Hepatocytes

Hepatocytes are liver epithelial cells used for both basic research and drug metabolism studies.

Fresh and cryopreserved primary hepatocytes contain all the major enzyme pathways for drug and xenobiotic biotransformation. These include the major phase I drug metabolism enzyme family (P450) and phase II enzymes (UGT, SULT, GST, and NAT). Hepatocytes also contain all the gene regulation pathways for P450 induction. Appropriate culture conditions are required to maintain hepatic P450 activity.

Hepatocytes can be cultured on collagen I1-4, BD Matrigel™ Matrix5-9 or BD™ PuraMatrix™10-11. BD BioCoat™ Collagen I Cultureware is a commonly used surface for cultures of both fresh and cryopreserved hepatocytes12-13 (Figure 1). Cells cultured on this surface maintain their biological activity, as shown by P450 induction (Figure 2). Sandwich cultures, such as hepatocytes grown on BD BioCoat Collagen I with BD Matrigel Matrix overlay, are used to assess bile canaliculi formation14. Choly-lysyl-fluorescein (CLF) is a fluorescein-labeled bile acid that is secreted into bile canaliculi by bile salt export pump (BSEP) which can be used to visualize bile canaliculi (Figure 3). BD Matrigel Matrix has been shown to suppress cell growth and prevent growth-associated dedifferentiation5, as well as maintain liver-specific functions in vitro longer than most collagen-based systems6-8. Hepatocytes cultured on BD Matrigel Matrix also have a more differentiated morphology than hepatocytes cultured on collagen I (Figure 4). Both BD Collagen I and BD Matrigel Matrix are animal-derived products; BD PuraMatrix, a synthetic peptide hydrogel, is a suitable alternative for assays that require a xeno-free culture environment. Therefore the appropriate culture surface depends on the experimental goals (e.g., drug metabolism, bile canaliculi formation or xeno-free environment).

Figure 1. BD Gentest Inducible-Qualified Human CryoHepatocytes Cultured on BD BioCoat Collagen I

Figure 1. BD Gentest Inducible-Qualified Human CryoHepatocytes Cultured on BD BioCoat Collagen I

BD Gentest™ Inducible-qualified human CryoHepatocytes were isolated using the BD Gentest CryoHepatocyte Purification Kit and resuspended in freshly prepared ISOMs seeding media at a concentration of 1x106 cells/ml. Cells were plated onto BD BioCoat Collagen I 24-well plates and incubated for approxiamately 2 hours, after which plating media was removed and replaced with supplemented BD™ Hepatocyte Culture Media.

Figure 2. Induction of BD Gentest Inducible-Qualified Human CryoHepatocytes

Figure 2. Induction of BD Gentest Inducible-Qualified Human CryoHepatocytes

BD Gentest™ Inducible-qualified Human CryoHepatocytes were isolated using the BD Gentest CryoHepatocyte Purification Kit and resuspended into freshly prepared ISOMs seeding media at a concentration of 1x106 cells/ml. Cells were plated onto BD BioCoat™ Collagen I 24-well Multiwell Plates and incubated for approximately 2 hours, after which plating media was removed and replaced with supplemented BD™ Hepatocyte Culture Media. Cells were monitored for degree of attachment at 18-24 hours after plating and daily during the experiment. Cells were induced with either 20 μM Rafampicin (A) or 20 μM β-Napthoflavone (B) over a 3-day period. Controls were treated with the appropriate solvent control. Metabolic activity was determined on day 5 of the experiment using 200 μM Testosterone as a substrate to measure CYP3A4 (left graph) activity and 100 μM Phenacetin as a substrate for CYP1A2 (right graph). Assays were run for 30 minutes and 60 minutes, respectively. Analysis was performed by HPLC and activity expressed per mg of protein.


 
Figure 3. BD Gentest Choly-LYSYL-Fluorescein Sequestered in Bile Canaliculi

Figure 3. BD Gentest Choly-LYSYL-Fluorescein Sequestered in Bile Canaliculi

CLF sequestered in the bile canaliculi of BD Gentest Inducible-qualified Human CryoHepatocytes cultured on BD BioCoat Collagen I overlaid with BD Matrigel Matrix.

Figure 4. Effects of ECM on Cell Morphology: Micrographs of Hepatocytes Cultured on Various Culture Substrata

Figure 4. Effects of ECM on Cell Morphology: Micrographs of Hepatocytes Cultured on Various Culture Substrata

Scanning electron micrographs of primary rat hepatocytes cultured for two days on collagen I (A), collagen I gel (B), or BD Matrigel Matrix (C). Note the clusters of spherical cells for hepatocytes cultured on BD Matrigel Matrix, typical of differentiated cells.

References

  1. Fahmi OA, Boldt S, Kish M, Obach RS, Tremaine LM. (2008) Prediction of drug-drug interactions from in vitro induction data: application of the relative induction score approach using cryopreserved human hepatocytes. Drug Metab Dispos. 36(9):1971.

  2. Healan-Greenberg C, Waring JF, Kempf DJ, Blomme EA, Tirona RG, Kim RB. (2008) A human immunodeficiency virus protease inhibitor is a novel functional inhibitor of human pregnane X receptor. Drug Metab Dispos. 36(3):500.

  3. Lee P, Peng H, Gelbart T, Beutler E. (2004) The IL-6- and lipopolysaccharide-induced transcription of hepcidin in HFE-, transferring receptor 2-, and β2 microglobulin-deficient hepatocytes. Proc Natl Acad Sci. 101(25):9263.

  4. DiPersio CM, Jackson DA, Zaret KS. (1991) The extracellular matrix coordinately modulates liver transcription factors and hepatocyte morphology. Mol Cell Biol. 11(9):4405.

  5. Rana B, Mischoulon D, Xie Y, Bucher NL, Farmer SR. (1994) Cellextracellular matrix interactions can regulate the switch between growth and differentiation in rat hepatocytes: reciprocal expression of C/EBP alpha and immediate-early growth response transcription factors. Mol Cell Biol. 14(9):5858.

  6. Schuetz EG, Li D, Omiecinski CJ, Muller-Eberhard U, Kleinman HK, Elswick B, Guzelian PS. (1988) Regulation of gene expression in adult rat hepatocytes cultured on a basement membrane matrix. J Cell Physiol. 134:309.

  7. Schuetz JD and Schuetz EG. (1993) Extracellular matrix regulation of multidrug resistance in primary monolayer cultures of adult rat hepatocytes. Cell Growth and Diff. 4:31.

  8. Mann DJ, Strain AJ, Bailey E. (1992) Hormonal induction of malic enzyme in rat hepatocytes cultured on laminin-rich gels. J Mol Endocrinol. 8(3):235.

  9. Kane RE, Tector J, Brems JJ, Li A, Kaminski D.(1991) Sulfation and glucuronidation of acetaminophen by cultured hepatocytes reproducing in vivo sex-differences in conjugation on Matrigel and type 1 collagen. In Vitro Cell Dev Biol. 27A:953.

  10. Wang S, Nagrath D, Chen PC, Berthiaume F, Yarmush ML. (2008) Three-dimensional primary hepatocyte culture in synthetic selfassembling peptide hydrogel. Tissue Eng. 14(2):227.

  11. Semino CE, Merok JR, Crane GG, Panagiotakos G, Zhang S. (2003) Functional differentiation of hepatocyte-like spheroid structures from putative liver progenitor cells in three-dimensional peptide scaffolds. Differentiation. 71(4-5):262.

  12. Dike LE, Haiyan X, Snodgrass BR, Patten CJ. (2006) Characteristics of replateable and inducible cryopreserved hepatocytes. Poster presented at the 14th North American ISSX Meeting, Rio Grande, PR, Poster No. 162.

  13. Weng Y, Stresser DM, Zhang JG. (2005) Characterization of CYP1A2, 2B6 and 3A4 induction in primary cultures of human hepatocytes by RT-PCR, Enzyme Activity and Western Blot Poster presented at the 8th International ISSX Meeting, Sendai, Japan.

  14. Bi YA, Kazolias D, Duignan DB. (2006) Use of cryopreserved human hepatocytes in sandwich culture to measure hepatobiliary transport. Drug Metab Dispos. 34(9):1658.

AlexaFluor is a trademark of Invitrogen Corporation.
CytoFlour is a trademark of Applied Biosystems.
MetaMorph is a registered trademark of Universal Imaging Corporation (UIC).
mTeSR1 and WiCell is the property of WiCell Research Institute.
PuraMatrix is a registered trademark of 3DM, Inc.
Victor2 is a trademark of PerkinElmer, Inc.


For Research Use Only. Not for use in diagnostic or therapeutic procedures.