Fa2N-4 Immortalized Human Hepatocytes
DESCRIPTION
PACKAGINGCHARACTERIZATION PROVIDEDSAFETY, HANDLING AND STORAGEPOSTERS
PRODUCTS 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.
Morphology of human hepatocytes (left) and FaN2-4 cells (right) at light microscopy level
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September 2006 FDA Draft Guidance (PDF).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 results of using 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)
Return to TopPACKAGING
Immortalized Human Hepatocytes are available in the following formats:
1.5 mL cryopreserved vials Freshly plated on 6, 12, 24 or 96 well plates 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. Return to Top
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.
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SAFETY, HANDLING AND STORAGE
Informed Consent Statement
A single organization regulates and oversees the use of human tissue intended for transplantation in the United States, namely the United Network for Organ Sharing (UNOS). Organ donors may elect to have their organs used either for transplantation only, or for transplantation or research. Thus, the donor (or the donor's family) has the right to prevent the use of the donor's organs for research. Regardless of the use of donated organs, no compensation is given to the donor’s family; any such compensation is illegal in the United States. In those cases where donors (or family members) elect to withhold organs from research uses, any organs that cannot be transplanted are discarded.
XenoTech receives hepatic, renal, intestinal, pulmonary, and other human tissue from various regional organ procurement organizations (OPOs) that obtain organs approved for research use. Regulations in the United States require that, regardless whether the organ is intended for transplantation or research purposes, the organ donor's identity be treated as highly confidential information. Organ procurement organizations maintain the informed consent records from each donor, and our Standard Operating Procedure requires that XenoTech personnel confirm the existence of informed consent for research purposes, prior to transport of organs to XenoTech. This procedure is intended to ensure that XenoTech manufactures human-derived products only when informed consent has been granted for research use of those specific organs. XenoTech does not, and, in consideration of confidentiality, cannot obtain the informed consent records from these organizations.
All human tissue accepted by XenoTech has been tested for the possible presence of various infectious diseases, and XenoTech does not accept human-derived material unless the donor has tested negative (non-reactive) for RPR, HIVAb, HTLVAb, HBsAg and HCVAb. All human tissue is also tested for CMVAb. However, due to the widespread (nearly ubiquitous) appearance of CMV in the population, and its relative insignificance as an infectious agent, tissues from donors reactive for CMVAb are accepted. The serology status of each donor is typically determined by ELISA by the organ procurement hospital.
XenoTech does not deal directly with - nor does it make any direct payments to - the surgeons who procure organs or the medical institutions where they work.
Cryopreserved immortalized human hepatocytes should be stored in liquid nitrogen vapor (-160°C or colder). They should be thawed in accordance with our thawing and plating protocol (PDF), which is shipped with your hepatocytes.
Plated immortalized human hepatocytes should be stored in an incubator at 37°C, 95% humidity, 5% CO2 until ready to replace media. Please follow our plating protocol (PDF), which is shipped with your hepatocytes.
Caution: These immortalized hepatocytes were derived from a donor who tested negative for HIV and hepatitis. However, we recommend that these samples be considered as potential biohazards and that universal precautions be used when working with human derived products. Cellular and subcellular fractions are intended for in vitro use only.
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POSTERS
Pfizer (2002): HTS screening for CYP and transporter induction (PDF)
Roche (2003): CYP and transporter induction (PDF)
XT (2003): Induction of major CYPs, compare fold induction to FHH (PDF)
XT (2004): Use in induction studies. CYP induction and Tox comparisons to FHH (PDF)
XT (2004): Induction of UGTs (PDF)
XT (2005): Improving lot-to-lot, dynamic range (supplement A), chromosome abnormalities (PDF)
KU/XT (2005): Induction of CYPs in IHH and FHH (PDF)
Pfizer (2005): Clinical Prediction (PDF)
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PRODUCTS
Product No. |
Product |
IFH15 |
Cryopreserved Immortalized Hepatocytes, Fa2N-4 cells, 1.5 mL/vial |
IFHP06 |
Fa2N-4 cells, 6 well collagen-coated plate |
IFHP12 |
Fa2N-4 cells, 12 well collagen-coated plate |
IFHP24 |
Fa2N-4 cells, 24 well collagen-coated plate |
IFHP96 |
Fa2N-4 cells, 96 well collagen-coated plate |
K4000 |
MFE Essential Plating Medium F, 1.0 L |
K4001 |
MFE Essential Plating Medium F, 100 mL |
K4005 |
MFE Essential Plating Medium F, 500 mL |
K4100.X |
MFE Essential Support Medium F 1.0 L w/MFE Culture Medium Supplement A |
K4105.X |
MFE Essential Support Medium F 500 mL w/MFE Culture Medium Supplement A |
|
Return to TopOrder Fax: 913.227.7171
Order E-mail: info@xenotechllc.com
Call 877.588.7530 (toll-free US and Canada) for pricing and availability.
If ordering outside of North America, please see our Distributor information.