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 border="1">
<tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Column1</td>
 </tr>
 <tr>
 <td>Earle'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's Modified Eagle'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'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'GAAGGAGGAGGAACU-CAAAdTdT) </td>
 <td>Annealed siRNA was purchased at Eurogentec (Luik, Belgium)</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>siRNA2 Cx43 (sense: 5'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'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>Piezoelectric 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>Microelectrode 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

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Yeah, so I'm Lauren Deon. I'm in charge of the Human Amgen stem cell co facility at MGH. Yeah, so I'm Lauren, I'm in charge of the Human AM stem cell co facility at MGH.So I've been working with human Amgen stem cells for years now since I was a postdoc in George De Lab before, from four, four years. And so right now we have two cell lines, H one and H nine, which are both from Y cell. And so the way we are expanding or maintaining these cells in culture is by, at least the way we are splitting these cells is by using collagenous.So what I usually do is for the first few passage after throwing the cells, I use a technical, the picking colonies. So in this case, I can check under the microscope which colonies are good and differentiate and nice shape, and I will collect only these colonies and I will cut them in few pieces and put them back in, in a fresh flat. And the, the colonies that look kind of differentiate it are just left them alone.The other way to pass the cells is by using trypsin. So I don't use this technique here. I think collagenase is a little bit safer because human being stem cells don't like to be individualized or they don't like to be alone.So if you do trypsin, if you use trypsin for a little bit too long, if you use it for like three to four minutes, which is the way we use strips in for other type of cells, human stem cells will be individualized and most of them will die or just differentiate or will not attach to the myth. So the idea is you have to keep the cells as clumps. You don't want them to be alone in the in, in the flask.So using collagenase, the advantage is to keep cells as plants. Using chipsy, you just select for cells that will survive this very, very harsh process of g opsonization. So these cells are usually very highly proliferative.So you get, you can adapt your cells to this tripsin process. The problem is that you have cells that they behave differently, they go much faster. And one risk is that you can get some commercial abnormality to, to this, to this very high division process.So you say that ization can cause selection of cells in culture for most proliferative cells.Right. More ative and probably also higher survival rates than other cells. So after proliferation, basically culture after proliferation in tripsin, after several passages become a different culture, these different properties.So it's, it's hard to figure out if they are exactly identical. If you do the test, the usual test, let's say you check markers, regular markers of end differentiated cells like OC four, nano one 60 SC four C, C3, all this molecule might still be expressed. If you do TER formation, this cells might still differentiate and be formed like al like normal cells.If you look at the karyotype, you might have some abnormality, but you might still have a normal karyotype. The, the, the selective advantage might be due to just a mutation that you cannot see by doing a karyotype. So the fact that they grow much faster is by itself I think a little bit worser.And if we want to use these cells for transplantation at some point, I would definitely not use this type of cells.Okay. So would you like to tell me what are the main technical problems with using this technique for, for passages of cells? So using S, yes.It's just a little bit longer and also it's, since the cells are not going as fast, it's just much longer to get a high number of cells to do some experiment. So it's just less convenient I would say. Would you like to tell me about few words about the goals of your experiment?How do you use CS cells? Okay, so my main project here is to get endothelial cells from human AB stem cells. And the idea is to be able to make blood.And we are working a lot with tissue bioengineering labs and one of their goal is to make tissue in vitro. But one of the limitation to this is the lack of blood vessels going through this tissues. So if we can bring endothelial cells or put endothelial cells in this tissue, we would have better growth of this tissue.So the idea few sources of cells are known for endothelial cells like cold blood or pressure blood, but it would be interesting to have another source of cells like human years. And the advantage is since human years can be expanded and most infinitely, you could modify these cells, make some inducible system and be able to use it. Then in your final product, your material cells, in this case.

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial cells regulate permeability of the endothelium, and many studies have indicated the important contribution of the actin cytoskeleton to determining junctional integrity1-5. A cortical actin belt is thought to be important for the maintenance of stable junctions1, 2, 4, 5. In contrast, actin stress fibers are thought to generate centripetal tension within endothelial cells that weakens junctions2-5. Much of this theory has been based on studies in which endothelial cells are treated with inflammatory mediators known to increase endothelial permeability, and then fixing the cells and labeling F-actin for microscopic observation. However, these studies provide a very limited understanding of the role of the actin cytoskeleton because images of fixed cells provide only snapshots in time with no information about the dynamics of actin structures5. 
Live-cell imaging allows incorporation of the dynamic nature of the actin cytoskeleton into the studies of the mechanisms determining endothelial barrier integrity. A major advantage of this method is that the impact of various inflammatory stimuli on actin structures in endothelial cells can be assessed in the same set of living cells before and after treatment, removing potential bias that may occur when observing fixed specimens. Human umbilical vein endothelial cells (HUVEC) are transfected with a GFP-&beta;-actin plasmid and grown to confluence on glass coverslips. Time-lapse images of GFP-actin in confluent HUVEC are captured before and after the addition of inflammatory mediators that elicit time-dependent changes in endothelial barrier integrity. These studies enable visual observation of the fluid sequence of changes in the actin cytoskeleton that contribute to endothelial barrier disruption and restoration. 
Our results consistently show local, actin-rich lamellipodia formation and turnover in endothelial cells. The formation and movement of actin stress fibers can also be observed. An analysis of the frequency of formation and turnover of the local lamellipodia, before and after treatment with inflammatory stimuli can be documented by kymograph analyses. These studies provide important information on the dynamic nature of the actin cytoskeleton in endothelial cells that can used to discover previously unidentified molecular mechanisms important for the maintenance of endothelial barrier integrity.

Microscopic imaging of live endothelial cells expressing GFP-actin allows characterization of dynamic changes in cytoskeletal structures. Unlike techniques that use fixed specimens, this method provides a detailed assessment of temporal changes in the actin cytoskeleton in the same cells before, during, and after various physical, pharmacological, or inflammatory stimuli.

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...