Unlike most hepatic cell lines, which by definition are altered or transformed cells, primary hepatocytes retain most, if not all, of their original biochemical and molecular signaling pathways and phenotypic gene expression profiles, including important transcription factors and nuclear receptor responses. These are typically required for more discerning studies to determine human-specific hepatic disposition and hepatic responses to compound exposure, especially species- or population-specific metabolism, uptake and toxicity outcomes.

Although theoretically possible, it is not recommended to submit primary hepatocytes to repeated freezing and thawing cycles due to the additional stress and damage imposed on the cells and their subcellular components.

Yes, but not all batches of cryopreserved hepatocytes will ‘plate’ under standard culture conditions (i.e., collagen-coated 96- or 24-well culture plates). It is important to identify those batches of cells that have been pre-qualified as ‘plateable’ lots based on the data in the individual CoA’s, such as their optimal seeding density, attachment efficiency, formation of confluent monolayers, and viability over time in culture.

Yes, all batches of cryopreserved hepatocytes can be utilized for suspension cultures for short-term, non-adherent experiments, such as drug metabolism or uptake studies. Optimal culture conditions for seeding density, recommended medium and incubation conditions are provided in the Instructions for Use. If experiments require the development of 3D aggregates, such as spheroid cultures, inquire with your technical representative to determine which lots are best suited for these applications.

If the cells are to be utilized for drug metabolism or uptake experiments, low-binding multi-well plates (i.e., Corning 96-well) are recommended for these applications. In some cases glass vials or culture tubes in a shaking water bath may be used as well.

There is significant lot-to-lot variability in how long primary hepatocytes stay viable and retain functionality over time in culture. Longevity and functionality over time in culture is also dependent on the culture conditions and format, such as ECM overlay, medium formulation, co-culture with stromal cells, or 3D aggregate culture. “Standard” monolayer culture conditions, such as ECM overlaid or non-overlaid, support most plateable hepatocyte batches for 1-2 weeks, depending on the lot, ECM composition and the medium formulation. More advanced cell culture systems, including co-cultures with stromal cells or 3D aggregate culture, have been shown to greatly extend the longevity and performance of primary hepatocytes out to several weeks.

For plateable hepatocytes, the optimal seeding density for achieving a confluent monolayer is typically between 125,000 and 150,000 cells per cm2. However, each batch of primary human hepatocytes exhibits different attachment efficiencies. Refer to the CoA documents for the optimal seeding density determined for individual plateable lots. The technical bulletins for plateable and suspension lots contain valuable information on the number of cells per incubation using different well formats

For drug metabolism and uptake experiments, the cells are ready for use after attachment (i.e., 2-4 hours). For induction testing, hepatocyte monolayers in standard multi-well plate formats are refractory to drug treatments during the initial 12-18 hours and treatments with drugs are typically started the following day after seeding. For hepatotoxicity and biliary excretion testing, it is recommended that cell-cell adhesions and cellular polarity be restored as much as possible, which can vary somewhat between lots, and experiments should be initiated after several days

in culture.

Compared to most hepatic cell lines, primary hepatocytes are larger in size, more fragile and generally should be treated more gently while handling. It is recommended that gentle thawing and resuspension methods be adopted when handling cell pellets and during pipetting and plating of hepatocytes. Harsher mechanical resuspension techniques, such as vortexing or rapid mixing with a pipette, should be avoided to preserve the integrity and viability of the cells.

It is important to understand whether a “simple” culture method, i.e., hepatocyte monolayers on collagen-coated plates, or a more “sophisticated” culture system, i.e., sandwich-culture, co-culture with stromal cells, or 3D spheroid aggregate cultures, is better suited for your particular application(s). For many standard metabolism and uptake experiments, a simple system will often suffice especially if throughput and efficiency is a major consideration. However, the more advanced models usually provide greater longevity, stability and performance over time and biologically relevant outcomes than the simple models. Please consult your technical representative for more information and to discuss your particular application requirements.

Plateable lots adhere to, and form, confluent monolayers when cultured on collagen-coated plates (24, 48, 96 wells). Plateable lots exhibit ≥ 70% post-thaw viability, ≥ 80% confluency, and ≥ 80% attachment efficiency in adhering to the plate. Suspension lots are not plateable and experiments are typically conducted in a vial, tube, or low binding plate with the cells in suspension culture.