Hepatocyte cell morphology
When thawed and plated as a monolayer, Def-HEP cells form hepatocytes with characteristic cobblestone morphology and tight cell junctions.
Figure 1. Overview of Def-HEP cell morphology. Def-HEP WT cells exhibit typical hepatocyte cobblestone morphology and bi-nucleation.
Hepatocyte maturation markers
QPCR analysis shows Def-HEP cells show key hepatocyte markers at similar levels to PHH. Functional characteristics including albumin secretion, A1AT production, glycogen storage and LDL uptake are also present.
Figure 2. Functional analysis of Def-HEP WT hepatocytes. (A) Albumin secretion, 10x magnification (B) Glycogen storage shown by PAS staining (C) LDL cholesterol uptake shown by fluoresceinated LDL incorporation.
Figure 3. Gene expression analysis demonstrates that Def-HEP express key hepatocyte markers at similar levels to PHH. AFP levels are extremely low in Def-HEP indicating the cells have attained a functional mature status.
Extended culture time
Figure 4. Cryopreserved Def-HEP cells are thawed, plated, and recovered over 7 days to ensure a functional hepatocyte monolayer forms. Subsequently the cells have a +20 day functional window to enable hepatitis, disease modelling, and toxicology studies to be undertaken over a longer timeframe than is possible with PHH.
Multiple Inducible CYP450 Activities
Def-HEP cells display CYP450 induced activity profiles that are similar to PHH (CYP1a2 EROD assay, inducer – omeprazole), (CYP3A4 PGlo assay, inducer – rifampicin).
Figure 5. Multiple CYP activities of cryopreserved Def-HEP hepatocyte cells. The results show Def-HEP cells have comparable CYP activity to PHH and induced activity profiles that are highly similar to PHH (CYP1a2 EROD assay, inducer – omeprazole), (CYP3A4 PGlo assay, inducer – rifampicin).
Hepatitis marker analysis
Figure 6. Gene expression analysis demonstrates that multiple batches of Def-HEP cells reproducibly express key hepatitis markers including CD81, SR-B1, Claudin-1 and Occludin. The levels of markers observed are similar to PHH (Hep G2 cells do not express all key hepatitis markers)
Metabolism – Absence Of Crabtree Effect
Dosing Def-HEP WT with +/- glucose media does not alter the hepatotoxic effect of chlorpromazine as assessed via the MTT cell proliferation assay. Therefore, it is highly unlikely that our cells exhibit the Crabtree effect, in contrast to immortalized lines.
Figure 7. Confirmation of absence of the Crabtree effect in Def-HEP WT.
As assessed by dosing Def-HEP WT with valinomycin (0.3-600nM) and papverine (0.03 – 60µM) for 48 hrs both compounds caused significant mitochondrial toxicity with concentration ranges recognized in literature. Cell viability was assessed via the CellTiter 96® Aqueous Non-Radioactive Cell. Both valinomycin and papaverine caused significant mitochondrial toxicity
Figure 8. Typical Def-HEP WT mitochondrial toxicology drug response to valinomycin and papverine.
Brochure: Definigen Hepatocytes Wild Type
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