Human Primary Hepatocytes

Product Description

The liver is a vital organ in mammals, and hepatocytes, the major cell type in liver, play critical roles in protein synthesis, detoxification of various metabolites, transformation of carbohydrates, and the production of biochemical necessary for digestion ^[1]. Primary hepatocyte culture is an important model for in vitro studies in drug development, including hepatotoxicity, drug transport, hepatitis virus infection, hepatic drug metabolism and hepatobiliary excretion ^[2].

iXCells Biotechnologies provides high quality human primary hepatocytes isolated from whole liver of human organ donors and cryopreserved on the day of isolation with ≥5 million viable cells in each vial. iXCells offers human hepatocytes in plateable & suspension categories and includes cells from both healthy and diseased tissue. Our cryoplateable primary human hepatocytes (CPHH) reach and maintain a confluent monolayer (>90% confluency) for 5-7 days (Fig 1), express general hepatocyte marker albumin, and show accumulation of lipid droplets (Fig 2). Each lot of human hepatocytes is QC tested & validated including key Cytochromes P450 (CYPs) enzyme activity and gene expression induction. They are negative for HIV-1/2, HBV, HCV, mycoplasma, bacteria, yeast, and fungi.

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Figure 1. Phase contrast images of CPHH. CPHH were plated in a 6-well culture dish precoated with Collagen type I and cultured for 5 days following iXCells protocol. CPHH reached and maintained monolayer with >90% confluency. Hepatocytes exhibited hexagonal shape with single or double nuclei and characteristic “chicken wire” like bile canaliculus formation between hepatocytes.

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Figure 2. Immunostaining and Oil Red O staining of cultured CPHH at D5. CPHH were plated in a 12-well culture dish and cultured for 5 days. (A) Albumin recognized by anti-human albumin antibody (green), and cytoskeleton marker F actin revealed by Alexa Fluor 555 Phalloidin (red), with cell nuclei counterstained by DAPI (blue). (B) Hepatocyte cytoskeletons were stained with anti-Cytokeratin 18 (CK18, green) antibody and intracellular lipid droplets were recognized by anti-ADRP/Perilipin 2 antibody (red). (C) Lipid droplet staining using Oil red O in CPHH.

P450 CYP Enzyme Induction

Table 1. Representative data of inducible hepatocytes. Cryopreserved human hepatocytes were thawed, plated in a collagen Type 1 coated 12-well culture plate (750,000 cells/well). The hepatocytes were incubated with appropriate inducers or vehicle control (0.1% DMSO) for 48 hours. For P450 CYP enzymatic activity induction, Luciferin-1A2 or Luciferin-IPA substrate was added to the plate and CYP1A2 or CYP3A4 enzymatic activities was detected using P450-Glo™ Assay (Promega, Cat. No. V8421; V9001). The fold changes of the drug induced P450 CYP enzyme activity over the basal level were calculated. For CYP gene expression induction, hepatocyte RNA was extracted after inducer treatment, and the mRNA expression levels of CYP1A2 and CYP3A4 were analyzed using TaqMan qRT-PCR.  The fold changes of the drug induced P450 CYP gene expression over the basal levels were calculated using theC_T method.

Product Details

References

[1] Schulze RJ, Schott MB, Casey CA, Tuma PL, McNiven MA. The cell biology of the hepatocyte: A membrane trafficking machine. J Cell Biol. 2019 Jul 1;218(7):2096-2112.
[2] Zeilinger K, Freyer N, Damm G, Seehofer D, Knöspel F. Cell sources for in vitro human liver cell culture models. Exp Biol Med(Maywood). 2016 Sep;241(15):1684-98.

Datasheet & Culture Protocol