In the Spotlight
Cell-based Assays and their Role in the Future of Toxicity Testing: Part I
Published: July 18, 2008
Last year marked the 100th anniversary of a major scientific milestone, the development of cell culture as a research tool (Ryan, 2007). Ross Granville Harrison, circa 1907, developed a hanging drop technique, previously used for the study of microorganisms, to observe the outgrowth of neurons from explanted frog embryonic neural tube fragments. Harrison’s pioneering studies “solved the basic cell culture problems of medium, culture vessel, observation, and culture contamination,” and paved the way for other researchers to use cell culture for research and for producing important cellular products such as antibodies and therapeutic proteins (Ryan, 2007). Today, cell culture is a fundamental and irreplaceable tool in many research, biotechnology, and testing laboratories. For more on the history of cell culture see the article “Celebrating a Century of Cell Culture“.
The recent report by the U.S. National Research Council, Toxicity Testing in the 21st Century: A Vision and Strategy, forecasts a shift in the direction of toxicity testing away from in vivo methods and towards in vitro and other non-animal methods. Cell-based assays, in the form of test batteries and/or as part of tiered testing schemes to predict human toxicity endpoints, will be a key technology used to achieve this goal. The role of cell-based assays in toxicology and toxicity testing has steadily increased over the past several decades; however, their validation and acceptance for regulatory testing purposes has been slow and problematic. Only stand-alone cell-based assays have been evaluated thus far in validation studies, and, although some have been recommended for use as screening assays or as a component in a tiered testing scheme, few have been endorsed as full replacements for an animal test method.
Cells are considered to be the basic ‘building blocks’ of living organisms. However, the identity, behavior, and survival of a cell depend not only on neighboring cells, but on many molecular pathways and biological processes occurring within the entire organism. This is the premise – the biological complexity and inter-connectedness of the pathways – which is used by whole animal toxicologists to suggest that cell-based assays will never be sufficient to predict human toxicity. On the other hand, it is precisely this complexity of the whole organism as well as the need to use animal models to obtain whole organism toxicity data which are problematic when it comes to understanding human and mechanistic toxicity responses. This ‘battle’ over ‘the best’ test model has existed since cell biologists, biochemists, and toxicologists developed their respective research specialties and preferences.
There is a great need for physiologically relevant human cell-based assays that can provide the biomedical and toxicity data that cannot be obtained from animal models or humans. With the current growth in new technologies, knowledge, and methods for complex data analysis, it now appears feasible that data from reductionist methods (of which cellular assays are an important component) will be able to be reassembled into models that can better predict human toxicity. Some of the recent developments and trends in cell-based assays that are relevant to the future of toxicity testing will be reviewed in this article series.
Cell Culture Basics
Living cells are isolated from human or animal tissues using a number of techniques. Many cell types are present in any tissue, so methods have been developed to isolate relatively pure populations of particular cell types. To maintain cells in culture (grow them over time in laboratory cabinets that control environmental factors), the cells must have access to essential nutrients (culture medium), be maintained at physiological temperature (typically 37°C), and be maintained in a sterile environment so that they are not overgrown by microorganisms (contamination). Specialized equipment, supplies, and cell culture reagents have been developed and are commercially available so that cells can be isolated and maintained without contamination. Most types of cells can also be frozen and stored for long periods of time, and then thawed and cultured again when needed for experiments.
Many types of cells have a characteristic morphology (their shape and appearance which can be viewed microscopically). However, to specifically identify the type of cell in a culture, the cells must be characterized. Techniques to characterize cells include: morphology, identification of cell-specific proteins within or on the surface of the cells, and genetic analysis.
To obtain more information or training on basic cell culture techniques, the reader is referred to the following sources:
Corning’s Online Cell Culture and Assay Training
SBS Online Cell-based Assays for Drug Discovery
NIH BIO-TRAC: Lecture and laboratory training
Good Cell Culture Practice
The principles of Good Laboratory Practice (GLP) are quality measures used to ensure the implementation of standardized procedures and record keeping for particular studies. Good Cell Culture Practice (GCCP) is the expanded application of the principles of GLP to cell-culture based studies. GCCP includes specifications for procedures that may not be encountered with traditional GLP studies. Special considerations relevant to GCCP are described in the presentation “Critical aspects in implementing the OECD Monograph 14 ‘The application of the principles of GLP to in-vitro studies‘”. Examples include: special attention for aseptic conditions, and characterization, maintenance, viability, and responsiveness of the test system.
Three key organizations involved in assessing the validation status of toxicity test methods, the Organisation for Economic Cooperation and Development (OECD), the European Centre for the Validation of Alternative Methods (ECVAM), and the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), have all participated in developing documents to define GCCP principles.
Individual mammalian cells are not visible to the human eye. Most scientists are familiar with using light and phase-contrast microscopy to view fixed and stained cells or tissues on slides, and to follow the growth of cells being propagated for experiments. New methodologies in imaging are now allowing us to move beyond static cell images, and to obtain data on functional molecules in 3-dimensional viable cell cultures over time.
New technologies and more complex instrumentation have led to the establishment of centralized cell imaging facilities at many institutions. These facilities may house complex workstations that perform functions such as confocal microscopy and/or live cell imaging. Newer confocal microscopy instruments coupled with digital imaging can provide high-resolution 3- and 4-dimensional images of cells by capturing images from laser scans through thin layers and then reconstructing the final image. Confocal systems have been developed that can be used to measure intracellular parameters such as ion concentration and signal transduction without the loss of fluorescence signal, and that can provide variable excitation and emission wavelengths so that different dyes can be optimally excited while minimizing tissue damage. Various features of Zeiss, Olympus, Nikon, and Leica confocal microscopes for live cell imaging have been reviewed by May (2007).
Changes within cells and their responses to stimuli can now be observed in real time using live cell imaging. Live cell imaging can be performed using various types of microscopy, including confocal, and requires an environmentally controlled chamber on the microscope stage for maintaining the cell’s environment as they are viewed (Swedlow & Platani, 2002). Cell environmental chambers may consist of static cultures such as microwell plates, or flow through systems (plates or microfluidic platforms) where reagents and media can be rapidly altered in the individual compartments. Live cell imaging is an extremely powerful tool that goes beyond mere visualization of cell structures; it has contributed to our knowledge of the location, function, and interactions of proteins in cells – more on this topic in Part II (high-content screening and cell signaling pathways).
For microscopic visualization, cells and cellular components are typically labeled in some manner. For live cell imaging, concerns about labeling include whether the technique affects cell viability and/or perturbs cellular function. The workhorse of cellular labeling has been the fluorescent tagged protein with its ease of use and wide availability, but also with technical limitations (autofluorescence and photobleaching) and the potential for phototoxic damage to the cells. In the earliest live cell imaging experiments, fluorescent-tagged proteins were microinjected into cells (Haraguchi, 2002). This method had many limitations, and has been replaced by the use of genetically encoded fluorescent proteins produced by the cells themselves. Molecular biology techniques were developed for transfecting cDNA into cultured cells that signaled the cells to express specific proteins tagged with the green fluorescent protein (GFP). Cells expressing GFP-tagged proteins can be imaged using real-time live cell microscopy. Limitations to this technique include potential artifacts and possible loss of function of the tagged protein, as well as the technical limitations inherent with using any fluorescent probe (Girotti & Banting, 1996; Swedlow & Platani, 2002). Even so, GFP labeling, digital imaging, and advances in microscopy instrumentation have revolutionized our ability to visualize and understand the functioning of living cells.
Quantum dots (QDs) are the ‘new kid on the block’ when it comes to live cell labeling for microscopic visualization. QDs are nanometer-sized particles composed of ceramic-coated semiconductor material, which provide a brighter and more stable fluorescent signal in a multitude of colors; the color varies with the dot size and semiconductor material (DePalma, 2007). These unique optical properties make fluorescent QDs useful for cell labeling applications “that require long-term, multi-target, and highly sensitive imaging” (Jaiswal & Simon, 2004). For intracellular labeling, QDs can be conjugated to proteins that are taken up by cells (Biju, et al., 2007; Tomlinson, 2007), or they can be carried across cell membranes by encasing them in cationic liposomes (Dudu, et al., 2008). Without conjugation or liposome delivery, QDs can be difficult to get into cells. Other shortcomings of QDs, such as their tendency to aggregate in aqueous solutions, are being resolved (Jaiswal & Simon, 2004; Netterwald, 2008). QD cytotoxicity has been reported in some studies, and lack of cytotoxicity has been reported in other studies. A review of QD toxicity suggests that cytotoxicity varies and can be modulated (Hardman, 2006). QDs for cellular imaging are therefore modified to minimize cytotoxicity and to have other favorable properties such as water solubility and low non-specific binding (Liu, et al., 2008).
An emerging application of quantum dots is their use in tracking the location and monitoring the survival of stem cells in vivo and in vitro. This technology has proved useful for stem cells implanted into animals. Mouse embryonic stem cells labeled with six different QDs provided multiplex imaging of the stem cells in the in vivo tissue (Lin, et al., 2007). The QDs did not appear to adversely affect stem cell viability, proliferation, or differentiation. Human mesenchymal stem cells have also been labeled using QDs and followed over time with different effects reported on their replication and differentiation (Hsieh, et al., 2006; Rosen, et al., 2007; Shah, et al., 2006). Muller-Borer, et al. (2007) followed the proliferation and differentiation of QD-labeled rat bone marrow MSC in an in vitro model, a cardiac myocyte and MSC co-culture. The labeled MSCs, observed using fluorescence confocal microscopy, were bright and photostable, but dose-dependent QD cytotoxicity was observed suggesting the need to optimize the protocol to the lowest QD concentration found useful.
Detection methods based on label-free imaging are being developed to overcome the limitations of using labeled proteins and to protect the viability of the cells being observed. Three types of spectroscopic microscopes based on infrared, near-infrared, and Raman spectroscopy can be used to view molecules within cells (Dove, 2008). Spectroscopic imaging of cultured cells is an emerging field, reported to have the potential to “reveal extremely detailed maps of the predominant molecular structures in different regions” (Dove, 2008).
Cell imaging technologies are now being combined with high-throughput and high-content cell screening assays. These types of multi-parameter assays are being developed by pharmaceutical companies as cell-based safety assessments (CBSA) for the early identification of problem compounds (Shaw, 2008). While pharma companies did not have much confidence in the ability of simpler cell-based assays to predict toxicity, many are investing in multi-parameter CBSA for toxicity screening. Because of new initiatives in cell-based toxicity testing, such as the US inter-agency high throughput toxicity screening initiative, it is timely to consider whether the new cell-based methods will be the disruptive technology needed to launch toxicity testing into a new generation. Cell-based assays, relevant technologies, and examples of particular assays used for toxicity testing will be discussed next month in Part II.
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