Liver Cells for Toxicology Studies

The liver is composed of five major cell types, and the hepatocyte is the most abundant cell type. Hepatocytes comprise approximately 70% of the liver cell population and are responsible for most metabolic and hormonal processes. The other four cell types, collectively known as hepatic non-parenchymal cells (NPCs), consist of resident macrophages called Kupffer cells, stellate cells, liver sinusoidal endothelial cells, and cholangiocytes. These cells serve to support the liver structure, transport molecules in and out, and communicate with the immune system.

One of the greatest challenges in drug development is the prediction of the safety and the metabolic fate of a new drug before it enters human clinical trials. Because the liver is the major site of metabolism and detoxification, in vitro cell systems that mimic the liver are significantly useful for predicting the consequences of drugs administered to humans in the clinic. The ability to isolate highly pure hepatocyte populations from non-transplantable, donated human livers to mimic the human liver environment has therefore become an integral part of the drug development pipeline.

Hepatocyte research

Introduction to liver cell biology

The liver plays a critical role in vertebrate biology. It has many important metabolic functions, including the regulation of glucose and cholesterol metabolism, the production of plasma proteins including clotting factors, and the detoxification of endogenous and exogenous compounds. The liver also produces various hormones involved in insulin regulation, blood pressure, and blood lipid levels. Because of the many physiological processes that depend on the liver, a fundamental understanding of liver biology and the ability to address these at the benchtop is essential for researchers involved in creating new, life-saving medicines.

Liver cell isolation

Hepatocytes are isolated using a two-step collagenase perfusion protocol. The isolation of human primary hepatocytes can yield either plateable or suspension hepatocytes. Both can be useful models for hepatoxicity studies. The cellular morphology, structure, and functionality is different for suspended and plated cells, and understanding these differences are important depending on the specific applications for the hepatocytes.

The functionality of primary hepatocytes in culture is very similar to that of in vivo hepatocytes, as indicated by albumin production, urea production, and a variety of metabolic enzyme activities.

Hepatocyte isolations also in the ability to obtain a mixed population of all the other major cell types from the liver. From this mixture, enrichment of Kupffer, stellate, and endothelial cells can be performed to enable the development of complex tissue-like models of the liver in vitro.

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Schematic of multiple liver functions

The liver has many physiological functions including cholesterol and amino acid synthesis as well as detoxification of various small molecules.

Selecting the right hepatocyte cell model

Many cell culture models have been used to simulate the liver environment for research and development. Some of these hepatocyte models include tumor cell lines such as Huh-7 and HepG2 cells. Tumor cell lines include the advantage of self-renewal, which saves resources and enables the more rapid production of genetic models for mechanistic research. However, the metabolic activity of tumor lines is 10-1,000 orders of magnitude less than that of primary hepatocytes and are not recommended for toxicity studies in the drug discovery and development process where appropriate drug metabolism might be important.

Lonza supplies a more biologically relevant hepatic cell line, HepaRG™ Cells. These cells are fully-functional, adult-phenotype, human hepatic cells. HepaRG™ Cells exhibit many characteristics similar to primary human hepatocytes and can be used for many of the same applications. The increased metabolic activity of HepaRG™ Cells compared to HepG2 enables more predictive value for hepatic toxicity assays conducted in early drug discovery.

The NoSpin HepaRG™ Cells are provided in a convenient, terminally differentiated, cryopreserved format that does not require the user to centrifuge, resuspend, or count the cells after thawing. This eliminates multiple steps that introduce qualitative and quantitative variability and benefits high-volume labs by reducing the time and effort needed to perform an assay. Each lot of HepaRG™has consistent yield, viability, and functionality across multiple applications.

NoSpin^TM HepaRG^TM (8.0M cells/vial)

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Differences between primary hepatocytes and hepatic cell lines

Primary hepatocytes are the gold standard for physiologically relevant in vitro liver models as they retain in vivo-like functions and morphologies. Hepatocytes directly isolated from liver tissue are called primary hepatocytes. Decades of research and development has resulted in a robust isolation process for primary hepatocytes, yielding cultures with purities over 98%. Once isolated from the tissue, the hepatocytes may be used right away or cryopreserved for later use. The functionality of primary hepatocytes in culture is very similar to that of in vivo hepatocytes, as indicated by albumin production, urea production, and a variety of metabolic enzyme activities.

Formats for primary hepatocyte use

Suspension
Hepatocytes are cultured in many formats. For short term metabolic assays (less than 4 hours), hepatocytes can be placed into a reaction vessel in suspension either directly following isolation or after reanimation from cryopreservation.
Plated monolayer
Hepatocytes are used in plated formats when drugs are poorly metabolized in short-term suspension assays, or when mechanistic data regarding drug-drug interactions and mechanistic toxicity is needed. Hepatocytes plated in a monolayer on a collagen matrix have an extended lifespan compared to cells in suspension and maintain their metabolic function for longer.
Sandwich culture
In vivo, hepatocytes have a polar morphology whereby cell surface proteins vary on the different 3D surfaces. This polar morphology can be replicated in vitro by providing an overlay of collagen or other basement membrane extracts on top of a hepatocyte monolayer. By “sandwiching” hepatocytes between these basement membrane protein mixtures, they form the correct basal surface structures and a small apical membrane pocket between cells known as bile canaliculi. These structures enable excretion of bile acids from the hepatocytes, replicating the situation in vivo. It is generally accepted that the sandwich culture method reflects the 3D in vivo shape of hepatocytes, while maintaining the functionality of a 2D format.
Spheroid
An emerging technique for recapitulating the in vivo physiology of the hepatocyte is to allow the cells to self-assemble into a spheroid shape. Using simple to use low attachment, round-bottom culture plates, hepatocytes adhere to each other more readily and over the course of 4-5 days will become a tight ball of cells which demonstrate polarity and tight junctions. These small spheroids with about 1500-3000 cells have been shown to function more like the in vivo liver tissue and be better predictors of chemical toxicity that standard hepatocyte monolayer cultures.