(39a) Rapid Formation of Chemical Gradients Combined with Cell Culture for Water Toxicity Testing

The ability to build pumpless fluidic devices that generate controllable chemical gradients while maintaining the portability and scalability of microfluidic systems is of significant benefit for field testing of water safety and security. This work will demonstrate a portable microfluidic device for rapid chemical concentration gradient generation combined with a cytotoxicity test. The concentration gradient profile in the test device can be easily altered and controlled by choosing the initial analyte concentration and by manipulating the timing of the forward and backward flow. This simple and rapid approach for gradient generation on a controllable centimeter-length scale combined with physiologically active cell culture is a promising platform for creating a fieldable water toxicity sensor. Integrating and interpreting cell function in the presence of contaminants changes the paradigm for field sensing where results are obtained on total sample toxicity rather than contaminant identification.

This presentation will describe two applications of this portable microfluidic device for cytotoxicity testing upon rapid concentration gradient generation. In the first application, a stabilized concentration gradient of a cardiac toxin, Alpha-cypermethrin, was used to test the toxicity response of HL-1 cardiac cells seeded within the channel. Three 2µL drops of medium containing 20 mM Alpha-cypermethrin were loaded consecutively into the HL-1-containing microdevice. A concentration gradient of the toxin was established by evaporation when the microdevice was left at ambient conditions for 5 minutes. This gradient was stabilized when the microdevice was transferred to a humidified incubator. HL-1 cells exposed to the toxin concentration gradient for 4h exhibited distinguishable morphologies along the channel, with more severe effects observed in the regions containing higher concentrations of toxin.

A second application incorporates dichlorofluorescein (DCF), a fluorescent marker of cellular oxidative stress, to generate a visual cytotoxicity scale along the microchannel. PC-12 rat pheochromocytoma cells were loaded with the non-fluorescent DCF precursor 2'7'-dichlorodihydrofluorescein diacetate (H2DCFDA) and seeded within the microchannel. A concentration gradient of 3-morpholinosydnonimine (SIN-1), a peroxynitrite generator, was established in the same manner as Alpha-cypermethrin in the previous application, beginning with 100µM toxin. PC-12 cells exposed to the SIN-1 concentration gradient over 4h exhibited decreasing and quantifiable fluorescent intensity along the channel, corresponding to the decrease in toxin concentration. Based upon these SIN-1 data, a toxicity scale was generated along which the relative toxicities of other analytes can be determined based on the length of the fluorescent response given by exposed cells seeded in the microdevice.

This simple and rapid approach for gradient generation on a controllable centimeter-length scale is a promising platform for applications such as drug testing and studying biological phenomena, such as chemotaxis. This represents an initial prototype for constructing a cell-based toxicity sensor that uses a combination of MEMS with a realistic and relevant bioresponse.