Hepatocytes are widely used to screen potential drug candidates and have allowed researchers to fast-track many new treatments into clinical trials. Quickly releasing new drug treatments that meet the high standards called for by patients and doctors is an immense challenge, the success of which is determined largely by how reliable the models used in testing are.
Here, we’ll explore how hepatocytes are accelerating this process giving hope for safer, cheaper, and more efficacious drug therapies. We’ll go through what they are, the roles they play in inflammation, why they’re reducing the need for animal models and what the various screens are for which they’re used.
What are hepatocytes?
Hepatocytes are specialized, polygonal cells that make up 60-80% of the mass of the liver. They stack on top of each other to form layers and are key cells in the diverse functions of the liver.
“Hepato-” means liver
“-cyte” means cell
Specialized cells: cells that are modified to carry out very specific functions/tasks.
They play a crucial role in liver immunity. Upon encountering a pathogenic insult, these eager cells secrete acute phase proteins, immune cell growth factors, and cytokines, such as IL-6 a pro- and anti-inflammatory cytokine. These recruit neutrophils and B cells and suppress T regulatory cells. This is important in allowing the liver to circumvent its normal immunosuppressive state.
Additionally, hepatocytes are involved in food metabolism, breaking down carbohydrates and lipids and secreting bile.
Lastly, hepatocytes also metabolize, detoxify, and inactivate exogenous compounds like drugs. Thus hepatocytes grown in suspension or culture can be used to explore mechanisms of drug metabolism and predict how the human liver with respond to the drug: be damaged by it and/or filter all/most/some of the drug out. This allows researchers to better theorize what dose might be suitable for human use.
But why this is so important in drug screening can only be understood by learning more about the liver, the second largest organ of the body (our skin being the first!).
The Liver: Home of the Hepatocytes
The liver is an incredible organ. Besides the role it plays in metabolism and storage, the liver mediates systemic inflammation – meaning if you’ve got a fever, your liver is the one running the show. It does this by a vast array of innate immune cells capable of responding to pathogen- and danger-associated molecular patterns. The total blood volume of the human body circulates through the liver approximately 360 times each day. Consequently the liver is continually exposed to pathogenic stresses, dietary and bloodborne antigens, and immunological signals, from the GI tract and the systemic circulation. Usually, the liver exists in an immunosuppressed state to avoid system inflammation; it can’t get ticked off every time it sees something it doesn’t like. Key to this balance is the response of hepatocytes and this is why these cells are the cells of choice for toxicological and pharmacological testing.
Hepatocytes and Drug Toxicology Screening
We’ll briefly list and explain some of the more old-school tests that have been around for a while as well as the cutting edge more exciting tests that are growing in popularity.
|1) Liver Slices||– Maintains liver structure.
– Same cell types as in vivo.
– Models xenobiotic metabolism well.
– Zone-specific cytochrome activity present.
– Culture length: 30 min to 5 days.
|– Necrosis after 48-72 hours in culture.
– Metabolic enzyme levels greatly reduced after 6-72 hours.
– Lower rates of drug metabolism and clearance than in isolated hepatocytes,
– Km values often higher than in isolated hepatocytes
– Possible gradient in chemical exposure resulting in not all hepatocytes participating in the metabolism of compounds.
|2) Immortalized Cell Lines||– Cell line-specific fidelity to certain aspects of in vivo cells.
– Relatively inexpensive to buy and maintain.
– Relatively easy to culture.
|– May not have the phenotypic characteristics of the liver tissue.
– Expression profile of genes can be inconsistent.
– Represent the phenotype of a single donor.
|3) Primary Hepatocytes Suspensions||– Easy to use for moderately high-throughput studies.
– Maintains functionality better than cultured cells.
|– Routine handling and isolation damages intra- and extracellular structures.
– Rapid loss of viability in culture.
|4) Primary Hepatocytes Cultures||– Considered gold standard.
– Functional activity stays for up to 24-72 hours.
– Ideal for enzyme studies,
– Easy to use for moderately high-throughput studies.
– Allows comparison of interspecies and inter-individual metabolism.
– Increasing dysfunction can be ameliorated somewhat using Matrigel® or Geltrex™ matrices.
|– Underlying assumptions that these cells will respond the same way alone as they would in vivo in very different surroundings.
– Cells change when cultured and become dysfunctional overtime.
– Begin to de-differentiate within the first 24-48 hours of culture.
|5) Cytokine Co-Culture||
– Allow you to better reproduce clinical drug hepatotoxicity responses.
– Provides information on which pathways play what roles in the response to the drug.
|Same as 3) and 4).|
- Three-dimensional Culture Systems: includes simple matrix-supported sandwiches and spheroids and more complex, multi-faceted systems.
- HμREL® Biochip: this is a microfluidic device that enables you to co-culture various cell types but the cell remain physically separated and communicate only through linking passages. You can use each separate compartments to simulate various organs enable you to create an in vivo-like set up.
- Hollow-Fiber Bioreactor: this system is based on maximizing surface area as well as creating an in vivo-like system. Cells grow outside long porous fibers that introduce nutrients, remove waste or release the drug to be tested.
- Single- and Multi-Well Perfused Bioreactor: this is similar in concept to what a cross between between (2) and (3) would look like.
- Bioartificial Livers: this is more of a category of different systems than one defined technology and it ranges from systems similar to (3) to more grown liver organoids.
- Stem Cells: embryonic and induced pluripotent stem cells (ESCs and iPSCs) can be used to make hepatocytes but these have thus far had the same problems as other cultured cells.
Why Not Just Use Animal Models?
In vivo models to screen potential new drug treatments come with a host of problems. These include:
- Cannot evaluate the small concentrations used as doses in humans. Rodent studies involve giving high doses to mice which are not comparable to normal human medical doses.
- Cannot test the vast combinations of chemicals humans are exposed to. It also isn’t feasible to test drug interactions on a large scale, but it is unusual that someone will be taking only one medication at a time and even more unusual that that person will be drug naive. The effects of co-exposure cannot be evaluated sufficiently in in vivo models as it would take a momentous amount of time.
- Do not give detailed data on how a drug works e.g. drug metabolism.
- Do not account for the diversity of human responses and susceptibilities.
- Are costly, time-consuming, resource-intensive and have a greater environmental impact.
- The adverse events predictive accuracy of in vivo testing is inconsistent as the human population is orders of magnitude more variable than the hyper-inbred mouse models we use to test drugs.
- Requires larger amounts of chemicals than in vivo models
- Lower throughput than in vivo models.
Hence, improving in vitro models has become a strong focus of the toxicology community backed up by the U.S. Environmental Protection Agency (EPA) and the National Research Council (NRC) (seen in NRC’s report Toxicology Testing in the 21st Century: a Vision and Strategy, written at the request of the EPA). With the advent of humanized and genetically engineered mice, there are of course benefits to using in vivo models, but there is clear advantage in lowering the numbers of animal studies needed.
Hepatocytes are resident liver cells that have a broad range of functions in the human liver. These include drug metabolism and hence they are of high importance in drug toxicology research. As technologies advance, the need for animal models will be reduced and largely replaced by increasingly in vivo-like systems.