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Drug Testing and Drug Development in Cell Culture
Abstract
Cell culture systems are one of the widely used systems implicated in drug testing and drug development. Recombinant cell lines, primary cells and animal models are widely used for drug development and drug testing. Stem cells have a potential use in the all stages of drug discovery processes including target identification, target validation, inhibitor screening and toxicology studies. Stem cells are capable of unlimited self-renewal and can generate physiological relevant cells through controlled differentiation. These two properties make stem cells an attractive alternative to currently used recombinant cell lines and primary cells. However, currently the use of stem cells is limited due to difficulties in directed differentiation and the cost of maintenance.
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In the embryonic stem cell test (EST), differentiation of mouse embryonic stem cells (mESCs) is used as a model to assess embryotoxicity in vitro. The test was successfully validated by the European Center for the Validation of Alternative Methods (ECVAM) and models fundamental mechanisms in embryotoxicity, such as cytotoxicity and differentiation. In addition, differences in sensitivity between differentiated (adult) and embryonic cells are also taken into consideration. To predict the embryotoxic potential of a test substance, three endpoints are assessed: the inhibition of differentiation into beating cardiomyocytes, the cytotoxic effects on stem cells and the cytotoxic effects on 3T3 fibroblasts. A special feature of the EST is that it is solely based on permanent cell lines so that primary embryonic cells and tissues from pregnant animals are not needed. In this protocol, we describe the ECVAM-validated method, in which the morphological assessment of contracting cardiomyocytes is used as an endpoint for differentiation, and the molecular-based FACS-EST method, in which highly predictive protein markers specific for developing heart tissue were selected. With these methods, the embryotoxic potency of a compound can be assessed in vitro within 10 or 7 d, respectively.
Stem cell biology offers advantages to investigators seeking to identify new therapeutic molecules. Specifically, stem cells are genetically stable, scalable for molecular screening, and function in cellular assays for drug efficacy and safety. A key hurdle for drug discoverers of central nervous system disease is a lack of high quality neuronal cells. In the central nervous system, alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA) subtype glutamate receptors mediate the vast majority of excitatory neurotransmissions. Embryonic stem (ES) cell protocols were developed to differentiate into neuronal subtypes that express AMPA receptors and were pharmacologically responsive to standard compounds for AMPA potentiation. Therefore, we hypothesized that stem cell-derived neurons should be predictive in high-throughput screens (HTSs). Here, we describe a murine ES cell-based HTS of a 2.4 x 10(6) compound library, the identification of novel chemical "hits" for AMPA potentiation, structure function relationship of compounds and receptors, and validation of chemical leads in secondary assays using human ES cell-derived neurons. This reporting of murine ES cell derivatives being formatted to deliver HTS of greater than 10(6) compounds for a specific drug target conclusively demonstrates a new application for stem cells in drug discovery. In the future new molecular entities may be screened directly in human ES or induced pluripotent stem cell derivatives.
The development of methods for growing human cancer cells in culture has raised the question of whether it might be possible to use culture methods to model all the features important for clinical antitumor activity. Cell culture techniques play a key role in the development of new anticancer drugs by imposing additional constraints on those of receptor interaction alone, such as drug uptake and efflux, interaction with other cellular receptors, and cellular metabolism. Clonogenic assays, integrating multiple cell death pathways, are particularly useful in measurement of cell survival following exposure to cytotoxic drugs. On the other hand, microcultures, combined with colorimetric and other methods for measuring antiproliferative effects, have provided the basis for large-scale screening of cytotoxic and cytostatic drugs. The U.S. National Cancer Institute program has made it possible to use 60 or more cell lines to compare the growth inhibition profiles of potential new drugs with those of tens of thousands of previously tested compounds, providing information on mechanism of action as well as antiproliferative activity. Some of the most promising new approaches include the use of three-dimensional cell cultures to allow the measurement of rates of drug diffusion, as well as the assessment of the effects of cell contact and tumor microenvironment on drug sensitivity.
Stem cells have enormous potential to revolutionise the drug discovery process at all stages, from target identification through to toxicology studies. Their ability to generate physiologically relevant cells in limitless supply makes them an attractive alternative to currently used recombinant cell lines or primary cells. However, realisation of the full potential of stem cells is currently hampered by the difficulty in routinely directing stem cell differentiation to reproducibly and cost effectively generate pure populations of specific cell types. In this article we discuss how stem cells have already been used in the drug discovery process and how novel technologies, particularly in relation to stem cell differentiation, can be applied to attain widespread adoption of stem cell technology by the pharmaceutical industry.
Induced pluripotent stem cell (iPSC) technology is revolutionizing medical science, allowing the exploration of disease mechanisms and novel therapeutic molecular targets, and offering opportunities for drug discovery and proof-of-concept studies in drug development. This review focuses on the recent advancements in iPSC technology including disease modeling and control setting in its analytical paradigm. We describe how iPSC technology is integrated into existing paradigms of drug development and discuss the potential of iPSC technology in personalized medicine.
Tumor cell cultures in drug development
- Bc Baguley
- Ko Hicks
- Wr Wilson
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