Development of a Microfluidics-Based Approach for Investigating Microtubule Polymer Mechanics

In this protocol, we describe the design and fabrication of a microfluidic device developed for the investigation of microtubule polymer mechanics. The design utilizes the intrinsic benefits of Polydimethylsiloxane (PDMS)-based microfluidic devices and introduces several features to enable a robust and customizable high-throughput experimental approach. The developed device incorporates redundant bubble-trapping capabilities to prevent the occurrence of detrimental air bubbles. Furthermore, the device interfaces with an automated flow control system to reduce manual intervention and enable high-throughput analyses. Commercial simulation software is utilized to better develop and understand the fluid transport using this system. Finally, we demonstrate the capability to conduct multiple experiments simultaneously within a single device by growing microtubule extensions with distinct fluorescent labels in different sections of the device. Overall, this microfluidic flow system can be used to probe microtubule polymer mechanics and provides improvements in experimental design for broader microtubule in vitro studies. The synthesis of microfabrication, automated flow control, and computational modeling approaches enables a flexible system ideally suited for probing the cellular cytoskeleton in vitro.