HomePrimary Cell CultureLifeNet Health® Primary Human Hepatocytes

LifeNet Health®
Primary Human Hepatocytes

We provide high viability cryoplateable and cryopreserved hepatocytes sourced from LifeNet Health® that predictably represent in vivo conditions. Applications for primary hepatocytes include in vitro evaluation of metabolism, drug-drug interactions, drug transporter activity, and toxicity of drug candidates. There are many other applications that encompass cryoplateable and cryopreserved hepatocytes.

LifeNet Health®, celebrating 35 years of saving lives, restoring health, and giving hope to thousands each year, is the world's most trusted provider of transplant solutions — from organ procurement to bio-implants and cellular therapies — and a leader in regenerative medicine, while always honoring the donors and healthcare professionals who enable healing.

LifeNet Health®’s primary human hepatocytes meet the specific needs of a wide range of scientific research applications, including drug development and risk assessment. Donated liver tissues are procured under state-of-the-art conditions using the highest standards for tissue recovery and preservation, which utilize enhanced tissue handling and transportation methods and minimize warm and cold ischemia times to optimize tissue processing outcomes and product integrity. These measures, combined with refined cell isolation techniques and advanced post-thaw characterization, represent a new industry standard for hepatocyte quality and performance.

Prior to release, each batch of cryopreserved hepatocytes is carefully characterized to determine the post-thaw results. The batch-specific functionality, donor medical history, and clinical data includes the following:

  • Cell viability and yield per vial
  • Morphological integrity and attachment efficiency
  • Optimal seeding density (based on a 24-well plate format)
  • CYP enzyme activity using prototype selective substrates for all the major enzymes pertinent to drug discovery and development
  • Additional functional testing can be performed upon request for specific cell culture applications
  • Each batch comes with a comprehensive Certificate of Analysis (CoA) with representative images, relevant donor demographics, BMI, pre-mortem liver function lab values, serological test results, and pertinent tobacco, alcohol, drug, and medication history
  • Liver biopsy histology images and pathology results may also be available upon request

The liver microanatomy: our guide for next gen model systems

Figure 1.The liver microanatomy: our guide for next gen model systems

  • Download a complete list of currently available hepatocyte lots from LifeNet Health®.

Figure 2.Hepatocytes

Effects of Cell Adhesion on Cellular Response to Enzyme Inducers

Effects of Cell Adhesion

Figure 3.Effects of Cell Adhesion

Categories of Cryopreserved Human Hepatocyte Lots

The following are the major categories of cryopreserved hepatocytes that are currently available through LifeNet Health®. Additional options or categories, not listed below, may be considered upon individual request:

Adult Suspension/Metabolism Qualified:
Primary adult human hepatocytes are considered the preferred method for determining the metabolic stability and intrinsic clearance of new compounds in development. LifeNet Health®’s suspension batches of cryopreserved hepatocytes are characterized for drug-metabolizing enzyme activity using prototype selective substrates for phase I and II metabolic pathways.

Adult Plateable/Induction Qualified:
Use of cultured primary human hepatocytes has become the accepted best practice for conducting in vitro testing of new drugs for their potential to be involved in unwanted drug-drug interactions due to induction of hepatic clearance pathways. LifeNet Health®’s induction qualified batches of cryopreserved hepatocytes are tested for response to prototype inducers of CYP1A2, CYP2B6, and CYP3A4. These prequalified lots are guaranteed to produce stable confluent monolayers for a minimum of 1 week and meet or surpass the induction specifications when used in conjunction with LifeNet Health®’s recommended culture conditions.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in Western countries with a wide disease spectrum. For LifeNet Health®’s Adult High BMI/NAFLD/NASH hepatocytes (suspension and plateable), a
histopathological evaluation of formalin-fixed, paraffin-embedded tissue sections after H&E and trichrome staining is performed by a certified pathologist. Representative histological images and a pathology report can be provided with each CoA upon request.

Donor LHuf15102

Figure 4.Donor LHuf15102: BMI = 37; Z1-2 microfat = 50%

It is becoming more and more important in risk assessment to understand the developmental changes in the expression of phase I and II enzymes and other clearance mechanisms at different life stages that determine the pharmaco- and toxicokinetics of chemicals.

‘Normal’ Human Hepatocytes

Figure 5a. ‘Normal’ Human Hepatocytes

‘Steatotic’ Human Hepatocytes

Figure 5b. ‘Steatotic’ Human Hepatocytes

Features and Benefits

  • Drug metabolism studies including stability, clearance, species comparison,
  • Metabolite profiling and ID
  • Metabolism of low clearance compounds
  • CYP induction (mRNA and enzyme activity)
  • Plated and suspension drug transporter assays (uptake and efflux)
  • Hepatotoxicity assays
  • High content imaging and screening
  • 3D culture technologies

Importance of Restoring Hepatocyte Physiology for In Vitro Cell Culture Studies

Importance of Restoring Hepatocyte Physiology for In Vitro Cell Culture Studies

Figure 6.Importance of Restoring Hepatocyte Physiology for In Vitro Cell Culture Studies

The most accurate picture for hepatic effects can be obtained with cultures of primary cells in which the native architecture and the natural orientation for linked enzymes, transporters and receptors are restored.

LifeNet Health Human Primary Hepatocyte FAQs

Why would you use primary human hepatocytes as opposed to cell lines?

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 corresponding technical bulletin for (MTOXH1000, MTOXH1002, MTOXH1001, MTOXH1005, MTOXH1008, MTOXH1011, MTOXH1012). 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 multiwell plates (e.g. 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 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 average seeding density typically is between 125,000 and 150,000 cells per cm2. However, each batch of primary human hepatocytes exhibits slightly 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 (MTOXH1000, MTOXH1002, MTOXH1001, MTOXH1005, MTOXH1008, MTOXH1011, MTOXH1012).

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 multiwell plate formats are refractory to drug treatments during the initial 12-18 hours, and treatments with drugs are typically started 1-2 days 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 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, e.g. hepatocyte monolayers on collagen-coated plates, or a more “sophisticated” culture system, e.g. 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.

Catalog item MTOXH1000, for example, are plateable on collagen-coated plates (24, 48, 96 wells). MTOXH1001 are not plateable and the experiment has to be conducted in a vial or tube with the cells in suspension culture. These suspension hepatocytes are rated for metabolic clearance studies because of their expression of phase I & II metabolic enzymes. MTOXH1005, for example, are not platable and the experiment has to be conducted in a vial or tube with the cells in suspension culture. These hepatocytes are not rated for metabolic clearance assays.

Cryoplateable means that the hepatocytes form a monolayer when cultured on collagen-coated plates. They have an ≥ 80% post-thaw viability, ≥ 80% confluency after 120 hours when plated and an ≥ 80% attachment efficiency in adhering to the plate.

No these are single-use consumables and are biologically not able to passage.

It is the most economical option for primary human hepatocytes, but these cells are not plateable and are in suspension format.