| Summary: | Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2013. === Cataloged from PDF version of thesis. === Includes bibliographical references (p. 100-104). === Fluid flow is an essential feature of every microsystem involving cell handling, culture or sorting. The particular application determines the relevant flow rates used in a device. Flows inevitably generate fluid shear stress (FSS) that may cause undesirable physiological cell stress. Simple assays of cell viability, morphology or growth are typically reported to indicate any gross disturbances to cell physiology. However, no straightforward metric exists to specifically evaluate physiological implications of FSS within microfluidic devices, or among competing microfluidic technologies).This thesis presents the first genetically encoded cell sensors that fluoresce in a quantitative fashion upon FSS pathway activation. A transcriptional sensor was chosen, meaning that fluorescence would be turned on when transcription of a relevant protein was initiated. Creating an effective transcriptional sensor requires identifying an appropriate inducible promoter to drive fluorescence expression upon FSS. To this end, the native mechanotransduction of a widely used and easy-to-culture cell line (NIH3T3s) was elucidated by culturing them in a microfluidic device and applying logarithmic FSS via microfluidic perfusion. A panel of shear-responsive genes was analyzed using qRT-PCR, which resulted in the choice of EGR-1 upregulation as the node for FSS detection. A reporter plasmid with a minimal EGR-1 promoter driving the expression of Turbo-RFP fluorescence was chosen and the cell sensor was created by stable transfection and clonal selection. Inducing the pathway with Phorbol-myristate-acetate resulted in fluorescence induction by both microscopy and flow cytometry, verifying the sensor functionality. The fluorescence activation was then characterized across PMA doses and durations. Next, the sensors were tested using multiple duration microfluidic perfusions, where it was noted that the mean induced fluorescence intensity correlated to applied FSS intensity, as desired. It is anticipated that these cell sensors will have wide application in the microsystems community, allowing the device designer to engineer systems with acceptable FSS, and allowing the end-user to evaluate impact of FSS upon their assay of interest. === by Sarvesh Varma. === S.M.
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