A European Commission-funded public-private consortium led by scientists from Commissariat á l’Énergie Atomique in France has published a paper demonstrating how they combined arrayed cell-based nanodroplet assays with high-content image-based screening to create a tool for high-throughput toxicity testing.
The project, called Toxdrop, is another example of the increasing use of cell-based assays as tools for assessing drug toxicity well before the animal testing stage and underscores the burgeoning use of high-content imaging in such testing.
The research may also benefit French biological imaging firm Imstar, which will have a hand in commercializing future versions of the “Cell-on-Chip” technology, including a combined cytotoxicological and genotoxicological assay.
The findings, published in the January 2007 issue of PLoS One, were the result of a two-year, €1.62-million ($2.1 million) Specific Targeted Research Project (STREP) grant awarded by the European Commision’s Sixth Framework Program. The project began in January 2005 and ended this past December.
Specific terms of the STREP funding program call for the participation of a minimum of three partners from three different EU member states or associated states, of which two must be member states or associated candidate countries. The goal of the STREP projects, according to the FP6 website, is to improve European competitiveness in specific areas or to meet the needs of society or community policies.
The specific goal of the Toxdrop project is to develop a new substrate for testing chemical toxicity in vitro, which falls in line with the EU’s recent Registration, Evaluation, and Authorization of Chemicals (REACH) legislation to develop toxicity testing techniques that “reduce, refine, and replace” the use of live animals.
Participants in the project include the Commissariat á l’Énergie Atomique, Université Claude Bernard Lyon, Institut National de la Santé et de la Recherche Médicale, and biotech firm Imstar, all located in France; the Joint Research Center in Belgium; mass spec vendor Tascon in Germany; and Ecole Polytechnique Fédérale de Lausanne in Switzerland.
As described in PLoS One, the researchers engineered a cell-based toxicity assay based on both stress promoter activation and morphological changes in genetically engineered cell lines. Specifically, they used a stress-inducible fluorescent HepG2 cell model in which heat shock promoters controlled enhanced green fluorescent protein expression.
To increase the throughput of the assay, they arrayed the cells inside several hundred individual nanoliter droplets on a small patterned glass substrate. This technology, dubbed Cell-on-Chip, has the effect of creating hundreds of individual miniature cell cultures, each of which can be interrogated differently or exposed to different chemicals.
The researchers then combined this device with the Pathfinder automated imaging platform made by Imstar to conduct high-resolution image-based phenotypic screens of multiple cellular parameters using three fluorescent markers. This allowed them to analyze cell viability, fluorescence intensities, and cell morphological changes, thereby “providing invaluable information on the behavior of individual cells in the presence of a compound,” the researchers wrote in the paper.
The scientists tested the platform by exposing cells to varying concentrations of the poisonous heavy metals sodium arsenate and cadmium chloride, as well as the organic herbicide paraquat.
They were able to observe chemical-specific responses, identified through differences in cell dynamics and fluorescence amplitudes, that agreed with previous studies of the same chemicals using a standard microwell format and cell-death assays. In addition, they were able to obtain IC50 values for the heavy metal toxicants that agreed with published data.
One of the key implications of the findings, the researchers wrote, is that their method was not only more sensitive than assays based on cell mortality alone, but it also allows them to obtain multiplexed information about the cells’ responses over time.
“This allowed them to analyze cell viability, fluorescence intensities, and cell morphological changes, thereby providing invaluable information on the behavior of individual cells in the presence of a compound.”
According to CEA’s Beatrice Schaack, who coordinated the Toxdrop project, the researchers are now turning their attention to commercializing the Cell-on-Chip.
“It is ready for commercialization,” Schaack wrote in an e-mail to CBA News this week. “We are talking to some industries right [now] about it. We believe it might take two years to get ready.”
Even while pursuing commercialization of the chip in its current format, the consortium has begun developing future iterations. Imstar will continue to have a hand in these developments, Schaack said.
Imstar, based in Paris, manufactures several high-content imaging platforms based on the Pathfinder core technology. This technology is a CCD-based automated imaging platform that the company originally developed for cytogenic applications such as karyotyping and fluorescence in situ hybridization, but has since adapted it for cell biology studies.
The company has flown under the radar for the last several years, at least in the high-content screening space, primarily because it has focused its instrumentation on the diagnostic and clinical markets, putting them more in line with companies such as Applied Imaging, Clarient, and even CompuCyte.
“Imstar’s high-content analysis tools are relatively cheap,” Schaack wrote in her e-mail. “We have [had] a strong collaboration together [for] four years.”
Specifically, Schaack said, the consortium has undertaken another project funded by the EC called COMICS, which seeks to increase the throughput of traditional Comet assays for DNA damage and combine it with the established cytotoxicological assays.
Schaack did not disclose the length or amount for funding for this project. She also said that additional projects will follow.
“We have suggested its use to screen the toxicity of nanoparticles,” she wrote in her e-mail. “The nanoparticles could be extended on the substrate of the chip before toxicity screening.”