Research work performed in the laboratory was supported by grants from the Research Foundation - Flanders (FWO; grant numbers G.0545.08 and G.0298.11), the Interuniversity Attraction Poles Program (Belgian Science Policy; grant number P6/28 and P7/13) and is embedded in an FWO-supported research community. CDH is a post-doctoral fellow of the Research Foundation - Flanders (FWO). The authors are very grateful to all current and former members of the Laboratory of Molecular and Cellular Signaling (KU Leuven), Dr. SP Srinivas (Indiana University School of Optometry, USA), the laboratory of Dr. Leybaert (Ghent University) and of Dr. Vinken (VUB) who provided helpful discussions, optimized procedures or were involved in the development of tools for the study of connexin hemichannels.

1. Isolation of Corneal Endothelial Cells
Before getting started: Isolate the cells from the fresh eyes, obtained from a local slaughterhouse, as soon as possible after enucleating the eye. Make sure that the eye was enucleated from a cow of maximal 18 months old, five minutes post mortem and preserved in Earle's Balanced Salt Solution - 1% iodine solution at 4 &deg;C for transportation to the laboratory.
<ol>
 <li>Take the eye out of the Earle's Balanced Salt Solution - 1% iodine solution and place it in a Petri dish (100 x 20 mm).</li>
 <li>Sterilize the eye with a solution containing 70% ethanol and rinse with Earle's Balanced Salt Solution containing 1% iodine.</li>
 <li>From this step on, work in a sterile hood. Carefully dissect the cornea from the eye and place it in a Petri dish (35 x 10 mm) containing Earle's Balanced Salt Solution, with the epithelial cell layer facing upward. Carefully remove any remaining iris tissue still attached to the cornea if needed.</li>
 <li>Transfer the cornea to another Earle's Balanced Salt Solution containing Petri dish with the endothelial cell layer upward and rinse twice with Earle's Balanced Salt Solution.</li>
 <li>Transfer the cornea with the endothelial layer facing upward to an hourglass, which is a cup-shaped dish, and cover it with growth medium. The growth medium consists of Dulbecco's Modified Eagle's Medium containing 25 mM glucose, 10% fetal bovine serum, 6.6% L-glutamine, 2.5 &mu;g/ml amphotericin-B and 1% antibiotic-antimycotic mixture containing 10,000 units/ml of penicillin, 10,000 &mu;g/ml of streptomycin, and 25 &mu;g/ml amphotericin B.</li>
 <li>Remove the medium with a suction pipette.</li>
 <li>Apply 300 &mu;l of a trypsin solution (0.5 g/L) to the endothelial layer of the cornea (all the steps that include trypsin are done using the same concentration).</li>
 <li>Place the hourglass containing the cornea in a covered Petri dish and put it in the incubator for 30 min at 37 &deg;C and 5% CO2.</li>
 <li>Gently scrape the endothelial cells away from the cornea with a fire-polished hook-shaped glass Pasteur pipette in a sterile hood.</li>
 <li>Suck off the solution containing the endothelial cells and add it to culture flasks (25 cm2) containing 4 ml of culture medium.</li>
 <li>Apply 300 &mu;l of growth medium to the cornea and repeat the scraping and add the solution containing the endothelial cells to the culture flask.</li>
 <li>Repeat this final step (1.11) once more.</li>
 <li>Place the culture flasks containing the corneal endothelial cells in the growth medium in the incubator at 37 &deg;C and 5% CO2.</li>
 <li>After two days, add 6 ml of culture medium.</li>
 <li>Refresh the growth medium every second day.</li>
</ol>
2. Cell Culture
<ol>
 <li>Remove the culture medium and wash the cells twice with Earle's Balanced Salt Solution, when confluency is reached (within about 10 days after isolation).</li>
 <li>Add 1.5 ml trypsin solution to the cells to detach them and place the flask in the incubator (37 &deg;C, 5% CO2) for 3 to 4 min.</li>
 <li>Add 12 ml of growth medium. Pipette the medium three times in and out to disperse the cells and count the cells.</li>
 <li>Seed the cells with a variable fraction depending on the cell density and put them in the incubator. Prepare two well-chambered slides (with an area of 4.2 cm2) with a cell count of 165,000 cells (cell density 39,286 per cm2). Prepare 80 cm2 culture flasks for a new passage at a density of 6,250 per cm2, and add fresh culture medium up to a total volume of 25 ml.</li>
 <li>Refresh the medium every two days.</li>
 <li>Confluency of the cell layer is reached after 3 to 4 days. Use cells for experiments.</li>
 <li>When confluency of the cell layer in the flasks is reached, repeat steps 2.1 to 2.6. Cell cultures up to passage 2 can be used for experiments.</li>
</ol>
3. Mechanical Stimulation for Inducing Calcium Wave
<ol>
 <li>Load the cells in the chambered slide with 10 &mu;M Fluo-4 AM in phosphate-buffered saline for 30 min at 37 &deg;C while gently shaking.</li>
 <li>Remove the Fluo-4 AM solution, wash the cells five times with phosphate-buffered saline, incubate the cells with phosphate-buffered saline and leave the cells for at least 5 min at room temperature before measurement.</li>
 <li>Excite at 488 nm with Argon laser and use beam splitter HFT 488, collect the fluorescence emission at 530 nm using a longpass emission filter LP 505, set the pinhole at minimum. Use an oil immersion 40X objective (Air, 1.2 N.A.). In experiments with ARL-67156, use a 10X objective (Air, 0.3 N.A.).</li>
 <li>Search for a field in which the cells are confluent on the confocal microscope.</li>
 <li>Position the pipette so that it is at 45&deg; in respect to the chambered slide and touching the cell membrane. Provoke a short (&asymp; 1 sec) mechanical stimulation to a single cell. The mechanical stimulation consists of an acute, short-lasting deformation of the cell by briefly touching less than 1% of the cell membrane with a glass micropipette (tip diameter &lt;1 &mu;m) coupled to a piezoelectric crystal nanopositioner, operated through an amplifier which is mounted on a micro-manipulator. The glass micropipettes are made with a microelectrode puller. Make sure that the nanopositioner is operated by a voltage between 0.2 and 1.5 V during the mechanical stimulation. A voltage higher than 1.5 V can result in cell damage. For each cell type and condition, the optimal voltage for mechanical stimulation without cell damage must be carefully determined by applying a series of voltages starting from low (0.2 V) to high (1.5 V) voltage. The voltage is a measure for the force of the stimulation since this voltage determines the mechanical stress and strain that are applied to the cell membrane. The force of the mechanical stimulation can be calculated by multiplying the mechanical stress with the area. Since both the area (&lt;3.14 &mu;m2) and the mechanical stress are very low, the force of the mechanical stimulation is low. (Note that when a cell is damaged, the fluorescence leaks out of the cell and the cell turns dark.)</li>
 <li>Measure spatial changes in [Ca2+]i following mechanical stimulation with the confocal microscope.</li>
 <li>Collect and store images.</li>
 <li>Draw a polygonal region of interest to define the total surface area of responsive cells (active area, AA) using the software of the confocal microscope.</li>
</ol>

Authors have nothing to disclose.

In this manuscript, we describe a simple method to measure intercellular Ca2+-wave propagation in monolayers of primary bovine corneal endothelial cells by providing a localized and controlled mechanical stimulation using a micropipette. Mechanically stimulated cells respond with a local increase in intracellular IP3 and Ca2+, both of which are essential intracellular signaling molecules that drive intercellular Ca2+-wave propagation 11,67 . IP3 is directl...

Intercellular communication and signaling are essential for the coordination of physiological processes in response to extracellular agonists at the tissue and whole-organ level 1,2 . The most direct way of intercellular communication is created by the occurrence of gap junctions. Gap junctions are plaques of gap junction channels, which are proteinaceous channels formed by the head-to-head docking of two connexin (Cx) hemichannels of adjacent cells 3,4 (Figure 1). Gap junctions allow the passage of small signaling molecules with a molecular weight of less than 1.5 kDa, including Ca2+ or IP3 5, causing and modulating Ca2+-release from the intracellular stores of the neighboring cells 6 (Figure 2). Gap junction channels are tightly regulated by intra- and intermolecular protein interactions and by cellular signaling processes, like redox modification and phosphorylation7. GJs facilitate the coordinated response of connected cells, thereby acting as a chemical and electrical syncytium. For example, the spreading of cardiac action potential across the atrial and ventricular myocytes is mediated by Cx-based GJ channels 85. Cxs not only have a role as gap junction channels, but also form unpaired hemichannels, thereby functioning as channels in membranes similarly to regular ion channels 8-10 (Figure 1). Hemichannels participate in paracrine signaling between neighboring cells by controlling the exchange of ions and signaling molecules between the intra- and extracellular environment.
In many cell types (like epithelial cells, osteoblastic cells, astrocytes, endothelial cells, etc.) and organs (like brain, liver, retina, cochlea and the vasculature), intercellular Ca2+-waves are fundamental for the coordination of multicellular responses 11. Increases in intracellular Ca2+ levels in a certain cell are not limited to this cell, but propagate to the surrounding neighboring cells, thereby establishing an intercellular Ca2+-wave 12,13 . These intercellular Ca2+-waves are important for normal physiological regulation of cell layers as a syncytium and their dysregulation has been associated with pathophysiological processes 11. In the corneal endothelium and epithelium, different groups 14-24, including our own 25-33, studied the mechanisms and roles of intercellular communication. In non-excitable cells, like corneal endothelial cells, two distinct modes of intercellular communication occur 28,29 , namely gap junctional intercellular communication and paracrine intercellular communication. Gap junctional intercellular communication involves a direct exchange of signaling molecules via gap junctions 7. Gap junctional intercellular communication is critical for maintaining tissue homeostasis, controlling cell proliferation, and establishing a synchronized response to extracellular stress 10,34,35 . In a number of pathologies, gap junction coupling is reduced due to defective Cxs, and hereby affecting gap junctional intercellular communication 36. This emphasizes the importance and influence of gap junctional intercellular communication in multicellular organisms. In contrast to gap junctional intercellular communication, paracrine intercellular communication is not dependent on cell-cell apposition, since it involves the release of diffusible extracellular messengers (Figure 2). Different types of signaling molecules are released in the extracellular space by signaling cells. The molecule is then transported to the target cell where it is detected by a specific receptor protein. Subsequently the receptor-signal complex induces a cellular response, which is terminated by removal of the signal, inactivation or desensitization. Released lipophilic extracellular signaling messengers penetrate the membrane and act on intracellular receptors. In contrast, hydrophilic messengers do not cross the plasma membrane of the responding cell, but act as a ligand that binds to surface-expressed receptor proteins, which then relay the signal to the intracellular environment. Three major families of cell surface receptor proteins participate in this process: ion-channel-linked, enzyme-linked, and G protein-linked. The released messenger molecule can act on receptors of the same cell (autocrine), on target cells in close proximity (paracrine), or on distant target cells that require the circulatory system (endocrine).
In many cell types, including corneal endothelium 28,29, ATP is one of the major hydrophilic, paracrine factors that drive the propagation of intercellular Ca2+-waves 37-40. During mechanical deformation, hypoxia, inflammation or stimulation by various agents, ATP can be released from healthy cells 41-44 in response to shear stress, stretch, or osmotic swelling 44,45. Different ATP-release mechanisms have been postulated, including vesicular exocytosis 44 and a plethora of transport mechanisms, such as ATP-binding cassette (ABC) transporters, plasmalemmal voltage-dependent anion channels 46, P2X7 receptor channels 47,48, as well as connexin hemichannels 49-52 and pannexin hemichannels 43,49,53. Extracellular ATP can be rapidly hydrolyzed to ADP, AMP and adenosine 54,55 by ectonucleotidases that are present in the extracellular environment. The extracellularly released ATP and its metabolite ADP 56 will spread through diffusion. The subsequent interaction of these nucleotides with purinergic receptors in the neighboring cells has been implicated in the propagation of intercellular Ca2+-waves 28,37,51. Two different classes of purinergic receptors are present: adenosine is the principal natural ligand for P1-purinoceptors, while both purine (ATP, ADP) and pyrimidine (UTP, UDP) nucleotides act on most P2-purinoceptors 57.
Intercellular communication can be investigated by different methods such as scrape loading, dye transfer, local uncaging of agonists like IP3 and Ca2+, mechanical stimulation, etc.. Here we describe the study of Ca2+-wave propagation elicited by mechanical stimulation of a single cell. The advantage of studying Ca2+-wave propagation by mechanical stimulation is that it provides an easy tool to quantify the spread of the Ca2+-wave over time and it allows quantitatively comparing different pretreatments of the cells. In the corneal endothelium, these intercellular Ca2+-waves allow a coordinated response from the monolayer, hereby acting as a possible defense mechanism of the non-regenerative corneal endothelium helping the endothelium to withstand extracellular stresses during intraocular surgery, or upon exposure to inflammatory mediators during immune rejection or uveitis 58,59 .

<table><tbody><tr><td>Earle&#039;s Balanced Salt Solution (EBSS)</td><td>Invitrogen-Gibco-Molecular Probes (Karlsruhe, Germany)</td><td>14155-048</td><td></td></tr><tr><td>Iodine</td><td>Sigma-Aldrich (Deisenhofen, Germany)</td><td>38060-1EA</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium (DMEM)</td><td>Invitrogen-Gibco-Molecular Probes (Karlsruhe, Germany)</td><td>11960-044</td><td></td></tr><tr><td>L-glutamine (Glutamax)</td><td>Invitrogen-Gibco-Molecular Probes (Karlsruhe, Germany)</td><td>35050-038</td><td></td></tr><tr><td>Amphotericin-B</td><td>Sigma-Aldrich (Deisenhofen, Germany)</td><td>A2942</td><td></td></tr><tr><td>Antibiotic-antimycotic mixture</td><td>Invitrogen-Gibco-Molecular Probes (Karlsruhe, Germany)</td><td>15240-096</td><td></td></tr><tr><td>Trypsin</td><td>Invitrogen-Gibco-Molecular Probes (Karlsruhe, Germany)</td><td>25300-054</td><td></td></tr><tr><td>Dulbecco&#039;s PBS</td><td>Invitrogen-Gibco-Molecular Probes (Karlsruhe, Germany)</td><td>14190-091</td><td></td></tr><tr><td>Fluo-4 AM</td><td>Invitrogen-Gibco-Molecular Probes (Karlsruhe, Germany)</td><td>F14217</td><td></td></tr><tr><td>ARL-67156 (6-N,N-Diethyl-b,g-dibromomethylene-D-ATP) </td><td>Sigma-Aldrich (Deisenhofen, Germany)</td><td>A265</td><td></td></tr><tr><td>Apyrase VI</td><td>Sigma-Aldrich (Deisenhofen, Germany)</td><td>A6410</td><td></td></tr><tr><td>Apyrase VII</td><td>Sigma-Aldrich (Deisenhofen, Germany)</td><td>A6535</td><td></td></tr><tr><td>Gap26 (VCYDKSFPISHVR)</td><td>Custom peptide synthesis</td><td></td><td></td></tr><tr><td>Gap27 (SRPTEKTIFII)</td><td>Custom peptide synthesis</td><td></td><td></td></tr><tr><td>Control Peptide (SRGGEKNVFIV)</td><td>Custom peptide synthesis</td><td></td><td></td></tr><tr><td>siRNA1 Cx43 (sense: 5&#039;GAAGGAGGAGGAACU-CAAAdTdT) </td><td>Annealed siRNA was purchased at Eurogentec (Luik, Belgium)</td><td></td><td></td></tr><tr><td>siRNA2 Cx43 (sense: 5&#039;CAAUUCUUCCUGCCGCAAUdTdT) </td><td>Annealed siRNA was purchased at Eurogentec (Luik, Belgium)</td><td></td><td></td></tr><tr><td>siRNA scramble: scrambled sequence of siCx43-1 (sense: 5&#039;GGUAAACG-GAACGAGAAGAdTdT) </td><td>Annealed siRNA was purchased at Eurogentec (Luik, Belgium)</td><td></td><td></td></tr><tr><td>TAT-L2 (TAT- DGANVDMHLKQIEIKKFKYGIEEHGK)</td><td>Thermo Electron (Ulm, Germany)</td><td></td><td></td></tr><tr><td>TAT-L2-H126K/I130N (TAT-DGANVDMKLKQNEIKKFKYGIEEHGK) </td><td>Thermo Electron (Ulm, Germany)</td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Two chambered glass slides</td><td>Laboratory-Tek Nunc (Roskilde, Denmark)</td><td>155380</td><td></td></tr><tr><td>Confocal microscope </td><td>Carl Zeiss Meditec (Jena, Germany)</td><td>LSM510</td><td></td></tr><tr><td>Piez&amp;#8211;lectric crystal nanopositioner (Piezo Flexure NanoPositioner)</td><td>PI Polytech (Karlsruhe, Germany)</td><td>P-280</td><td></td></tr><tr><td>HVPZT-amplifier</td><td>PI Polytech (Karlsruhe, Germany)</td><td>E463 HVPZT-amplifier</td><td></td></tr><tr><td>Glass tubes (glass replacement 3.5 nanoliter)</td><td>World Precision Instruments, Inc. Sarasota, Florida, USA</td><td>4878</td><td></td></tr><tr><td>Micr&amp;#8211;lectrode puller </td><td>Zeitz Instrumente (Munchen, Germany)</td><td>WZ DMZ-Universal Puller</td><td></td></tr></tbody></table>

mechanical stimulation-induced calcium wave propagation in cell monolayers: the example of bovine corneal endothelial cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 &mu;m. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

All experiments are executed in compliance with all relevant guidelines, regulations and regulatory agencies and the protocol being demonstrated is performed under the guidance and approval of the animal care and use committee of the KU Leuven.
In bovine corneal endothelial cells (BCEC), functional gap junctions are expressed and both gap junctional intercellular communication and paracrine intercellular communication contribute significantly to intercellular communication in an interactive wa...

Watch this Scientific Journal Video about Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells at JoVE.com

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

mechanical-stimulation-induced-calcium-wave-propagation-cell

Research

JoVE Journal

Biology

16.2K Views.  KU Leuven. Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

Video: Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema and diminished visual acuity. However, human CECs are prone to compromised proliferative potential. Furthermore, stimulation of cell growth is often complicated by gradual endothelial-mesenchymal transition (EnMT). Therefore, understanding the mechanism of EnMT is necessary for facilitating the regeneration of CECs with competent function. In this study, we prepared a primary culture of bovine CECs by peeling the CECs with Descemet's membrane from the corneal button and demonstrated that bovine CECs exhibited the EnMT process, including phenotypic change, nuclear translocation of &#946;-catenin, and EMT regulators snail and slug, in the in vitro culture. Furthermore, we used a rat corneal endothelium cryoinjury model to demonstrate the EnMT process in vivo. Collectively, the in vitro primary culture of bovine CECs and in vivo rat corneal endothelium cryoinjury models offers useful platforms for investigating the mechanism of EnMT.

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

A primary culture of bovine corneal endothelial cells was used to investigate the mechanism of corneal endothelial-mesenchymal transition. Furthermore, a rat corneal endothelium cryoinjury model was used to demonstrate corneal endothelial-mesenchymal transition in vivo.

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema ...

Developmental Biology

Growth of Human and Sheep Corneal Endothelial Cell Layers on Biomaterial Membranes

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is deleterious for the cells as it reduces their ability to form a mature and functional layer. Here, we present a method for establishing and subculturing human and sheep corneal endothelial cell cultures that minimizes the loss of cell-to-cell contact. Explants of corneal endothelium/Descemet&#39;s membrane are taken from donor corneas and placed into tissue culture under conditions that allow the cells to collectively migrate onto the culture surface. Once a culture has been established, the explants are transferred to fresh plates to initiate new cultures. Dispase II is used to gently lift clumps of cells off tissue culture plates for subculturing. Corneal endothelial cell cultures that have been established using this protocol are suitable for transferring to biomaterial membranes to produce tissue-engineered cell layers for transplantation in animal trials. A custom-made device for supporting biomaterial membranes during tissue culture is described and an example of a tissue-engineered graft composed of a layer of corneal endothelial cells and a layer of corneal stromal cells on either side of a collagen type I membrane is presented.

This protocol describes the critical steps required to establish and grow corneal endothelial cell cultures from explants of human or sheep tissue. A method for subculturing corneal endothelial cells on membranous biomaterials is also presented.

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is ...

Bioengineering

Silicon Nanowires and Optical Stimulation for Investigations of Intra- and Intercellular Electrical Coupling

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These cell-silicon hybrids offer leadless optical modulation capabilities with minimal perturbation to normal cell behavior. The optical capabilities are obtained by the photothermal and photoelectric properties of SiNWs. These hybrids can be harvested using standard tissue culture techniques and then applied to different biological scenarios. We demonstrate here how these hybrids can be used to study the electrical coupling of cardiac cells and compare how myofibroblasts couple to one another or to cardiomyocytes. This process can be accomplished without special equipment beyond a fluorescent microscope with coupled laser line. Also shown is the use of a custom-built MATLAB routine that allows the quantification of calcium propagation within and between the different cells in the culture. Myofibroblasts are shown to have a slower electrical response than that of cardiomyocytes. Moreover, the myofibroblast intercellular propagation shows slightly slower, though comparable velocities to their intracellular velocities, suggesting passive propagation through gap junctions or nanotubes. This technique is highly adaptable and can be easily applied to other cellular arenas, for in vitro as well as in vivo or ex vivo investigations.

This protocol describes the use of silicon nanowires for intracellular optical bio-modulation of cell in a simple and easy to perform method. The technique is highly adaptable to diverse cell types and can be used for in vitro as well as in vivo applications.

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These ...

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.&#160;endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g.&#160;nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic ...

Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the optical breakdown at the laser focus. Acoustic shock waves and cavitation bubbles are generated, causing tissue rupture. Bubble sizes and pressure amplitudes vary with pulse energy and position of the focal point. In this study, enucleated porcine eyes were positioned in front of a commercially available Nd:YAG laser. Variable pulse energies as well as different positions of the focal spots posterior to the cornea were tested. Resulting lesions were evaluated by two-photon microscopy and histology to determine the best parameters for an exclusive detachment of corneal endothelial cells (CEC) with minimum collateral damage. The advantages of this method are the precise ablation of CEC, reduced collateral damage, and above all, the non-contact treatment.

Here, we present a protocol to detach corneal endothelial cells (CEC) from Descemet&#8217;s membrane (DM) using a neodymium:YAG (Nd:YAG) laser as an ex vivo disease model for bullous keratopathy (BK).

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the ...

Medicine

A Cardiac Microphysiological System for Studying Ca2+ Propagation via Non-genetic Optical Stimulation

In vitro cardiac microphysiological models are highly reliable for scientific research, drug development, and medical applications. Although widely accepted by the scientific community, these systems are still limited in longevity due to the absence of non-invasive stimulation techniques. Phototransducers provide an efficient stimulation method, offering a wireless approach with high temporal and spatial resolution while minimizing invasiveness in stimulation processes. In this manuscript, we present a fully optical method for stimulating and detecting the activity of an in vitro cardiac microphysiological model. Specifically, we fabricated engineered laminar anisotropic tissues by seeding human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) generated in a 3D bioreactor suspension culture. We employed a phototransducer, an amphiphilic azobenzene derivative, named Ziapin2, for stimulation and a Ca2+ dye (X-Rhod 1) for monitoring the system&#39;s response. The results demonstrate that Ziapin2 can photomodulate Ca2+ responses in the employed system without compromising tissue integrity, viability, or behavior. Furthermore, we showed that the light-based stimulation approach offers a similar resolution compared to electrical stimulation, the current gold standard. Overall, this protocol opens promising perspectives for the application of Ziapin2 and material-based photostimulation in cardiac research.

Using light to control cardiac cells and tissue enables non-contact stimulation, thereby preserving the natural state and function of the cells, making it a valuable approach for both basic research and therapeutic applications.

In vitro cardiac microphysiological models are highly reliable for scientific research, drug development, and medical applications. Although widely ...

Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte monolayers. HL-1 cells have been widely used as a 2-Dimensional cellular model for studying diverse aspects of cardiac physiology. However, it has been a great challenge to optically map calcium (Ca) transients and action potentials simultaneously from the same field of view in a cultured HL-1 atrial cell monolayer. This is because special handling and care is required to prepare healthy cells that can be electrically captured and optically mapped. Therefore, we have developed an optimal working protocol for dual channel optical mapping. In this manuscript, we have described in detail how to perform the dual channel optical mapping experiment. This protocol is a useful tool to enhance the understanding of action potential propagation and Ca kinetics in arrhythmia development.

This article describes the technique used to perform dual channel optical mapping in cultured HL-1 atrial cell monolayers. This unique protocol allows the simultaneous visualization of both calcium (Ca) and voltage (Vm) activity in the same area for the detailed detection and analysis of electrophysiological properties of culture monolayers.

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte ...

Multifunctional, Micropipette-based Method for Incorporation And Stimulation of Bacterial Mechanosensitive Ion Channels in Droplet Interface Bilayers

MscL, a large conductance mechanosensitive channel (MSC), is a ubiquitous osmolyte release valve that helps bacteria survive abrupt hypo-osmotic shocks. It has been discovered and rigorously studied using the patch-clamp technique for almost three decades. Its basic role of translating tension applied to the cell membrane into permeability response makes it a strong candidate to function as a mechanoelectrical transducer in artificial membrane-based biomolecular devices. Serving as building blocks to such devices, droplet interface bilayers (DIBs) can be used as a new platform for the incorporation and stimulation of MscL channels. Here, we describe a micropipette-based method to form DIBs and measure the activity of the incorporated MscL channels. This method consists of lipid-encased aqueous droplets anchored to the tips of two opposing (coaxially positioned) borosilicate glass micropipettes. When droplets are brought into contact, a lipid bilayer interface is formed. This technique offers control over the chemical composition and the size of each droplet, as well as the dimensions of the bilayer interface. Having one of the micropipettes attached to a harmonic piezoelectric actuator provides the ability to deliver a desired oscillatory stimulus. Through analysis of the shapes of the droplets during deformation, the tension created at the interface can be estimated. Using this technique, the first activity of MscL channels in a DIB system is reported. Besides MS channels, activities of other types of channels can be studied using this method, proving the multi-functionality of this platform. The method presented here enables the measurement of fundamental membrane properties, provides a greater control over the formation of symmetric and asymmetric membranes, and is an alternative way to stimulate and study mechanosensitive channels.

Bacterial mechanosensitive channels can be used as mechanoelectrical transducers in biomolecular devices. Droplet interface bilayers (DIBs), cell-inspired building blocks to such devices, represent new platforms to incorporate and stimulate mechanosensitive channels. Here, we demonstrate a new micropipette-based method of forming DIBs, allowing the study of mechanosensitive channels under mechanical stimulation.

MscL, a large conductance mechanosensitive channel (MSC), is a ubiquitous osmolyte release valve that helps bacteria survive abrupt hypo-osmotic shocks. ...

Evaluating Calcium Activity in Retinal Ganglion Cells Layer

Calcium Imaging In Electrically Stimulated Flat-Mounted Retinas

Retinal dystrophies are a leading cause of blindness worldwide. Extensive efforts are underway to develop advanced retinal prostheses that can bypass the impaired light-sensing photoreceptor cells in the degenerated retina, aiming to partially restore vision by inducing visual percepts. One common avenue of investigation involves the design and production of implantable devices with a flexible physical structure, housing a high number of electrodes. This enables the efficient and precise generation of visual percepts. However, with each technological advancement, there arises a need for a reliable and manageable ex vivo method to verify the functionality of the device before progressing to in vivo experiments, where factors beyond the device's performance come into play. This article presents a comprehensive protocol for studying calcium activity in the retinal ganglion cell layer (GCL) following electrical stimulation. Specifically, the following steps are outlined: (1) fluorescently labeling the rat retina using genetically encoded calcium indicators, (2) capturing the fluorescence signal using an inverted fluorescence microscope while applying distinct patterns of electrical stimulation, and (3) extracting and analyzing the calcium traces from individual cells within the GCL. By following this procedure, researchers can efficiently test new stimulation protocols prior to conducting in vivo experiments.

Author Spotlight: An Innovative Approach to Neural Electrical Stimulation Using Calcium Imaging 

Retinal prostheses have the ability to generate visual perceptions. To advance the development of new prostheses, ex vivo methods are needed to test devices before implantation. This article provides a comprehensive protocol for studying calcium activity in the retinal ganglion cell layer when subjected to electrical stimulation.

Retinal dystrophies are a leading cause of blindness worldwide. Extensive efforts are underway to develop advanced retinal prostheses that can bypass the ...

Neuroscience

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca2+ [Ca2+]i increases and subsequent activation of small- and intermediate conductance Ca2+-activated K+ (SKCa/IKCa) channels.
Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 &#176;C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial &#34;tubes&#34; are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca2+]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Summary

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Multifunctional, Micropipette-based Method for Incorporation And Stimulation of Bacterial Mechanosensitive Ion Channels in Droplet Interface Bilayers

Calcium Imaging In Electrically Stimulated Flat-Mounted Retinas

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium