A drug-compatible and temperature-controlled microfluidic device for live-cell imaging

Abstract

Monitoring cellular responses to changes in growth conditions and perturbation of targeted pathways is integral to the investigation of biological processes. However, manipulating cells and their environment during live-cell-imaging experiments still represents a major challenge. While the coupling of microfluidics with microscopy has emerged as a powerful solution to this problem, this approach remains severely underexploited. Indeed, most microdevices rely on the polymer polydimethylsiloxane (PDMS), which strongly absorbs a variety of molecules commonly used in cell biology. This effect of the microsystems on the cellular environment hampers our capacity to accurately modulate the composition of the medium and the concentration of specific compounds within the microchips, with implications for the reliability of these experiments. To overcome this critical issue, we developed new PDMS-free microdevices dedicated to live-cell imaging that show no interference with small molecules. They also integrate a module for maintaining precise sample temperature both above and below ambient as well as for rapid temperature shifts. Importantly, changes in medium composition and temperature can be efficiently achieved within the chips while recording cell behaviour by microscopy. Compatible with different model systems, our platforms provide a versatile solution for the dynamic regulation of the cellular environment during live-cell imaging.

Thermoplastic elastomer with advanced hydrophilization and bonding performances for rapid (30 s) and easy molding of microfluidic devices

“…The absorptive properties of Flexdym TM and PDMS were also tested using a more sensitive cell$based assay. 54 We used fission yeast cells whose proliferation is solely driven by a fusion protein between the cell cycle cyclin$ 25 dependent kinase Cdc2 and the cyclin B Cdc13. 55 For these experiments, DC450 cells ( 54 were grown in minimal medium plus supplements (EMM6S) at 32 °C.…”

“…54 We used fission yeast cells whose proliferation is solely driven by a fusion protein between the cell cycle cyclin$ 25 dependent kinase Cdc2 and the cyclin B Cdc13. 55 For these experiments, DC450 cells ( 54 were grown in minimal medium plus supplements (EMM6S) at 32 °C. These 30 cells are sensitive to dose$dependent and reversible inhibition of Cdc2 activity by the ATP analog 3-MBPP1 (A602960, Toronto Research Chemicals Inc.).…”

“…We then ascertained the absorptive properties of Flexdym TM using a cell$based assay, as our previous study demonstrated that the rhodamine test is not sufficiently sensitive to precisely evaluate this parameter. 54 To 30 this end, fission yeast cells operating with an analog$sensitive fusion protein of the cell cycle cyclin$dependent kinase Cdc2 and the B$type cyclin Cdc13 55 were treated with 1 M of the ATP analog 3-MBPP1 (a small hydrophobic molecule) and grown in contact with either a glass substrate, Flexdym TM or PDMS (see…”

Whole-head recording of chemosensory activity in the marine annelid Platynereis dumerilii

“…The trap design could also be adapted for other marine larvae or small organisms, some of which are suited for microfluidic experiments (TF Chartier 2015, personal observations). Though our conclusions would not have been altered, absorption issues documented for PDMS devices may have reduced effective stimulant concentrations [ 88 ]; hence, alternative materials such as COC [ 89 ] or sTPE [ 90 ] may be preferred to PDMS for future experiments. Microfluidic set-ups have been successfully used to explore neuronal and motor activity in nematodes and fish larvae [ 4 , 91 ], and our study shows that similar experiments are possible with Platynereis juveniles.…”

CDK activity provides temporal and quantitative cues for organizing genome duplication

“…Importantly, previous studies using this system have demonstrated a tight quantitative relationship between CDK activity and the level of inhibitor to which cells are exposed. These notably reported differences in substrate phosphorylation [ 28 ], periodic gene expression [ 29 ], cell cycle progression [ 13 ], and cell size at division [ 30 ] that were dependent on the concentration of inhibitor applied. This powerful approach thus allows us to impose precise changes in a single CDK activity in vivo in order to explore the roles of the dynamics and levels of CDK activity in origin selection.…”