Electroporation Loading and Dye Transfer: A Safe and Robust Method to Probe Gap Junctional Coupling

Decrock, Elke; Bock, Marijke De; Baere, Diego De; Hoorelbeke, Delphine; Wang, Nan; Leybaert, Luc

doi:10.1007/978-1-4939-3664-9_11

Cited by 5 publications

(4 citation statements)

References 42 publications

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“…Commonly performed assays to probe gap junctional coupling involve the transfer of a reporter dye from donor cells to acceptor cells. The dye can be mechanically incorporated into donor cells by electroporation, scrape-loading or micropipette injection performed on conventional culture dishes [97][98][99]. These methods are low-throughput, lack spatial organization of cell-cell contacts and mechanically harm the cells [99,100].…”

Section: Discussionmentioning

confidence: 99%

“…The dye can be mechanically incorporated into donor cells by electroporation, scrape-loading or micropipette injection performed on conventional culture dishes [97][98][99]. These methods are low-throughput, lack spatial organization of cell-cell contacts and mechanically harm the cells [99,100]. The more gentle parachute assay consists in incubating the donor cells with the reporter dye and dropping them on a monolayer of acceptor cells, which still leads to cell-cell contacts randomly distributed throughout the culture dishes [101].…”

Section: Discussionmentioning

confidence: 99%

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Development of bone cell microarrays in microfluidic chips for studying osteocyte-osteoblast communication under fluid flow mechanical loading

Yvanoff¹,

Willaert²

2022

Biofabrication

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Bone tissue remodels throughout life in response to mechanical loads. Impaired activities of bone cells (osteocytes, osteoblasts and osteoclasts) result in a disruption of the bone remodelling cycle, which eventually leads to bone disorders such as osteoporosis. To develop efficient therapeutic strategies against bone disorders, new tools are needed to unravel the bone remodelling cycle at the molecular level. Here, we developed a microfluidic platform, which should allow understanding the bone remodelling cycle in much more detail and ultimately be used to discover new therapeutic compounds. We focused specifically on studying cell-cell communication between osteocytes and osteoblasts cells via connexin 43-gap junctions. Therefore, a new cell printing method was developed to create living cellular bone cell arrays in a microfluidic channel. Several cell printing designs where osteocytes and osteoblasts heterotypically interacted at localized interfaces were evaluated. Physical contacts between the bone cells were characterised at high resolution by correlative atomic force microscopy (AFM) - fluorescence microscopy. We demonstrated that the platform is compatible with single-cell mechanostimulation by AFM nanoindentation and subsequent fluorescent analysis of the mechanoresponse. As a proof of concept, we showed the functionality of the platform by analysing the induced in vivo-like Ca++ wave in the printed osteocyte-osteoblast network upon mechanical stimulation by fluid flow shear stress.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Development of bone cell microarrays in microfluidic chips for studying osteocyte-osteoblast communication under fluid flow mechanical loading

Yvanoff¹,

Willaert²

2022

Biofabrication

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“…Gap junctions of all sorts are permeable to ions and small metabolites, and most are permeable to a wide range of molecular probes. Assessing dye permeability is one of the most common methods of identifying cells coupled by gap junctions in vivo or in cell culture (e.g., Hanani, ; Decrock et al, ), often leading to detailed characterization of exogenously expressed channels (Weber et al, ) or a better understanding of channel regulation (Spray et al, ). Although there is great diversity in the cut‐off limit for molecular probes within vertebrates and invertebrates, gap junctions of invertebrates are generally permeable to larger molecules than those of vertebrates (Fig.…”

Section: Functional Aspects Of Vertebrate and Invertebrate Gap Junctimentioning

confidence: 99%

A structural and functional comparison of gap junction channels composed of connexins and innexins

Skerrett

Williams

2016

Developmental Neurobiology

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Methods such as electron microscopy and electrophysiology led to the understanding that gap junctions were dense arrays of channels connecting the intracellular environments within almost all animal tissues. The characteristics of gap junctions were remarkably similar in preparations from phylogenetically diverse animals such as cnidarians and chordates. Although few studies directly compared them, minor differences were noted between gap junctions of vertebrates and invertebrates. For instance, a slightly wider gap was noted between cells of invertebrates and the spacing between invertebrate channels was generally greater. Connexins were identified as the structural component of vertebrate junctions in the 1980s and innexins as the structural component of pre‐chordate junctions in the 1990s. Despite a lack of similarity in gene sequence, connexins and innexins are remarkably similar. Innexins and connexins have the same membrane topology and form intercellular channels that play a variety of tissue‐ and temporally specific roles. Both protein types oligomerize to form large aqueous channels that allow the passage of ions and small metabolites and are regulated by factors such as pH, calcium, and voltage. Much more is currently known about the structure, function, and structure–function relationships of connexins. However, the innexin field is expanding. Greater knowledge of innexin channels will permit more detailed comparisons with their connexin‐based counterparts, and provide insight into the ubiquitous yet specific roles of gap junctions. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 522–547, 2017

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“…Similarly, paired electrophysiological recordings 32 , 33 , even though offering a superior temporal resolution, are limited to studying GJIC between a pair of cells. Electroporation, which uses electric pulses to disrupt cell membrane, can load dye molecules into a large number of cells 34 ; but suitable electrodes and protocols are needed for single cell operations and minimizing cell detachment/death.…”

Section: Introductionmentioning

confidence: 99%

Visualization and quantification of dynamic intercellular coupling in human embryonic stem cells using single cell sonoporation

Fan

Xue

et al. 2020

Sci Rep

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Gap junctions (GJs), which are proteinaceous channels, couple adjacent cells by permitting direct exchange of intracellular molecules with low molecular weights. GJ intercellular communication (GJIC) plays a critical role in regulating behaviors of human embryonic stem cells (hESCs), affecting their proliferation and differentiation. Here we report a novel use of sonoporation that enables single cell intracellular dye loading and dynamic visualization/quantification of GJIC in hESC colonies. By applying a short ultrasound pulse to excite single microbubbles tethered to cell membranes, a transient pore on the cell membrane (sonoporation) is generated which allows intracellular loading of dye molecules and influx of Ca2+ into single hESCs. We employ live imaging for continuous visualization of intercellular dye transfer and Ca2+ diffusion in hESC colonies. We quantify cell–cell permeability based on dye diffusion using mass transport models. Our results reveal heterogeneous intercellular connectivity and a variety of spatiotemporal characteristics of intercellular Ca2+ waves in hESC colonies induced by sonoporation of single cells.

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Electroporation Loading and Dye Transfer: A Safe and Robust Method to Probe Gap Junctional Coupling

Cited by 5 publications

References 42 publications

Development of bone cell microarrays in microfluidic chips for studying osteocyte-osteoblast communication under fluid flow mechanical loading

Development of bone cell microarrays in microfluidic chips for studying osteocyte-osteoblast communication under fluid flow mechanical loading

A structural and functional comparison of gap junction channels composed of connexins and innexins

Visualization and quantification of dynamic intercellular coupling in human embryonic stem cells using single cell sonoporation

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