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Use of custom-cell products: Research off the beaten path
Tara Williamson, Celsis In Vitro Technologies
The need for custom cell products
Pharmaceutical research is a complex process of melding development of new chemical entities (NCEs) with therapeutic efficacy in an effort to produce promising drug candidates. Successful development of chemicals with drug-like properties such as low molecular weight, permeability, solubility, metabolic stability and minimized drug-drug interactions (DDI) requires specialized tests. Success hinges on the ability to determine failures in drug discovery and early stage development in order to eliminate unsuitable NCEs before they ever reach clinical trials, the most cost and resource intensive portion of the drug development process. The use of in vitro models and methods to evaluate compounds for absorption, distribution, metabolism and excretion (ADME) and toxicity allows more accurate in vivo predictive modeling. Traditional ADME-Tox testing focuses on the liver, but other organs may be important depending on the route of administration, e.g., subcutaneous injection, inhalation and oral administration. Diversified in vitro testing systems may help pharmaceutical researchers better predict in vivo interactions, leading to efficient and effective drug development.
Efficiently weeding out unsuitable NCEs in the preclinical laboratory requires researchers to employ dependable, reproducible tools and products that reduce technical and financial risks yet meet the researcher's performance criteria. Custom-cell products, customized characterization, and defined cell culture environments are all becoming critical components in drug discovery and cell biology research. One manner in which scientists have managed to mitigate risk has been with the use of more physiologically relevant cells. With ready access to the precise models, researchers are accelerating the drug discovery process —from target and hit identification, assay development, lead optimization and into preclinical development.
Common custom-cell products
Custom-cell products are finding widespread use throughout many areas of the preclinical arena and can be used to monitor the entire cycle of drug administration, metabolism and excretion. The demand for such specific yet varied products originated with Big Pharma seeking new means to rapidly bridge in vitro to in vivo research. Commonly employed in vitro products include freshly isolated and cryopreserved primary cells, homogenates, and subcellular fractions, such as microsomes, cytosol, and S9. These in vitro products are generally created from a list of standard, toxicologically relevant species, including human, rodents and dog. However, custom products can be tailored specifically for the end user, allowing for applications that exceed standard research expectations. These products promise greater harmonization of research models throughout the pharmaceutical industry and more economical use of human, animal and material resources such that unnecessary delays in new treatment development are diminished while safeguards on quality, safety and efficacy endure.
Serving up specified species
Human, rodents and dog are the most commonly utilized species for ADME-Tox testing. However, other species may be employed depending on the animal used in the therapeutic model, like primates for studying Parkinson's disease or the chinchilla animal model for hearing loss. In vitro testing in the same species as in vivo studies may be used to refine in vivo protocols. This can reduce the number of animals used by eliminating unnecessary testing, optimizing dosing ranges and producing pertinent data. The availability of custom-cell products created from many different types of mice, rats or monkeys enables researchers to expand study-design horizons beyond commonly used, readily-available species. The expanded selection may also afford certain economic benefits by not having to repeat experiments to bridge inter-species differences. Utilizing the same species for in vitro and in vivo studies may be enhanced by focusing on a specific sub-species, like Han Wistar, and further by sourcing animals from the same colony. This minimizes experimental variability that may stem from genetic variations among animal facilities or from treatment differences with regard to feeding or housing.
Yet, species selection is just one element of the specialization possible with custom-cell products. Some custom-cell providers are also able to isolate products from treated animals supplied by the pharmaceutical laboratory. This affords the researchers to directly correlate in vivo observations with sensitive in vitro methods. Other conditions like gender and age of the animal or type of diet (fasted versus ad libitum) may be specified. Additionally, custom-cells may be supplied fresh in a particular time frame from a donor animal or they can potentially be cryopreserved to fit experimental needs in today's accelerated research timelines.
Availability of optimal tissue type and whole organs
Today's leading custom-cell manufacturers also provide cell culture models from many different tissues that are useful for predictive toxicological modeling. Fresh and cryopreserved cells are employed to monitor dynamic changes initiated by an NCE. Traditionally, hepatocytes are used to study metabolic stability, to monitor drug-drug interactions or determine metabolite formation. Cryopreserved hepatocytes can be banked from isolations to ensure the availability of multiple research tools from the same donor while offering the long-term storage and convenience of cryopreserved cells. Increasingly, manufacturers are further processing the liver to produce other cell populations, such as stellate, kupffer, and sinusoidal endothelial cells. These subtypes can be studied in their purified form or in reconstituted co-cultures that better mimic physiological conditions. Likewise, sub-populations of the kidney, such as proximal and distal tubules, can be purified and cultured in a specific format. Published literature demonstrates that transwell-plated in vitro assay formats utilizing proximal and distal tubule cells can be used to study renal cell responses to injury, mechanisms of toxicity and xenobiotic transport.1, 2
In parallel with the availability of specific tissue sub-types, the increasing availability of whole organs from animals is greatly facilitating toxicology and mechanism of action research. Whole organs and whole organ systems are allowing drug developers to better understand drug metabolism and how it is affected by different routes of administration or within different species. For instance, testing the ADME of an orally-administered NCE may require an organ system of intestinal mucosa products to adequately demonstrate absorption and metabolism across intestinal biological membranes. Products can be prepared from functionally distinct areas of the intestine, such as the jejunum, duodenum and ileum of the small intestine and the cecum and colon of the large intestine, to fully understand absorptive patterns. Analyzing each segment of an organ system in a single format allows researchers to observe in vitro how the naturally coordinated system of enzymes and transporters impacts a given NCE. Such complete and biologically interconnected ADME-Tox data had previously been limited to in vivo study.
Selecting cell types
With such a variety of custom-cell products available, the process of selecting the appropriate cell type requires assessment of research objectives. For the analysis of cytochrome P450 enzyme activity, microsomes are the gold standard. The availability of purified cell fractions from a range of tissues (kidney, lung, large and small intestine, skin, heart, brain, testes, prostate, spleen, muscle) allows researchers to evaluate the metabolism profile of the NCE during all phases to predict the in vivo response. For example, the kidneys may play a major role in Phase II drug metabolism and can be highly susceptible to drug-induced toxicity. Additionally, both lung and intestinal tissue are involved in first-pass metabolism and can create toxic metabolites through bioactivation of the compound. The metabolism profile of the NCE can be further analyzed using homogenates, S9 fractions and cytosol. These products provide complementary data to fully understand non-specific binding and the influence of non-P450 enzyme activity on metabolism. Finally, custom-cell products specific to mitochondrial and/or DNA fractions can be used to further explore an NCE's impact on portions of cellular machinery.
Advantages and new frontiers
Understandably, researchers are shifting focus to the growing scope, control and design opportunity afforded by working with custom-cell products. Likewise, these products also provide an unprecedented level of control for assay media, plating, seed density and co-culture specifications. Along with defining the requirements of the cell assay, users can specify packaging, volumes and vial size to enhance the end user's convenience. As an illustration of the level of customization possible, products can be created using integrated enzyme database information with algorithms to configure custom products to the exact specifications of a previously existing lot or to design a custom product containing high levels of specificity by "dialing in" the particulars of enzyme activities or donor specifications.
With the range of advantages and opportunities afforded by custom cell products, the essential key is the selection of a provider with the requisite capability, techniques and expertise to prepare and characterize cell samples. Towards this end, production, quality control and R&D all work together to achieve quality results. A dynamic long-lasting partnership between cell product provider and user is the final ingredient to ensure that investment in custom cell products is a cost-effective approach to accelerating the pace of successful drug development.
1Taub M. Primary kidney cells. Methods Mol Biol. 1997;75:153-61.
2 Brown CD, Sayer R, Windass AS, Haslam IS, De Broe ME, D'Haese PC, Verhulst A. Characterisation of human tubular cell monolayers as a model of proximal tubular xenobiotic handling. Toxicol Appl Pharmacol. 2008 Dec 15;233(3):428-38. Epub 2008 Oct 1.
Tara Williamson is an R&D Scientist and Custom Cell Specialist for Celsis In Vitro Technologies. She is co-author of several posters and scientific papers on hepatocytes and hepatic transporters, and has developed a number of protocols for the company's QC and production departments.
Tara joined Celsis IVT in 2005 as production assistant and was subsequently promoted to test article coordinator, assistant scientist and associate scientist. A graduate of North Carolina State University with a double major in Animal Science and Zoology, Tara is currently pursuing an advanced academic degree in Biotechnology at Johns Hopkins University.