In the Spotlight
Cell-based Microfluidic Devices for Toxicity Testing
Cell-based microfluidic devices, the application of microfluidic technology to cell culture-based assays, are also described as “cell chips,” “cell biochips,” or “microbioreactors.” These microscale cell assay devices are now becoming practical tools for the rapid screening of chemicals and drugs, and several have been developed specifically as toxicity screening assays.
Cell biochip devices are based on methods developed for larger scale cell assays, such as cytotoxicity or inflammatory mediator release, which are then miniaturized as very small chambers in the flow-through device. Multiple chambers are typically used to serve as media reservoirs, air chambers, and cell repositories. A device may contain one cell type in one or more chambers, or different types of cells from various organs in different chambers. Devices are adaptable to the use of animal or human cells, primary cells or cell lines, adherent or non-adherent cells, and cells cultured as monolayers (2D or 2-dimensional cultures) or as 3-dimensional (3D) stratified, multilayered, or aggregated cultures.
Chamber configurations and connections are modified to carry out different types of experiments, such as recirculation of media or reagents to different chambers in a specific sequence. In many cases, researchers have microfluidic devices manufactured to their specifications, but cell-free commercial devices are available (for example, CellASIC Corporation). Possible variations on microfluidic testing platforms and their applications are enormous – delimited only by the creative talents of the developer as well as current technological capabilities.
Assay detection methods may be limited by the device and by the small number of cells (Kim, et al., 2007). Transparent plastic is used so the optical properties of microfluidic devices are compatible with non-destructive optical detection methods and provide for monitoring cell morphology and survival. Yang, et al. (2007) described electrochemical and optical assay detection methods as those most often used in cell-based high-throughput assays.
Cell-based microfluidic devices for toxicity testing are still in the research and developmental stages, and are being developed by various academic researchers and biotech companies. Cell-based microfluidic applications for toxicity testing involve drug screening assays intended to increase throughput while reducing the time and cost of the drug discovery process, and chemical toxicity screening that benefits similarly from increased throughput. Cell chip platforms are currently being designed as screening assays, which like larger-scale cell screening assays, may reduce animal testing in early drug discovery or by prioritizing chemicals for further testing, but will not replace animals in tests required by regulatory authorities.
The purpose of this article is to highlight some recent applications of cell-based microfluidic technology for toxicity testing. References are provided for those wishing to explore the technology and applications in greater detail.
A microfluidic device for pharmacokinetic/metabolism screening was developed by the Schuler lab at Cornell (Viravaidya, et al., 2004). Their early “animal-on-a-chip” model was composed of cultured cells to mimic the organ systems of an animal, and included target organ cells (lung), liver cells, and several empty chambers proposed for the future addition of other tissues and fat cells. Monolayer cultures of liver and lung cell lines were maintained in separate chambers, which were connected so that fluid could be pumped between the chambers. The liver cells were exposed to a chemical and the media containing the metabolites were transported by fluid flow to the lung cells where their toxic effect was assessed. This type of assay was designed to replicate the toxic effect of a chemical/drug in the same manner it occurs within the human body. The goal for this device was to add other organs to create an in vitro ADMET (absorption, distribution, metabolism, excretion/ toxicity) assay system. This same concept for testing the toxicity of metabolized compounds has been used successfully with standard-sized cell cultures (Li, et al., 2004). The primary purpose in miniaturization is increased throughput – more compounds can be screened faster and at reduced cost. The microfluidic cell chip technology developed at Cornell is under further development by the Hµrel Corporation (Beverly Hills, CA, US), where it will be offered as a testing service after the method is validated.
Microscale perfusion devices have also been developed using cells from specific organs. A renal microchip was developed using the MDCK kidney cell line as an in vitro model for chronic toxicity testing of chemicals (Baudoin, et al., 2007b). Sivaraman, et al. (2005) developed a micro-perfused system of 3D rat liver cells and demonstrated they retained function similar to the in vivo tissue to a greater extent than the same cells cultured by other methods. Applications they proposed for their device included testing of drugs and chemicals for acute and chronic liver toxicity. Primary human keratinocytes (skin cells) were used for cytotoxicity testing in a microfluidic device engineered to create linear dilutions of the test substance, an essential feature for a device to be used for high-throughput screening (HTS) (Walker, et al., 2007).
Stem cells have also been used to seed perfused microbioreactors for toxicity testing applications. Cui, et al. (2007) compared the toxicity responses of human bone marrow cells cultured as 2D monolayers and on 3D scaffolds in a microfluidic device. They observed significant differences in the toxicity responses of the cells cultured in 2D versus 3D formats, and concluded that their microbioreactor platform was “an efficient and standardized alternative testing method” for drug and toxicity testing.
Studies comparing the toxicity responses of monolayer and 3D cultured cells in microscale perfusion devices overwhelmingly claim that the 3D cultures more closely resemble cell responses observed in vivo. Yang, et al. (2008) published a review of cells, toxicity assays, and assay endpoints useful for high-throughput cytotoxicity analysis in microfluidic devices, and concluded that 3D cell cultures that mimic the in vivo tissue are essential for obtaining results comparable to the in vivo response. Their review also discusses potential applications of microbioreactor cultures for chronic and systemic toxicity testing, and the use of high-density microfluidic arrays for high-throughput and high-content screening.
A common feature among the microscale perfusion systems reviewed here is the claim of improved functionality of the systems in replicating the in vivo state of the cells. This was an advantage cited in addition to the miniaturization benefits of cost and time savings. Explanations for the claim of the superior cell environment provided by microfluidic devices included the following: the small volume of liquid in the microperfused system more closely replicates the in vivo extracellular volume; the close proximity of different types of cells; the continuous perfusion of media; and the fluidic control of reagent delivery and recirculation. Cell chips using 3D culture formats provided results more representative of the in vivo tissue, analogous to results reported for larger-scale 3D cell cultures.
Developers of cell-based microfluidic devices suggest the devices may provide faster, more cost effective, and technically superior tests that will expedite drug and chemical screening. However, many technical challenges remain before the devices will become widely used for toxicity testing. In addition to the technical challenges, the same limitations encountered with standard cell culture test methods also apply to the miniaturized test platforms; for example, the lack of compatibility with non-aqueous soluble samples. The micro-methods also face the same challenges for assay validation and regulatory acceptance as their larger counterparts.
Specific technical issues with the use of microscale perfusion chambers for cell studies, such as cell shear stress, bubble formation, and inter-assay reproducibility, are being addressed. It is well known that shear stress affects the growth and behavior of adherent cultured cells. Variability in the results obtained with cells cultured in flow chambers has been attributed to the design of the chambers, including the spacing between the chamber plates and the gasket geometry (Anderson & Knothe-Tate, 2007). A flow cell with “predictable and well defined mechanical forces at the surface of a cell monolayer” was designed by Anderson & Knothe-Tate (2007), which provided repeatable and reproducible results not attainable with some of the commercial flow chambers.
The potential benefits of a testing platform that can screen thousands of drug candidates or chemicals per day will certainly address a major bottleneck in drug development and chemical safety testing. Cell microfluidic methods adapted to HTS platforms have the potential to provide rapid testing using physiologically-relevant cell models. When microfluidic testing platforms are validated and marketed, they may become the eagerly awaited technological breakthrough needed to significantly reduce regulatory reliance on animal testing.
This short article has been able to highlight only a few of the microfluidic devices for toxicity testing that have been reported in the literature. Review articles that are recommended to the reader looking for more detailed information include: Baudoin, et al., 2007a; Kim, et al., 2007; and Yang, et al., 2008.
Baudoin, R., Corlu, A., Griscom, L., et al. (2007a). Trends in the development of microfluidic cell biochips for in vitro hepatotoxicity. Toxicol. In Vitro. 21, 535-544.
Baudoin, R., Griscom, L., Monge, M., et al. (2007b). Development of a renal microchip for in vitro distal tubule models. Biotechnol. Prog. 23, 1245-1253.
Cui, Z.F., Xu, X., Trainor, N., et al. (2007). Application of multiple parallel perfused microbioreactors and three-dimensional stem cell culture for toxicity testing. Toxicol. In Vitro. 21, 1318-1324.
Floriano, P.N. (Ed.). (2007). Microchip Based Assay Systems. Humana Press: Totawa, NJ.
Kim, L., Toh, Y.C., Voldman, J. & Yu, H. (2007). A practical guide to microfluidic perfusion culture of adherent mammalian cells. Lab Chip. 7, 681-694.
Li, A.P., Bode, C. & Sakai, Y. (2004). A novel in vitro system, the integrated discrete multiple organ cell culture (IdMOC) system, for the evaluation of human drug toxicity: comparative cytotoxicity of tamoxifen towards normal human cells from five major organs and MCF-7 adenocarcinoma breast cancer cells. Chem. Biol. Interact. 150, 129-136.
Sivaraman, A., Leach, J.K., Townsend, S., et al. (2005). A microscale in vitro physiological model of the liver: predictive screens for drug metabolism and enzyme induction. Curr. Drug Metab. 6, 569-591.
Viravaidya, K., Sin, A. & Shuler, M.L. (2004). Development of a microscale cell culture analog to probe naphthalene toxicity. Biotechnol. Prog. 20, 316-323.
Walker, G.M., Monteiro-Riviere, N., Rouse, J. & O’Neill, A.T. (2007). A linear dilution microfluidic device for cytotoxicity assays. Lab Chip. 7, 226-232.
Yang, S.T., Zhabng, X. & Wen, Y. (2008). Microbioreactors for high-throughput cytotoxicity assays. Curr. Opin. Drug Discov. Devel. 11, 111-127.