DESCRIPTION
The immortalized hepatocyte cell line (designated Fa2N-4)was isolated from normal human hepatocytes transformed with SV40 virus large T-antigen. The cell line is homozygous for the wild type alleles (*1/*1) of CYP2D6, CYP2C9 and CYP2C19, three cytochrome P450 enzymes that show a high degree of genetic polymorphism. Fa2N-4 hepatocytes attach to collagen and adopt the well-defined, cuboidal shape that is characteristic of normal hepatocytes. The morphology of these immortalized cells in culture closely resembles that of primary cultures of human hepatocytes, as shown in the following figure.


ENZYME INDUCTION STUDIES
Studies conducted at XenoTech and two major pharmaceutical companies have established that cytochrome P450 enzymes – including CYP1A2, 2B6, 2C9 and 3A4 – are inducible in the immortalized hepatocytes, which distinguishes the Fa2N-4 cells from other hepatic cell lines. The immortalized hepatocytes express sufficient cytochrome P450 for enzyme induction to be assessed based on measurements of enzymatic activity, as well as mRNA levels. These immortalized human hepatocytes demonstrate acceptable performance as a test system for in vitro drug induction studies, as defined in the September 2006 FDA Draft Guidance (PDF).

TOXICITY STUDIES
Fa2N-4 cells provide a human hepatocyte-derived system for conducting cell-based toxicity assays in vitro. The following figure illustrates the use of Fa2N-4 cells in toxicity testing. Treatment of cells with toxic concentrations (up to 100 mM) of several agents (namely 3-methylcholanthrene, methotrexate, menadione, rotenone and troglitazone) caused a loss of membrane integrity, resulting in the release into the medium of an intracellular enzyme, namely alpha-glutathione S-transferase (a-GST), which was measured with Biotrin High Sensitivity Alpha GST EIA (Biotrin International, Dublin, Ireland). In contrast, little or no a-GST was released from Fa2N-4 cells treated with non-toxic concentrations of omeprazole, acetaminophen, probenecid, felbamate or rifampin. It should be noted that some of these agents, such as acetaminophen, cause clinically significant liver toxicity, but only at high doses (and hence at much higher concentrations than those used in the study depicted in the following figure).

Utility of Fa2N-4 cells in differentiating toxicants from non-toxicants (measuring release of a-GST into media following 72-hour exposure to compounds)

PACKAGING

Immortalized Human Hepatocytes are available in the following formats: 1.5mL cryopreserved vials

These products are intended for one-time use only. A shrink wrap agreement (PDF) will be provided with each shipment and by breaking the seal on the package you are agreeing to the terms of the agreement.

CHARACTERIZATION PROVIDED

Induction data from a representative lot of Fa2N-4 cells (plated from cryopreserved vial)

Enzyme	Prototypical Inducer	Marker Substrate Reaction	Fold Induction*
CYP1A2	Omeprazole (100 µM)	Phenacetin O-deethylation	19.0
CYP2B6	Phenobarbital (750 µM)	Bupropion hydroxylation	1.61
CYP2C9	Rifampin (10 µM)	Diclofenac 4'-hydroxylation	2.53
CYP3A4/5	Rifampin (10 µM)	Atorvastatin hydroxylation	16.3

* Refers to the increase in pmol metabolite formed when compared to control.

We recommend using MFE Essential Plating, Support and Culture Medium Supplement A to obtain best results with the immortalized cells.