High throughput assay of diffusion through Cx43 gap junction channels with a microfluidic chip

Area of Science:

• Cell biology
• Biophysics
• Microfluidics

Background:

• Gap junctions mediate direct cell-to-cell communication.
• Measuring gap junction function is crucial for understanding cellular processes and disease.
• Existing methods for assessing dye diffusion can be time-consuming or lack throughput.

Purpose of the Study:

• To develop and validate a microfluidic assay for quantifying gap-junction mediated dye diffusion.
• To enable real-time monitoring of dye movement between adjacent cell compartments.
• To establish a high-throughput screening platform for inhibitors of gap junction communication.

Main Methods:

• Utilized a microfluidic chip with multistream laminar flow to control cell exposure to different environments.
• Implemented a system for continuous cell loading in one compartment and real-time dye diffusion monitoring in a neighboring compartment.
• Applied a one-dimensional diffusion model to determine diffusion coefficients of various fluorescent dyes.

Main Results:

• Successfully measured the diffusion coefficients of four different dyes (5-(6)-carboxyfluorescein, 5-chloromethylfluorescein, Oregon green 488 carboxylic acid, and calcein).
• Demonstrated the ability to assay the inhibitory effects of different compounds on dye coupling.
• Validated the chip's capacity for parallel screening of multiple inhibitors in a single cell preparation.

Conclusions:

• The microfluidic assay provides a convenient and efficient method for measuring gap-junction mediated diffusion.
• The developed technique shows significant potential for high-throughput screening of drugs that modulate gap junction communication.
• This assay can accelerate the discovery of novel therapeutics targeting cell communication pathways.