Preparation and Testing of Impedance-based Fluidic Biochips with RTgill-W1 Cells for Rapid Evaluation of Drinking Water Samples for Toxicity

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial (RTgill-W1) cells for use in a field portable water toxicity ...

U.S. Army Center for Environmental Health Research

In 1996, the U.S. Congress mandated the development of a screening program for endocrine-disrupting chemicals (EDCs) using validated test systems. Subsequently, the Endocrine Disruptor Screening and Testing Advisory Committee recommended the development of a standardized amphibian assay for tier 2 testing of EDCs. For that reason, a tier 2 testing protocol using Xenopus (Silurana) tropicalis and a 30-week, flow-through exposure to the antiandrogen flutamide from stage 46 tadpoles through sexually mature adult frogs were developed and evaluated in this pilot study. The endpoints for this study included measurements of frog body lengths and weights, liver weights, ovary/egg mass weights, testicular and ovarian histopathology, plasma vitellogenin levels, and notes on any abnormalities observed at necropsy. Increasing exposure concentrations to flutamide caused significant increases in frogs with no recognizable gonadal tissue and increased body and liver weights in male frogs, whereas the body lengths and weights decreased significantly in female frogs. Important issues must be resolved before a tier 2 amphibian assay can be further developed and validated, including the establishment of baseline values in the controls for the parameters under study; the maintenance, measurement, and timing of exposure concentrations; and the development of additional biomolecular markers of effect. This study demonstrated the feasibility of conducting long-term EDC exposure studies using X. tropicalis.

A novel amphibian tier 2 testing protocol: a 30-week exposure of Xenopus tropicalis to the antiandrogen flutamide.

A major limitation to using mammalian cell-based biosensors for field testing of drinking water samples is the difficulty of maintaining cell viability and sterility without an on-site cell culture facility. This paper describes a portable automated bench-top mammalian cell-based toxicity sensor that incorporates enclosed fluidic biochips containing endothelial cells monitored by Electric Cell-substrate Impedance Sensing (ECIS) technology. Long-term maintenance of cells on the biochips is made possible by using a compact, self-contained disposable media delivery system. The toxicity sensor monitors changes in impedance of cell monolayers on the biochips after the introduction of water samples. The fluidic biochip includes an ECIS electronic layer and a polycarbonate channel layer, which together reduce initial impedance disturbances seen in commercially available open well ECIS chips caused by the mechanics of pipetting while maintaining the ability of the cells to respond to toxicants. A curve discrimination program was developed that compares impedance values over time between the control and treatment channels on the fluidic biochip and determines if they are significantly different. Toxicant responses of bovine pulmonary artery endothelial cells grown on fluidic biochips are similar to cells on commercially-available open well chips, and these cells can be maintained in the toxicity sensor device for at least nine days using an automated media delivery system. Longer-term cell storage is possible; bovine lung microvessel endothelial cells survive for up to four months on the fluidic biochips and remain responsive to a model toxicant. This is the first demonstration of a portable bench top system capable of both supporting cell health over extended periods of time and obtaining impedance measurements from endothelial cell monolayers after toxicant exposure.

A portable cell-based impedance sensor for toxicity testing of drinking water.

Rainbow trout gill epithelial cells (RTgill-W1) are used in a cell-based biosensor that can respond within one hour to toxic chemicals that have the potential to contaminate drinking water supplies. RTgill-W1 cells seeded on enclosed fluidic biochips and monitored using electric cell-substrate impedance sensing (ECIS) technology responded to 18 out of the 18 toxic chemicals tested within one hour of exposure. Nine of these chemical responses were within established concentration ranges specified by the U.S. Army for comparison of toxicity sensors for field application. The RTgill-W1 cells remain viable on the biochips at ambient carbon dioxide levels at 6°C for 78weeks without media changes. RTgill-W1 biochips stored in this manner were challenged with 9.4μM sodium pentachlorophenate (PCP), a benchmark toxicant, and impedance responses were significant (p<0.001) for all storage times tested. This poikilothermic cell line has toxicant sensitivity comparable to a mammalian cell line (bovine lung microvessel endothelial cells (BLMVECs)) that was tested on fluidic biochips with the same chemicals. In order to remain viable, the BLMVEC biochips required media replenishments 3 times per week while being maintained at 37°C. The ability of RTgill-W1 biochips to maintain monolayer integrity without media replenishments for 78weeks, combined with their chemical sensitivity and rapid response time, make them excellent candidates for use in low cost, maintenance-free field-portable biosensors.

Long-term storage and impedance-based water toxicity testing capabilities of fluidic biochips seeded with RTgill-W1 cells.

The US Army's need for a reliable and field-portable drinking water toxicity sensor was the catalyst for the development and evaluation of an electric cell-substrate impedance sensing (ECIS) device. Water testing technologies currently available to soldiers in the field are analyte-specific and have limited capabilities to detect broad-based water toxicity. The ECIS sensor described here uses rainbow trout gill epithelial cells seeded on fluidic biochips to measure changes in impedance for the detection of possible chemical contamination of drinking water supplies. Chemicals selected for testing were chosen as representatives of a broad spectrum of toxic industrial compounds. Results of a US Environmental Protection Agency (USEPA)-sponsored evaluation of the field portable device were similar to previously published US Army testing results of a laboratory-based version of the same technology. Twelve of the 18 chemicals tested following USEPA Technology Testing and Evaluation Program procedures were detected by the ECIS sensor within 1 h at USEPA-derived human lethal concentrations. To simplify field-testing methods further, elimination of a procedural step that acclimated cells to serum-free media streamlined the test process with only a slight loss of chemical sensitivity. For field use, the ECIS sensor will be used in conjunction with an enzyme-based sensor that is responsive to carbamate and organophosphorus pesticides.

Evaluation and refinement of a field-portable drinking water toxicity sensor utilizing electric cell-substrate impedance sensing and a fluidic biochip.

Linda M. Brennan

U.S. Army Center for Environmental Health Research

Preparation and Testing of Impedance-based Fluidic Biochips with RTgill-W1 Cells for Rapid Evaluation of Drinking Water Samples for Toxicity