Assay for molecular transport across gap junction channels in one-dimensional cell arrays

Abstract

Transport across gap junction channels (GJCs) between neighboring cells is critical to synchronizing cell's electrical and metabolic activities and maintaining cell homeostasis. Here we present a non-invasive microfluidic method to measure molecular diffusion across GJCs in multiple 1D cell arrays in real time. Using the chip, selective loading of a membrane permeant fluorescence dye (carboxyfluorescein) in Normal Rat Kidney (NRK) cells shows that the dye was able to diffuse through three cells along single cell chains in ∼35 minutes. Application of 100 µM 2-aminoethoxydiphenyl borate (2-APB) reversibly inhibits connexin-43 gap junctions in NRK cells; 0.8 mM 1-heptanol inhibits the diffusion partially. The method offers rapid exchange of reagents, enabling sequential screening of multiple gap junction specific drugs with only one preparation of cells. It is capable of measuring gap junction mediated diffusion between single cells.

Microfluidic Platforms for the Investigation of Intercellular Signalling Mechanisms

“…• Allows studying temporal dynamics of cell signalling mechanisms [32,77] • Facilitates tuning the diffusion to convection ratio to distinguish between autocrine and paracrine signalling mechanisms [39] • Allows precise control over the surface chemistry of the microfl uidic structures [8] • Enables precise control over shear stress induced cell signalling mechanisms [8,23] • Provides scalability in cell signalling analysis [8] • Allows mimicking of in-vivo conditions [8,48] Precise cell patterning • Enables formation of customised cell patterns [5b , 8] • Allows mimicking of in-vivo conditions [8,48] • Allows studying cell signalling down to the single cell level [10] Integration with other components and technologies • Enables customised microdevices capable of sequential processes such as fi ltering, mixing and heating [78] • Allows mimicking of in-vivo conditions [8,48] Parallelisation and automation • Enables multiple assays either under the same or different conditions to be run at the same time [54,55,76] • Enables high throughput experiments [8[ • Eliminates time consuming, labour-intensive processes [76] • Much cheaper than conventional fl uid-handling robots [76] small 2014, DOI: 10.1002/smll.201401444 as stochastic optical reconstruction microscopy (STORM), capable of recording dynamic processes in live cells with nanometer resolution. [ 80 ] Detection at the single-molecule level would be very useful in signalling investigations, although on-chip STORM is still in development.…”

“…[ 22 ] Correspondingly, Ye and colleagues. [ 23 ] developed a microfl uidic chip on which they performed dye transfer experiments to assess gap junction mediated diffusion. Additionally, Bathany and associates [ 22 ] utilised a similar approach, although using a tristream laminar fl ow, with cells in a single stream loaded with fl uorescent dye and the dye diffusion via GJCs to unloaded cells in adjacent streams measured.…”

Analysis of intercellular communication by flexible hydrodynamic gating on a microfluidic chip

“…The microfluidic chip was fabricated according to the rapid prototyping and replica molding method as previously reported [22][23][24]. In brief, SU-8 (GM 1070, Gersteltec, Switzerland) mold was fabricated on a silicon wafer using standard soft-lithography technique.…”

A microfluidic platform for measuring electrical activity across cells

“…The results are consistent with our previous studies using one dimensional cell arrays. 26 As expected, electrical coupling and molecular diffusion processes are correlated but, the electrical measurement is more sensitive and faster.…”

“…This approach allows observation of dye loading and diffusion between different regions. 25,26 Here, we show that the tri-stream chip can be used to measure electrical coupling across gap junctions. The chip is designed in a generic form so that it can be used to measure both electrical conductance and molecular diffusion in real time.…”