Construction Of Microfluidic Platform For In Vitro Gairdia Lamblia Culturing And Drug Screening

Objective: Giardia lamblia (syn. Giardia) is a unicellular parasitic protozoa leading toGiardiasis, which was worldwide distribution. However, most of the targeted drugs hadserious side-effects. Due to the high risk of contamination and the need for standardizedlow-oxygen environment in traditional drug screening, the new drug researches wereinefficiency, low throughput and high cost. Here, an integrated microfludic device for in vitroculturing and screening of Giardia was constructed, which simulated the in vivo growth environment of Giardia. It would provide a new platform for drug screening.Methods: The microfluidic device in our study integrated a polydimethylsiloxane(PDMS) chip with an anaerobic culture system. The PDMS chip mainly consists of anupstream CGG and a downstream diffusible culture module. A series of solutions withdifferent concentrations of drug were formed at the outlets of CGG. Then the flow channelsexchanged medium by diffusible channels with the culture module to maintain the in vivoenvironment. An anaerobic culture system was employed as it can remain in an air-tightenvironment flushed with gas mixtures as wish. Rhodamine123was used as an indicator forestimating the gradient produced by CGG and the diffusion condition of the diffusible culturechamber. The gas condition was optimized using the microfluidic device. And then, underoptimized gas condition, Giardia trophozoites were respectively inoculated into themicrofluidic chambers or a classic96-well microplate for72hour. Lastly, the drugsusceptibility assay of metronidazole and tinidazole were conducted on the microfluidic chipor96-well microplate, respectively.Results: The results proved that, eight concentrations of toxicants were generatedprecisely/rapidly in30s by the CGG module. The fluorescence intensity of Rh123inside thechambers could be uniform and close to that of the parallel flow channels within twenty fiveminutes. Microfluidic cultures did not support adequate growth under3%O2and were totallyunsatisfactory under5%O2. In contrast, strict anaerobiosis (100%N2) and flushing with themicroaerophilic gas mixture (1%O2,5%CO2, and94%N2) resulted in better growth(P<0.05). The best growth was observed under microaerophilic gas mixture in microfluidics.What’s more, two methods both supported rapid growth of trophozoites at48h (P>0.05). At72h, noticeable34×increase was found in microfluidic culture, but only23×increase wasfound in100μL plate. This implied that microfluidic culture supply significantly support longer growth period than96-well microplate (P<0.05). The correlation between microfluidicdevice and96-well microplate drug susceptibility assay results exhibited the same dosedependent inhibition trend and obtained values were comparable. For metronidazole, the IC50values were5.13μM and4.52μM, respectively; for tinidazole, the IC50values were2.00μMand2.85μM, respectively.Conclusions: The device simulated the in vivo growth environment of Giardia andcould be used for in vitro culturing and screening of Giardia. The application of this devicerepresents the first step in developing a completely integrated microfluidic platform forhigh-throughput screening and might be expanded to other assays based on in vitro culture ofG. lamblia with further tests.