepatocyte membrane integrity and cytotoxicity, with the readout being LDH activity in the media detected by colorimetric methods.
•LDL uptake assay tests the ability of hepatocytes to internalize low-density lipoproteins (LDL) via LDL receptors, reflecting liver function in cholesterol clearance and lipoprotein metabolism. The readout is typically the amount of fluorescently labeled LDL (e.g., Dil-LDL) taken up by cells, measured by fluorescence microscopy or plate-based fluorometric assays.
•Lipids and phospholipids accumulation assay tests the hepatocyte’s ability to synthesize and store neutral lipids and phospholipids, which reflects lipid homeostasis and can indicate steatosis when excessive. The readout is usually intracellular lipid staining (such as Oil Red O, Nile Red, or BODIPY for neutral lipids; specific fluorescent probes for phospholipids) or quantitative measurement of lipid and phospholipid content by biochemical assays.
•Lipolysis assay tests the ability of hepatocytes (or hepatocyte–adipocyte co-cultures) to break down stored triglycerides into free fatty acids and glycerol. The readout is usually the amount of glycerol and free fatty acids released into the culture medium, measured using colorimetric or fluorometric biochemical kits.
•Lipid peroxidation assay tests the extent of oxidative damage to membrane lipids in hepatocytes, which reflects oxidative stress and potential liver injury. The readout is usually the detection of peroxidation by-products, most commonly malondialdehyde (MDA) or 4-hydroxynonenal (4-HNE), measured by colorimetric/fluorometric assays (e.g., TBARS assay).
•Mitochondrial function assays test hepatocyte energy metabolism and mitochondrial health, with the readout being ATP production assays or oxygen consumption rate measured by Seahorse analysis.
•Oxidative stress assays are used to evaluate the level of oxidative damage and the cellular redox balance between reactive oxygen species (ROS) and antioxidants. The readouts often include fluorescence intensity, absorbance, or luminescence signals that correlate with the amount of ROS or oxidative damage present.
•Urea synthesis assay evaluates the detoxification capacity of hepatocytes through the urea cycle, and the readout is urea concentration in the media determined by colorimetric or enzymatic methods.