Name: a device for performing cell migration/wound healing in a 96-well plate
Uploaded: 2017-03-07T15:00:00
Description: Watch this Scientific Journal Video about A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate at JoVE.com

Les auteurs tiennent à remercier M. Tam Po Leung, M. Wong Chi Kin et le personnel technique de la Faculté des sciences Atelier, Hong Kong Baptist University, pour leurs compétences techniques et des conseils pour rendre le prototype de ce wounder.

1. Introduction aux parties du wounder (Figure 1) <ol><li> Préparer le porte-broche en fixant les broches blessantes à des distances égales. Tenez les embouts de pipette et d&#39;ajuster la hauteur de la pointe en déplaçant les broches blessantes réglables de haut en bas. </li><li> Monter le guidage-bar ajustable sur différentes marques de plaques de culture de 96 puits et de veiller à ce que la zone de la blessure est dans une position fixe. </li><li> Fixer la tige de blessure réglable en serrant la vis à t...

<ol>
	<li>Friedl, P., Wolf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour-cell+invasion+and+migration:+diversity+and+escape+mechanisms.">Tumour-cell invasion and migration: diversity and escape mechanisms.</a> Nat Rev Cancer. 3 (5), 362-374 (2003).</li><li>Friedl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prespecification+and+plasticity:+shifting+mechanisms+of+cell+migration.">Prespecification and plasticity: shifting mechanisms of cell migration.</a> Curr Opin Cell Biol. 16 (1), 14-23 (2004).</li><li>Lampugnani, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration+into+a+wounded+area+in+vitro.">Cell migration into a wounded area in vitro.</a> Methods Mol Biol. 96, 177-182 (1999).</li><li>Sholley, M. M., Gimbrone, M. A., Cotran, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+migration+and+replication+in+endothelial+regeneration:+a+study+using+irradiated+endothelial+cultures.">Cellular migration and replication in endothelial regeneration: a study using irradiated endothelial cultures.</a> Lab Invest. 36 (1), 18-25 (1977).</li><li>Gotlieb, A. I., Spector, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+into+an+in+vitro+experimental+wound:+a+comparison+of+porcine+aortic+endothelial+and+smooth+muscle+cells+and+the+effect+of+culture+irradiation.">Migration into an in vitro experimental wound: a comparison of porcine aortic endothelial and smooth muscle cells and the effect of culture irradiation.</a> Am J Pathol. 103 (2), 271-282 (1981).</li><li>Chen, Y. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+migration+chip+for+chemotaxis-based+microfluidic+selection+of+heterogeneous+cell+populations.">Single-cell migration chip for chemotaxis-based microfluidic selection of heterogeneous cell populations.</a> Sci Rep. 18 (5), 9980 (2015).</li><li>Yarrow, J. C., Periman, Z. E., Westwood, N. J., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+cell+migration+assay+using+scratch+wound+healing,+a+comparsion+of+image-based+readout+methods.">A high-throughput cell migration assay using scratch wound healing, a comparsion of image-based readout methods.</a> BMC Biotechnol. 4, 21 (2004).</li><li>Lauder, H., Frost, E. E., Hiley, C. R., Fan, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+the+repair+process+involved+in+the+repair+of+a+cell+monolayer+using+an+in+vitro+model+of+mechanical+injury.">Quantification of the repair process involved in the repair of a cell monolayer using an in vitro model of mechanical injury.</a> Angiogenesis. 2 (1), 67-80 (1998).</li><li>Yue, P. Y. K., Leung, E. P. Y., Mak, N. K., Wong, R. N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+method+for+quantifying+cell+migration/+wound+healing+in+96-well+plates.">A simplified method for quantifying cell migration/ wound healing in 96-well plates.</a> J. Biomol Screen. 15 (4), 427-433 (2010).</li><li>Yue, P. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elucidation+of+the+mechanisms+underlying+the+angiogenic+effects+of+ginsenoside+Rg(1)+in+vivo+and+in+vitro.">Elucidation of the mechanisms underlying the angiogenic effects of ginsenoside Rg(1) in vivo and in vitro.</a> Angiogenesis. 8 (3), 205-216 (2005).</li><li>Kwok, H. H., Chan, L. S., Poon, P. Y., Yue, P. Y., Wong, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ginsenoside-Rg1+induces+angiogenesis+by+the+inverse+regulation+of+MET+tyrosine+kinase+receptor+expression+through+miR-23a.">Ginsenoside-Rg1 induces angiogenesis by the inverse regulation of MET tyrosine kinase receptor expression through miR-23a.</a> Toxicol Appl Pharmacol. 287 (3), 276-283 (2015).</li></ol>

Notre wounder cellulaire a plusieurs caractéristiques uniques pour résoudre les problèmes des tests traditionnels de migration cellulaire. La wounder mécanique à 8 canaux cellule est réalisée en acier inoxydable de haute qualité (acier 304) avec une longue durée de vie qui peut être stérilisé par autoclavage. Presque toutes les marques commerciales de plaques de culture à 96 puits disponibles sur le marché peuvent être utilisés avec ce wounder mécanique des cellules en raison de la barre de guidage rég...

La migration cellulaire joue un rôle important dans les processus cellulaires, tels que l&#39; embryogenèse, la neurogenèse, l&#39; angiogenèse, la cicatrisation, la réparation des muqueuses, épithélio-mésenchymateuse transitions en cours de développement normal et la situation de la maladie 1. Il est un processus complexe qui implique la coordination de nombreux événements internationaux et intra-cellulaires , y compris la signalisation des interactions moléculaires, la polarisation de la cellule, la réorganisation du cytosquelette, remodelage de la matrice, protrusion de la membrane et cellule-cellule dynamique adhérence modulation 2. En étudiant la migration cellulaire, la découverte et la validation des produits chimiques ou des biomolécules conduisant à un mouvement cellulaire et les voies biochimiques apparentées peuvent être déterminées. Ce processus fondamental peut être utilisé avantageusement en tant que proxy pour diverses applications, telles que le traitement de la maladie et le ciblage de médicaments. L&#39;essai est blessant l&#39; un des essais de migration cellulaire 3. Ici, unéchantillon individuel de cellules sera partiellement éliminé par des moyens mécaniques pour produire une zone dénudée où les cellules migrent pour couvrir la zone. Le pourcentage de récupération à un point de temps particulier sera surveillé. Lors de la manipulation grand nombre d&#39;échantillons, des questions telles que le coût expérimental et la contamination croisée dans les échantillons peuvent être problématiques. Bien que de nombreux outils commerciaux et des essais sont disponibles pour l&#39; étude de la migration des cellules 4, 5, 6, beaucoup d&#39;entre eux nécessaire un équipement coûteux et sophistiqué et des améliorations sont encore nécessaires. Pour ces raisons, un wounder cellulaire mécanique à 8 canaux (figure 1) est développée. Notre wounder mécanique 8 canaux cellulaire a incorporé plusieurs caractéristiques uniques pour résoudre les problèmes mentionnés ci-dessus. Il offre la possibilité d&#39;effectuer la migration des cellules / blesser les dosages dans le format de plaque de culture à 96 puits. Le réglage gubar design Iding veille à ce que la zone de zéro est dans la position centrale de chaque puits. En outre, la hauteur des barres de guidage peut être ajustée de sorte que la wounder est applicable pour les différentes marques de plaques de culture. Enfin, la conception de la broche de blessure réglable permet un contact même des pointes de pipette à la surface de la plaque de culture pour obtenir une blessure simultanée et reproductible 7, 8.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-well cell culture plate</td>
			<td>Nunc</td>
			<td>167008</td>
			<td>Other brands of 96-well cell culture plate can also be used</td>
		</tr>
		<tr>
			<td>P10 pipette tips</td>
			<td>Axygen</td>
			<td>301-03-051</td>
			<td>Short P10 pipette tip is more easy to create a clear wound</td>
		</tr>
		<tr>
			<td>Wounder</td>
			<td>R&#38;P Technology Limited</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Medium 199</td>
			<td>Sigma</td>
			<td>M2520-1L</td>
			<td>For cell culture of human umbilical vein endothelial cells. 
			Use appropirate culture medium and condition for other type of cells.</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>26140079</td>
			<td>For cell culture of human umbilical vein endothelial cells. 
			Use appropirate culture medium and condition for other type of cells.</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>For cell culture of human umbilical vein endothelial cells. 
			Use appropirate culture medium and condition for other type of cells.</td>
		</tr>
		<tr>
			<td>Heparin sodium salt from porcine intestinal mucosa</td>
			<td>Sigma</td>
			<td>H3393</td>
			<td>For cell culture of human umbilical vein endothelial cells. 
			Use appropirate culture medium and condition for other type of cells.</td>
		</tr>
		<tr>
			<td>Gelatin from bovine skin</td>
			<td>Sigma</td>
			<td>G9391</td>
			<td>For cell culture of human umbilical vein endothelial cells. 
			Use appropirate culture medium and condition for other type of cells.</td>
		</tr>
	</tbody>
</table>

a device for performing cell migration/wound healing in a 96-well plate

La migration cellulaire / blessure dosage est une méthode couramment utilisée pour étudier la migration cellulaire et d&#39;autres processus biologiques tels que l&#39;angiogenèse et les métastases tumorales. Dans cet essai, les cellules sont cultivées pour former une monocouche confluente et une blessure mécanique est créée par grattage avec un périphérique. Ensuite, le taux des cellules vers la zone dénudée de migration peut être surveillé par imagerie. Notre wounder mécanique à 8 canaux est conçu pour traiter la plupart des problèmes liés à la détermination de la migration cellulaire. Tout d&#39;abord, notre wounder peut être facilement stérilisé par autoclavage ou avec des désinfectants courants. Deuxièmement, les broches individuels réglables permettent même le contact avec la plaque de culture cellulaire de telle sorte que les plaies vives et reproductibles peuvent être créés. Troisièmement, les barres de guidage des deux côtés de la position de wounder assurent une blessure constante dans chaque puits. L&#39;utilisation de jetables conseils de pipette en plastique pour blesser peut en outre assurer une meilleure gestion de la wounder ainsi que pour minimiser les contre-contamination. En conclusion, notre wounder cellulaire peut fournir aux chercheurs un dispositif convivial et reproductible utilisateur pour effectuer le test de migration des cellules en utilisant la plaque standard de culture de 96 puits.

Cette wounder mécanique 8 canaux est construit de façon à gratter une monocouche cellulaire afin d&#39;effectuer le test de migration des cellules. Il est un dispositif convivial qui peut effectuer des essais grattage de cellules dans des plaques à 96 puits en moins d&#39;une minute sans formation spéciale. Ce wounder peut introduire des zones enroulées sur des monocouches de cellules avec une largeur uniforme d&#39;environ 600 pm et présentant des arêtes vives (figures 5...

Watch this Scientific Journal Video about A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate at JoVE.com

A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate

Ici, nous présentons un protocole pour effectuer un test de migration des cellules semi-haut débit sur une plaque de culture cellulaire de 96 puits. Ce protocole est une méthode rapide, simple et économique pour créer des blessures à gratter cohérentes sur une monocouche cellulaire.

Un dispositif pour effectuer la migration cellulaire / Wound Healing dans une plaque à 96 puits

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor ...

The overall goal of this eight-channel mechanical cell wounder is to quantify the effect of treatment on cell migration using the wound-healing assay in a rapid and semi-high throughput manner without the use of expensive and complicated equipment. Cell migration is an important cellular process in different physiological and powerful physiological conditions. In vitro wound-healing assays are used in different studies including cancer, rescular, and dermatologic studies.The main advantage of this method is that the effect on cell migration can be monitored on a 96-rail page, enabling different treatment conditions to be observed simultaneously. Demonstrating the procedure will be Ka Chun Ng, a grad student from our laboratory. To begin the experiment, prepare the pinholder by fixing the wounding pins at equal distances.Hold the pipet tips and move the adjustable wounding pins up and down to adjust the tip height. Next, fit the adjustable guiding bar on different brands on 96-well culture plates and ensure that the wounding area is in a fixed position. Fix the adjustable wounding pin by tightening the hex screw.Fix the guiding bar in position by tightening the hex socket head caps on both ends. Use an M5 hex wrench to loosen the hex socket head caps. Insert a suitable number of adjusting rings to both sides of the pinholder so that the guiding bars fit perfectly with the width of the 96-well culture plate.Then tighten the hex socket head caps to fix the guiding bar. Begin adjusting the guiding bars by fitting the wounding pins with sterile 10-microliter plastic pipet tips. Next, loosen the hex socket head caps with the M5 hex wrench.Hold the wounder and dip the wounding pins into one column of a 96-well culture plate. Then adjust the height of the guiding bars until all of the tips barely touch the bottom of the wells. Tighten the hex socket head caps.Ensure that the wounder now perfectly fits on the culture plate and that the pins are well-positioned in the middle of the wells. Hold the wounder perpendicular to a flat, sterile surface, such as a Petri dish and loosen all the hex screws with the M3 hex wrench. Tap the wounder until all of the tips evenly touch the surface.After the tips evenly touch the surface, tighten the hex screws again to lock the pins in position. Then check the evenness of each tip by tapping the wounder gently on the flat sterile surface. Seed human umbilical vascular endothelial cells on a 0.1%gelatin-coated 96-well cell culture plate.Culture cells in medium 199 supplemented with 20%heat-inactivated fetal bovine serum, 1%penicillin streptomycin, and 0.09 grams per liter heparin. Maintain cells in a humidified incubator with 5%carbon dioxide at 37 degrees Celsius overnight before being used. Once cells have received 100%confluency, place the wounding tips at the leftmost side of each well within the same column of the culture plate.Slide the wounder across to the other side of the well. Make sure the wounding tips are touching the bottom of the well. To ensure the wound in every well of cell molinar is centered and in same wave, swing the wounder spanning across the well to the other end.Repeat the scratching for all columns. After wounding, discard the medium in each well and replace it with fresh medium containing testing compounds. Then move onto data acquisition and image analysis.Representative images of a row and a column of wells immediately after scratching enable quantitative analysis of the width of each wound. Wound images were obtained from human umbilical vein endothelial cells and human dermal fiber blasts. The dotted line indicates the wound area and the solid line indicates the cell migration area.After watching this video, you should have a good understanding of how to use this device to perform a wound-healing assay in a 96-well page in a semi-high throughput manner and to ensure every well is scratched by the wounder.

a-device-for-performing-cell-migrationwound-healing-in-a-96-well-plate

Research

JoVE Journal

Developmental Biology

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

Comprendre organogenèse précoce aide d&#39;un simplifié<em&gt; In Situ</em&gt; Protocole hybridation in<em&gt; Xenopus</em

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

Recherche

Biologie du développement

Automated Quantification of Hematopoietic Cell &#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data.

Automated Quantification of Hematopoietic Cell &amp;#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

A strategy to quantitatively analyze histological data in the bone marrow is presented. Confocal microscopy of fluorescently labeled cells in tissue sections results in 2-dimensional images, which are automatically analyzed. Co-localization analyses of different cell types are compared to data from simulated images, giving quantitative information about cellular interactions.

Automated quantification des cellules hématopoïétiques - Interactions cellulaires stromales dans les images histologiques de décalcifiées os

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. ...

Scalable 96-well Plate Based iPSC Culture and Production Using a Robotic Liquid Handling System

Continued advancement in pluripotent stem cell culture is closing the gap between bench and bedside for using these cells in regenerative medicine, drug discovery and safety testing. In order to produce stem cell derived biopharmaceutics and cells for tissue engineering and transplantation, a cost-effective cell-manufacturing technology is essential. Maintenance of pluripotency and stable performance of cells in downstream applications (e.g., cell differentiation) over time is paramount to large scale cell production. Yet that can be difficult to achieve especially if cells are cultured manually where the operator can introduce significant variability as well as be prohibitively expensive to scale-up. To enable high-throughput, large-scale stem cell production and remove operator influence novel stem cell culture protocols using a bench-top multi-channel liquid handling robot were developed that require minimal technician involvement or experience. With these protocols human induced pluripotent stem cells (iPSCs) were cultured in feeder-free conditions directly from a frozen stock and maintained in 96-well plates. Depending on cell line and desired scale-up rate, the operator can easily determine when to passage based on a series of images showing the optimal colony densities for splitting. Then the necessary reagents are prepared to perform a colony split to new plates without a centrifugation step. After 20 passages (~3 months), two iPSC lines maintained stable karyotypes, expressed stem cell markers, and differentiated into cardiomyocytes with high efficiency. The system can perform subsequent high-throughput screening of new differentiation protocols or genetic manipulation designed for 96-well plates. This technology will reduce the labor and technical burden to produce large numbers of identical stem cells for a myriad of applications.

The protocols described allow laboratories to perform scalable, adherent stem cell culture in high throughput with minimal labor, experience and equipment investment cost using a programmable liquid handling robot and 96-well plates. iPSCs passaged more than 20 times on this system maintained pluripotency, normal karyotypes and differentiated into cardiomyocytes.

Scalable plaque de 96 puits Basé iPSC Culture et production en utilisant un système robotique Liquid Handling

Continued advancement in pluripotent stem cell culture is closing the gap between bench and bedside for using these cells in regenerative medicine, drug ...

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to follow and manipulate the wound response ex vivo in an environment that closely mimics that of epithelial tissue injury in vivo. This issue was addressed by creating a clinically relevant epithelial ex vivo injury-repair model based on cataract surgery. In this culture model, the response of the lens epithelium to wounding can be followed live in the cells&#8217; native microenvironment, and the molecular mediators of wound repair easily manipulated during the repair process. To prepare the cultures, lenses are removed from the eye and a small incision is made in the anterior of the lens from which the inner mass of lens fiber cells is removed. This procedure creates a circular wound on the posterior lens capsule, the thick basement membrane that surrounds the lens. This wound area where the fiber cells were attached is located just adjacent to a continuous monolayer of lens epithelial cells that remains linked to the lens capsule during the surgical procedure. The wounded epithelium, the cell type from which fiber cells are derived during development, responds to the injury of fiber cell removal by moving collectively across the wound area, led by a population of vimentin-rich repair cells whose mesenchymal progenitors are endogenous to the lens1. These properties are typical of a normal epithelial wound healing response. In this model, as in vivo, wound repair is dependent on signals supplied by the endogenous environment that is uniquely maintained in this ex vivo culture system, providing an ideal opportunity for discovery of the mechanisms that regulate repair of an epithelium following wounding.

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

Described here is the establishment of a clinically relevant ex vivo mock cataract surgery model that can be used to investigate mechanisms of the injury response of epithelial tissues within their native microenvironment.

Création d&#39;une importance clinique<em&gt; Ex Vivo</em&gt; Mock Chirurgie de la cataracte modèle pour étudier épithéliale Wound Repair dans un microenvironnement maternelle

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to ...

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often located distally from the regulated gene in intergenic regions or even within the body of another gene. The position independent nature of enhancer activity makes it difficult to match enhancers with the genes they regulate. Deletion of an enhancer region provides direct evidence for enhancer activity and is the gold standard to reveal an enhancer's role in endogenous gene transcription. Conventional homologous recombination based deletion methods have been surpassed by recent advances in genome editing technology which enable rapid and precisely located changes to the genomes of numerous model organisms. CRISPR/Cas9 mediated genome editing can be used to manipulate the genome in many cell types and organisms rapidly and cost effectively, due to the ease with which Cas9 can be targeted to the genome by a guide RNA from a bespoke expression plasmid. Homozygous deletion of essential gene regulatory elements might lead to lethality or alter cellular phenotype whereas monoallelic deletion of transcriptional enhancers allows for the study of cis-regulation of gene expression without this confounding issue. Presented here is a protocol for CRISPR/Cas9 mediated deletion in F1 mouse embryonic stem (ES) cells (Mus musculus129 x Mus castaneus). Monoallelic deletion, screening and expression analysis is facilitated by single nucleotide polymorphisms (SNP) between the two alleles which occur on average every 125 bp in these cells.

Experimental validation of enhancer activity is best approached by loss-of-function analysis. Presented here is an efficient protocol that uses CRISPR/Cas9 mediated deletion to study allele-specific regulation of gene transcription in F1 ES cells which contain a hybrid genome (Mus musculus129 x Mus castaneus).

CRISPR Génération / cas9 Mediated monoallélique Suppressions pour étudier la fonction Enhancer dans souris cellules souches embryonnaires

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often ...

Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and behavioral phenotypes. The remarkably diverse phenotypes that have evolved as marine populations adapt to countless freshwater environments, combined with the ability to cross marine and freshwater forms, provide a rare vertebrate system in which genetics can be used to map genomic regions controlling evolved traits. Excellent genomic resources are now available, facilitating molecular genetic dissection of evolved changes. While mapping experiments generate lists of interesting candidate genes, functional genetic manipulations are required to test the roles of these genes. Gene regulation can be studied with transgenic reporter plasmids and BACs integrated into the genome using the Tol2 transposase system. Functions of specific candidate genes and cis-regulatory elements can be assessed by inducing targeted mutations with TALEN and CRISPR/Cas9 genome editing reagents. All methods require introducing nucleic acids into fertilized one-cell stickleback embryos, a task made challenging by the thick chorion of stickleback embryos and the relatively small and thin blastomere. Here, a detailed protocol for microinjection of nucleic acids into stickleback embryos is described for transgenic and genome editing applications to study gene expression and function, as well as techniques to assess the success of transgenesis and recover stable lines.

Transgenic manipulations and genome editing are critical for functionally testing the roles of genes and cis-regulatory elements. Here a detailed microinjection protocol for the generation of genomic modifications (including Tol2-mediated fluorescent reporter transgene constructs, TALENs, and CRISPRs) is presented for the emergent model fish, the threespine stickleback.

Microinjection pour transgénèse et Génome Édition dans Épinoche à trois épinoches

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and ...

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.

An example of adenoviral gene therapy in the human diabetic organ-cultured corneas is presented towards the normalization of delayed wound healing and markedly reduced epithelial stem cell marker expression in these corneas. It also describes the optimization of this process in stem cell-enriched limbal epithelial cultures.

Adénovirus thérapie génique diabétique Kératopathie: Effets sur la cicatrisation des plaies et des cellules souches Marker Expression dans Human cornées Organ-culture et de cellules épithéliales limbiques

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene ...

Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own body. The ability to grow bone de novo using a patient's own cells would allow bony defects to be filled with adequate tissue without the morbidity of harvesting native bone. There is interest in the use of adipose-derived stromal cells (ASCs) as a source for tissue engineering because these are obtained from an abundant source: the patient's own adipose tissue. However, ASCs are a heterogeneous population and some subpopulations may be more effective in this application than others. Isolation of the most osteogenic population of ASCs could improve the efficiency and effectiveness of a bone engineering process. In this protocol, ASCs are obtained from subcutaneous fat tissue from a human donor. The subpopulation of ASCs expressing the marker BMPR-IB is isolated using FACS. These cells are then applied to an in vivo calvarial defect healing assay and are found to have improved osteogenic regenerative potential compared with unsorted cells.

Adipose-derived stromal cells may be useful for engineering new tissue from a patient's own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.

Isolement rapide de BMPR-IB + Adipose-Derived cellules stromales pour utilisation dans un calvarial Defect Modèle de guérison

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

Une nouvelle technique de transplantation de graisse mammaire mammaire pour visualiser la génération de vaisseaux de cellules souches endothéliales vasculaires

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

Visualiser le nœud et la notochorde plaque en Gastrulating des embryons de souris à l’aide de la microscopie électronique à balayage et toute monture Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...