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Cryopreserved Hepatocytes

Cryopreserved hepatocytes are shipped in the vapor phase of liquid nitrogen contained in a non-hazardous container called a dry liquid nitrogen (LN2) vapor shipper or dewar. The internal temperature of the dewar is maintained between –140 degrees C and –160 degrees C, generally for up to 7–10 days if unopened. Once hepatocytes are received and transferred to a long term LN2 dewar in the laboratory, dewars are returned to the address listed on the pre-paid return shipping label for reuse. 

Upon receipt of a shipment of cryopreserved hepatocytes, carefully and quickly transfer the vials to the vapor phase of liquid nitrogen and keep at –135 degrees C or below until use. Any increase in the temperature of the cryovials before an experiment threatens the viability, functionality, and activity of the hepatocytes.

The HepExtend Media Kit contains the HepExtend Supplement as well as Williams E Medium, 500 mL and the Hepatocyte Maintenance Supplement Packs which are all the components needed to make the complete medium for hepatocyte culture.

If properly stored, cryopreserved hepatocytes can be maintained in the vapor phase of liquid nitrogen (–135 degrees C or below) for several years. This makes them ideal for a series of experiments conducted over many months.

Unlike immortalized cell lines, hepatocytes are primary cells that cannot be cultured indefinitely. The use of thawed suspension hepatocytes should be limited to short-term experiments with a maximum of 4–6 hour incubations. Plateable hepatocytes, which attach to collagen-coated plasticware in culture media, are generally metabolically active for anywhere from 2–7 days depending on which assay they are qualified for use with.

We source liver tissue from a network of hospitals in North America that perform surgical resections on living donors, as well as from deceased donors whose organs were rejected for transplant.

Yes, we comply with country-specific legal and ethical standards for procurement of human liver tissue, including the global ICH Guidelines, and the US’s HIPAA, Uniform Anatomical Gift Act, National Organ Transplant Act, and Hospital Internal Review Board (IRB) approval processes.

A service level agreement (SLA) is required ONLY if you are working in a laboratory in the European Union. An SLA is designed to comply with the UK’s Human Tissue Authority (HTA) regulations and outlines the scope of use for human biologicals. 

No. The overlay will interfere with transfection. For transfection assays, we recommend that you perform the transfection first, and then proceed to overlay the cells.

Our hepatocytes are prequalified and sold according to application, such as induction, short-term metabolism, and transporter uptake. If you require cells with particular P450 values or donor specifications, please contact our Hepatic Biology Tech Support Team to help with specific lot recommendations. 

The “5-Donor” Human Plateable Hepatocytes are a pooled population of plateable primary hepatocytes produced by combining cells from 5 individual donors in a single vial. The cells are provided in a cryopreserved format similar to single-donor plateable hepatocytes and have been shown to have approximately equal representation of each donor when plated. Pooling donors helps to minimize lot-to-lot variation and improves predictability of drug metabolism studies and other biological assays.

Hepatocytes need to be plated on collagen I-coated culture vessels.

HepExtend Supplement (50X) is an enriched supplement designed to enhance the culture lifespan (cell viability, function, and number of days in culture) of cryopreserved primary hepatocytes. We recommend using the supplement in conjunction with William’s E Medium and the Hepatocyte Maintenance Supplement Pack following the recommended hepatocyte culture methods. When cultured in the presence of the HepExtend Supplement, primary human hepatocyte lots will live and be functional for 10 days or longer depending on the lot of cells and other assay conditions. HepExtend Supplement does not contain growth factors, cytokines, fetal bovine serum, or pharmaceutical small molecules that are known to interfere with primary hepatocyte function.

We recommend putting the HepExtend Supplement through no more than two freeze/thaw cycles. Therefore, we recommend that HepExtend Supplement be thawed and divided into single-use aliquots, which are then stored in a non–frost-free freezer at –20°C to –5°C.

HepExtend Supplement does not contain growth factors, cytokines, FBS, or pharmaceutical small molecules. However, it does contain small amounts of BSA (0.12375g/mL at 50X).

HepExtend Supplement is compatible with current hepatocyte culturing methods and instrumentation and therefore, we recommend following the Thawing and Plating Cryopreserved Hepatocytes protocol.

We recommend that HepExtend Supplement be used to keep hepatocytes in culture for up to 10 days, and it has been shown to work on approximately 80% of human lots tested—including metabolism-qualified, induction-qualified, and transporter-qualified lots.

At this time, HepExtend Supplement has only been tested on cryopreserved human hepatocytes; we therefore cannot recommend using this supplement on other species.

The HepExtend Media Kit contains the HepExtend Supplement as well as Williams E Medium, 500 mL and the Hepatocyte Maintenance Supplement Packs which are all the components needed to make the complete medium for hepatocyte culture.

Transporter Products

Prepared from Sf9 cells which have been engineered to over-express specific ABC transporters, ATP-binding cassette (ABC) Transporter Vesicles are “inside-out” vesicles designed for use in vesicular transport assays. They allow the investigation of possible interactions between specific ABC transporters and test compounds. ABC Transporter Vesicles provide high levels of transporter activity with low background, giving a clear signal if the test compound is a substrate or inhibitor of a specific efflux transporter.

While ABC transporters typically mediate the efflux of substrates from cells, transporters expressed on these inside-out vesicles import substrates into the vesicles. It is therefore possible to quantitatively evaluate transport activity for the test compound by determining the amount incorporated into the vesicles.

Note: One vial of vesicle product can perform 50 vesicular transport assays.

We recommend storing them at –80 degrees C.

The quantity supplied is 2.5 mg total protein/500 µL per vial. The protein concentration is 5 mg/mL. When our recommended protocol is followed, 50 vesicular transport assays or 125 ATPase assays can be performed with one vial of ABC Transporter Vesicles product.

TRANSiPORT human SLC transporter cells are HEK293 cells that transiently overexpress a solute carrier (SLC) transporter protein. Solute carriers are one of two superfamilies of membrane transporters that can affect pharmacokinetics and drug exposure by governing the transport of solutes into and out of cells. Membrane transporters are found in hepatic cells, kidney proximal tubule cells, intestinal cells, and brain cells.

Domestic shipments are on dry ice and shipped overnight. We ask our customers to store the product under liquid nitrogen after they receive it. International shipments are under liquid nitrogen conditions.

One vial contains 8–12 million cells with high viability, designed to seed one 24-well plate for single use.

Yes, the protocol is available here.

Cell-based assays can be performed using radioisotope-labeled compounds, fluorescence-labeled compounds, or non-labeled compounds. The amount of substrate transported into the cells can be measured directly using a liquid scintillation counter, fluorescence plate reader, or LC-MS/MS, thereby allowing direct evaluation of SLC transporter activity.

Mock HEK293 cells that do not overexpress the SLC transporter are available for use as a negative experimental control. Some compounds may demonstrate high background levels of SLC transport due to the presence of endogenous transporters or non-specific binding.

HepaRG Cells

HepaRG cells are a human hepatic progenitor cell line that retains many characteristics of primary human hepatocytes. HepaRG cells are terminally differentiated and provided in a convenient cryopreserved format. For scientists who need reproducible metabolism data, HepaRG cells are an in vitro tool that provides reproducible results in a metabolically complete and scalable system.

The HepaRG cells are available as cryopreserved cells in vials. They are shipped in dry ice for an overnight delivery or in a vapor phase nitrogen container for longer shipments.

Upon receipt, cryovials must be stored in liquid or vapor phase nitrogen.

The cells are licensed for single-use applications.