This work was supported by National Institutes of Health Grant to A.S.M. (EY021784).

Il seguente protocollo è conforme alle Istituzionale Animal Care and Use Committee linee guida Thomas Jefferson University e con la dichiarazione ARVO per l&#39;uso di animali in Vision Research. 1. Configurazione e preparazione di lenti per la Cultura Ex Vivo Wound <ol><li> Mettere tre 100 piatti mm Petri in una, cappa a flusso laminare sterile. Riempire due dei piatti petri metà con tampone Tris / destrosio (buffer TD; 140 mM NaCl, KCl 5 mM, 0,7 mM Na 2 PO 4, 5 mM D-glucosio, 8,25 mM Tris Base, pH a 7,4 con HCl) a RT , lasciando il terzo vuoto. Terreni di coltura pre-caldo (Media 199 integrato con, 1% di L-glutamina e 1% di penicillina / streptomicina) a 37 ° C. Nota: La ferita norma di guarigione di coltura è senza siero, come avviene in vivo; tuttavia, le culture ferita di riparazione possono essere coltivate con successo in condizioni di media definiti che includono siero o altri fattori. </li><li> Rimuovere fertile d embrionaleay 15 bianco Livorno pulcino uovo da incubatrice (tenutasi a 37,7 ° C, con dolce dondolio) </li><li> Inserire uovo selezionato nella cappa a flusso laminare e pulita all&#39;esterno della scocca con etanolo al 70% da una bottiglia di lavaggio. Condurre tutte le procedure qui di seguito, in condizioni asettiche in cappa a flusso laminare, con soluzioni e strumenti sterili. </li><li> Crack uova e posizionare il contenuto in 100 millimetri Petri vuota. Decapitare embrione con pinze standard e forbici sottili. Mettere la testa pulcino embrione in una capsula di Petri contenente tampone TD e smaltire il resto dell&#39;embrione. Opzionalmente evitare che le testine di embrione di pollo in tampone TD per un breve periodo di tempo, non più di 15 min. </li><li> Mettere la testa embrionali di pollo su un coperchio capsula di Petri. Utilizzando pinze alta precisione, rimuovere l&#39;obiettivo insieme al suo vitreo allegata dall&#39;occhio nella seguente sequenza. Pizzicare la parte posteriore dell&#39;occhio con la pinza per creare una piccola apertura nella parte posteriore dell&#39;occhio. </li><li> Quindi, cogliere l&#39;umore vitreo wesimo pinze e tirare delicatamente il vitreo, con un moto di rollio, il vitreo con l&#39;obiettivo montato saranno sloggiati dall&#39;occhio. Luogo obiettivo / vitreo nella capsula di Petri rimanente contenente tampone TD. Lasciare le lenti di rimanere in tampone TD per non più di 30 minuti. </li><li> Spostare la lente per un nuovo coperchio Petri sotto un microscopio da dissezione. Da questo punto in poi eseguire tutti i passaggi sotto un microscopio da dissezione. Con pinze ad alta precisione lavarsi attentamente tutta corpo ciliare (cellule pigmentate) che sono stati sloggiato con la lente utilizzando il bordo della pinza, facendo attenzione a non danneggiare il tessuto dell&#39;obiettivo. Nota: Rimozione del corpo ciliare assicura che i tipi di cellule che non sono endogeni alla lente non sono inclusi nella cultura riparazione della ferita. </li><li> Separare l&#39;obiettivo dalla vitreo con una pinza ad alta precisione pizzicando fuori dal corpo vitreo dalla sua associazione con la capsula posteriore. </li><li> Usando l&#39;alta precisione Pinza trasferire l&#39;obiettivo in una piccola gocciadi tampone TD (circa 200 ml) in un piatto di coltura di tessuti 35 mm. </li></ol> 2. Esecuzione Mock chirurgia della cataratta <ol><li> Orientare la lente nella goccia di tampone TD nel piatto da 35 mm, con la faccia anteriore della lente rivolta verso l&#39;alto. Nota: Il anteriore della lente è facilmente identificabile dalla presenza di un anello denso nel tessuto che rileva il confine tra anteriore e regione equatoriale dell&#39;epitelio lente. Al contrario, vi è una mancanza di marcature sul posteriore della capsula del cristallino a cui le cellule di fibre lente sono allegati. </li><li> Utilizzando due pinze alta precisione fanno una piccola incisione (circa 850μm) al centro della capsula anteriore della lente, la membrana basale spessa che circonda il tessuto dell&#39;obiettivo, e la sua associata epitelio della lente anteriore, afferrando il tessuto con una pinza in ogni mano e tirando delicatamente in direzioni opposte. </li><li> Rimuovere la massa cellulare fibra, che costituisce la maggior parte del tessuto dell&#39;obiettivo, dislodging dai suoi allegati al epitelio lente e circostante capsula del cristallino da idro-eluizione (un approccio utilizzato nella chirurgia della cataratta classico, modellato in Figura 1A). </li><li> Riempire una siringa da 1 ml con una punta dell&#39;ago 27,5 G con 300 ml di tampone TD. Inserire la punta dell&#39;ago nella incisione fatta nella capsula anteriore del cristallino, e circa a metà strada nella lente. </li><li> Deprimere delicatamente la siringa per iniettare il buffer di TD nella massa cellulare fibra lente. Iniettare fra 50 e 200 microlitri TD, e mai più di 300 microlitri. Osservare la massa cellulare fibra stessa allentando dalla capsula epitelio e la lente. </li><li> Utilizzando pinze ad alta precisione, rimuovere la massa cellulare fibra allentato dalla lente attraverso il sito di incisione anteriore. Nota: Questa procedura lascia il posteriore della lente membrana basale capsula per cui le cellule della fibra erano stati allegati denudati delle cellule, e di un epitelio lente infortunato proprio adiacente a questo sito. </li></ol> 3. Preparing dell&#39;obiettivo Ferito per Ex Cultura Vivo <ol><li> Appiattire il sacco capsulare obiettivo che risulta dalla chirurgia della cataratta sopra descritto sul piatto di coltura, lato della cella su, facendo cinque tagli nella faccia anteriore del sacco capsulare. </li><li> Tagliare perpendicolare al sito di incisione originale attraverso all&#39;equatore della lente. Appiattire le risultanti cinque &quot;sportelli&quot; di capsula del cristallino con epitelio attaccato sul lato cultura piatto capsula giù, cellula verso l&#39;alto. Nota la lente ex vivo ferito ora dovrebbe assumere una stella o una forma a fiori (vedi Figura 1B). </li><li> Per fissare la capsula al piatto, premere dolcemente con le pinze in ogni punto della stella. Questo farà una piccola rientranza a cinque punte più esterne del espianto e il risultato in attacco sostenuto al piatto. Nota: È possibile danneggiare la capsula durante questa procedura e quindi è importante garantire la capsula al piatto più vicino alle punte del flaps possibile, così da rendere il minor numero di punti di fissaggio come possibile (in genere due, massimo di tre per falda). </li><li> Rimuovere il buffer TD dal piatto 35mm e sostituirlo con 1,5 ml di mezzi di pre-riscaldato. Coprire il piatto 35 millimetri con il suo coperchio e posto in incubatrice (37 ° C, 5% di CO 2). </li></ol> 4. Separazione della centrale migrazione Zone (CMZ), dove riepitelizzazione dell&#39;Area ferito della capsula posteriore si verifica, dal Attachment Originale della Zona (OAZ) delle cellule epiteliali lente, per l&#39;analisi quantitativa. Nota: le cellule iniziano a muoversi nella regione CMZ immediatamente in risposta al danno. Di giorno una cultura abbastanza cellule migrano attraverso la CMZ per l&#39;analisi molecolare e biochimica, dopo la separazione della CMZ e la OAZ da micro-dissezione 23. Questo protocollo comporta la rimozione di un lembo (OAZ) alla volta dalla zona ferita della capsula. <ol><li> Osservare una demarcatione, chiaramente visibili al microscopio da dissezione, tra OAZ e CMZ (vedi Figura 2A). Utilizzando due pinze alta precisione, afferrare con entrambe le pinze a bordo della linea OAZ / CMZ, quella appena adiacente all&#39;altra, su entrambi i lati della linea (vedere la Figura 2A, B, freccia). </li><li> Usando una mano / una pinza sul lato CMZ continuano a tenere su la cultura feriti, mentre con l&#39;altra mano / pinza, tirare delicatamente il OAZ lungo la linea OAZ / CMZ. La CMZ separa facilmente dalla OAZ lungo questa linea. Continuare lungo questa linea intorno all&#39;intera cultura finché le due regioni sono completamente separati. </li><li> Studiare i separati OAZ e CMZ frazioni per l&#39;analisi molecolare, come 24 o biochimica analisi di RNA-Seq quali Western Blot o co-immunoprecipitazione 23,25. </li></ol>

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The authors declare that they have no competing financial interests.

Here is described a technique for preparing a culture model of wound repair that involves performing an ex vivo cataract surgery on chick embryo lenses after their removal from the eye. The lens epithelium responds to this clinically relevant wounding with a repair process that closely mimics that which occurs in vivo, and shares features with wound repair in other epithelial tissues2,4. While the protocol is straightforward and simple to follow, performing mock cataract surgery with embryoni...

La clinicamente rilevante, la chirurgia della cataratta finto, ex vivo epiteliale modello guarigione delle ferite qui descritto è stato sviluppato per fornire uno strumento per indagare i meccanismi che regolano la riparazione dei tessuti epiteliali in risposta ad un infortunio. Le caratteristiche principali che sono state finalizzate per nella creazione di questo modello inclusi 1) creare condizioni che strettamente replicato la risposta in vivo a ferire in un ambiente culturale, 2) la facilità di modulare gli elementi regolatori di riparazione, e 3) capacità di immagine il processo di riparazione, nella sua interezza, in tempo reale. La sfida, quindi, è stato quello di creare un modello di cultura in cui è stato possibile studiare e manipolare, riparazione della ferita epiteliale in microambiente nativo delle cellule. La disponibilità di questo modello ferita riparazione apre nuove possibilità per individuare gli spunti di segnalazione endogeni di matrice proteine, citochine e chemochine che regolano il processo di riparazione. Inoltre, il modello è ideale per l&#39;esame come unn epitelio è in grado di muoversi come un foglio collettivo per ri-epithelialize della ferita 2,3, e per la determinazione della stirpe di cellule mesenchimali capo sul bordo della ferita che funzionano nel dirigere la migrazione collettiva dell&#39;epitelio feriti 4. Questo modello offre anche una piattaforma con cui identificare terapie che potrebbero promuovere efficace la guarigione delle ferite e prevenire ferita aberrante riparazione 5. Ci sono già un certo numero di modelli disponibili ferita riparazione, sia nella cultura e in vivo, che hanno fornito la maggior parte di ciò che si conosce il processo di riparazione delle ferite oggi. In modelli animali di lesioni, come cornea 6-12 e 13-17 della pelle, vi è la possibilità di studiare la risposta del tessuto al ferimento nel contesto di tutti i mediatori riparazione che potrebbero essere coinvolti nel processo, compresi i contributi dal vascolarizzazione e del sistema nervoso. Tuttavia, vi sono limitazioni per manipolare il expericondizioni mentali in vivo, e non è ancora possibile condurre studi di imaging della risposta di riparazione in vivo, continuamente nel tempo. Al contrario, la maggior parte dei modelli di coltura in vitro ferita riparazione, come la ferita graffiatura, possono essere facilmente manipolati e seguiti nel tempo ma mancano contesto ambientale di studiare guarigione delle ferite nel tessuto in vivo. Mentre ex vivo modelli offrono il vantaggio di studiare il processo di riparazione pregiudizio continua nel tempo nel contesto del microambiente delle cellule associata alla capacità di modulare i regolatori molecolari di riparazione in qualsiasi punto di tempo nel processo, ci sono alcuni modelli che si adattano questi parametri. Qui è descritto una procedura per generare altamente riproducibile ex vivo epiteliali guarigione della ferita culture che riproducono la risposta di un tessuto epiteliale di un ferimento fisiologico. Utilizzando la lente pulcino embrione come fonte di tessuto, un ex vivo mocviene eseguita la chirurgia della cataratta k. L&#39;obiettivo è un tessuto ideale da utilizzare per questi studi dal momento che è indipendente all&#39;interno di una spessa capsula membrana basale, avascolare, non è innervato, e priva di qualsiasi stroma associato 18,19. Nella malattia umana, chirurgia della cataratta Indirizzi perdita della vista a causa opacizzazione della lente, e comporta la rimozione della massa cellulare fibre lente, che comprende la maggior parte della lente. Dopo visione chirurgia della cataratta viene ripristinata mediante l&#39;inserimento di una lente intraoculare artificiale. La procedura di chirurgia della cataratta, attraverso la rimozione di cellule di fibre, induce una risposta lesioni nell&#39;epitelio lente adiacente, che risponde riepitelizzazione dell&#39;area posteriore della capsula del cristallino che era stato occupato dalle cellule della fibra. Nella chirurgia della cataratta, come nella maggior parte delle risposte di riparazione della ferita, si verifica talvolta un esito fibrotico aberrante risposta guarigione della ferita, associata alla comparsa di miofibroblasti, che nella lente è noto come posteriore capsule opacizzazione 20-22. Per generare il modello guarigione delle ferite chirurgia della cataratta, una procedura di chirurgia della cataratta è imitato in lenti rimosso dall&#39;occhio pulcino embrione per produrre una lesione fisiologica. Rimozione microchirurgica delle fibre lente risultati cellule in una ferita circolare molto consistente circondata dalle cellule epiteliali lente. Questa popolazione di cellule rimane fermamente attaccata alla membrana capsula del cristallino seminterrato ed è ferito dalla procedura chirurgica. Le cellule epiteliali migrano sulla zona dissodata della membrana basale endogena per guarire la ferita, guidata da una popolazione di cellule mesenchimali ricche di vimentina noti nel processo di riparazione come cellule capo 1. Con questo modello la risposta di un epitelio di lesione può essere facilmente visualizzata e seguita con il tempo nel contesto del microambiente delle cellule. Le cellule sono facilmente accessibili per le modifiche dell&#39;espressione o l&#39;attivazione di molecole che dovrebbero svolgere un ruolo nella riparazione delle ferite. Una potente funzione di thè il modello è la capacità di isolare e studiare i cambiamenti specifici di migrazione nel quadro di guarigione della ferita. La capacità di preparare un gran numero di ex vivo cicatrizzanti culture abbinati invecchiato per studi è un altro vantaggio di questo modello. Così, questo modello di sistema offre l&#39;opportunità unica di prendere in giro a parte i meccanismi di riparazione delle ferite e terapie di prova per il loro effetto sul processo di guarigione delle ferite. Il finto modello ex vivo chirurgia della cataratta dovrebbe avere ampia applicabilità, fornendo una risorsa fondamentale per lo studio dei meccanismi di riparazione del pregiudizio.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Fisher Scientific</td>
			<td>S271-3</td>
			<td>Use at 140mM in TD Buffer</td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Fisher Scientific</td>
			<td>P217-500</td>
			<td>Use at 5mM in TD Buffer</td>
		</tr>
		<tr>
			<td>Sodium Phosphate (Na2HPO4)</td>
			<td>Sigma</td>
			<td>S0876</td>
			<td>Use at .7mM in TD Buffer</td>
		</tr>
		<tr>
			<td>D-glucose (Dextrose)</td>
			<td>Fisher Scientific</td>
			<td>D16-500</td>
			<td>Use at 0.5mM in TD Buffer</td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Fisher Scientific</td>
			<td>BP152-1</td>
			<td>Use at 8.25mM in TD Buffer</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Fisher Scientific</td>
			<td>A144-500</td>
			<td>Use to pH TD buffer to 7.4</td>
		</tr>
		<tr>
			<td>Media 199</td>
			<td>GIBCO</td>
			<td>11150-059</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Corning/CellGro</td>
			<td>25-005-CI</td>
			<td>Use at 1% in Media199</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Corning/CellGro</td>
			<td>30-002-CI</td>
			<td>Use at 1% in Media199</td>
		</tr>
		<tr>
			<td>100mm petri dishes</td>
			<td>Fisher Scientific</td>
			<td>FB0875711Z</td>
			<td></td>
		</tr>
		<tr>
			<td>Stericup Filter Unit</td>
			<td>Millipore</td>
			<td>SCGPU01RE</td>
			<td>Use to filter sterilize Media</td>
		</tr>
		<tr>
			<td>Dumont #5 forceps (need 2)</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>35mm Cell Culture Dish</td>
			<td>Corning</td>
			<td>430165</td>
			<td></td>
		</tr>
		<tr>
			<td>27 Gauge 1mL SlipTip with precision glide needle</td>
			<td>BD</td>
			<td>309623</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors</td>
			<td>Fine Science Tools</td>
			<td>14058-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Forceps</td>
			<td>Fine Science Tools</td>
			<td>91100-12</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Other Items Needed: General dissection instruments,&#160; fertile white leghorn chicken eggs,&#160;</td>
		</tr>
		<tr>
			<td colspan="4">check egg incubator (humidified, 37.7&#176;C), laminar flow hood, binocular stereovision dissecting&#160;</td>
		</tr>
		<tr>
			<td>microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Ex Vivo Modello creato per studiare il processo di guarigione delle ferite nei microambiente nativo le cellule &#39; Per studiare i meccanismi coinvolti nella regolazione guarigione delle ferite di un epitelio all&#39;interno microambiente nativo delle cellule, un clinicamente rilevante ex vivo finto modello chirurgia della cataratta è stato creato. Questo modello è stato creato da tessuto lente che offre molti vantaggi per le sue proprietà intrinseche: 1) l&#39;obiettiv...

Watch this Scientific Journal Video about Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment at JoVE.com

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.

Istituzione di un clinicamente rilevanti<em&gt; Ex Vivo</em&gt; Mock chirurgia della cataratta modello per indagare epiteliale Wound Repair in un microambiente Native

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

establishment-clinically-relevant-ex-vivo-mock-cataract-surgery-model

Research

JoVE Journal

Developmental Biology

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

6.5K Views.  Thomas Jefferson University. 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. 

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

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

Catturare Tissue Repair in Zebrafish Larve con Time-lapse Brightfield stereomicroscopia

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

Ricerca

Biologia dello sviluppo

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing cardiovascular progenitors during development and disease. While a number of factors have been implicated in epicardium-derived cell (EPDC) mobilization, the mechanisms governing their subsequent migration and differentiation are poorly understood. Here, we present in vitro and ex vivo strategies to study EPDC motility and differentiation. First, we describe a method of obtaining primary epicardial cells by outgrowth culture from the embryonic mouse heart. We also introduce a detailed protocol to assess three-dimensional migration of labeled EPDC in an organ culture system. We provide evidence using these techniques that genetic deletion of myocardin-related transcription factors in the epicardium attenuates EPDC migration. This approach serves as a platform to evaluate candidate modifiers of EPDC biology and could be used to develop genetic or chemical screens to identify novel regulators of EPDC mobilization that might be useful for cardiac repair.

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

Here, we describe methods for isolating primary mouse epicardial cells by an outgrowth culture assay and assessing the functional migration of epicardial-derived cells (EPDC) using an ex vivo heart culture system. These protocols are suitable for identifying genetic and chemical modulators of epicardial epithelial-to-mesenchymal transition (EMT) and motility.

Epicardico escrescenza Cultura Assay e<em&gt; Ex Vivo</em&gt; La valutazione della migrazione delle cellule di derivazione epicardico

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing ...

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.

Adenovirale Terapia genica per diabetici cheratopatia: Effetti sulla guarigione delle ferite e Stem Cell Marker Expression in Human Organ cornee-coltura e le cellule epiteliali Limbal

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

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of morphogenetic events, such as gastrulation2, neurulation3-5, somitogenesis6, heart bending7,8, and brain formation9-13, during early embryogenesis. Key to understanding morphogenetic processes is to follow them dynamically by time-lapse imaging. The acquisition of time-lapse movies of chick embryogenesis ex ovo has been limited either to short time windows or to the need for an incubator to control temperature and humidity around the embryo14. Here, we present a new technique to culture chick embryos ex ovo for high-resolution time-lapse imaging using transmitted light microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique (EC-culture)1 and allows for the culturing of chick embryos without the need for a climate chamber. The embryo is sandwiched between two identical filter paper carriers and is kept fully submerged in a simple, temperature-controlled medium covered by a layer of light mineral oil. Starting from the primitive streak stage (Hamburger-Hamilton stage 5, HH5)15 up to at least the 28-somite stage (HH16)15, embryos can be cultured with either their ventral or dorsal side up. This allows the acquisition of time-lapse movies covering about 30 hr of embryonic development. Representative time-lapse frames and movies are shown. Embryos are compared morphologically to an embryo cultured in the standard EC-culture.
The submerged filter paper sandwich provides a stable environment to study early dorsal and ventral morphogenetic processes. It also allows for live fluorescence imaging and micromanipulations, such as microsurgery, bead implantation, microinjection, gene silencing, and electroporation, and has a strong potential to be combined with immersion objectives for laser-based imaging (including light-sheet microscopy).

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Here, we present a new technique to culture chick embryos ex ovo for time-lapse imaging using transmitted light and fluorescence microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique1 that allows for the culturing of chick embryos fully-submerged in a liquid culture medium.

Un sommerso Sandwich carta da filtro per il lungo termine<em&gt; Ex Ovo</em&gt; Time-lapse imaging di Chick primi embrioni

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of ...

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka and zebrafish larvae are widely used to analyze the behavior of bone cells during bone formation, degeneration, and repair. Their optical clarity allows the visualization of fluorescently labeled bone cells and fluorescent dyes bound to the mineralized skeletal matrix. Our lab has generated transgenic medaka fish that express the osteoclast-inducing factor Receptor Activator of Nuclear-factor &#954;B Ligand (RANKL) under the control of a heat&#160;shock-inducible promoter. Ectopic expression of RANKL results in the excess formation of activated osteoclasts, which can be visualized in reporter lines with nlGFP expression under the control of the cathepsin K (ctsk) promoter. RANKL induction and ectopic osteoclast formation leads to severe osteoporosis-like phenotypes. Compound transgenic medaka lines that express ctsk:nlGFP in osteoclasts, as well as mCherry under the control of the osterix (osx) promoter in premature osteoblasts, can be used to study the interaction of both cell types. This facilitates the in vivo observation of cellular behavior under conditions of bone degeneration and repair. Here, we describe the use of this system to test a drug commonly used in human osteoporosis therapy and describe a protocol for live imaging. The medaka model complements studies in cell culture and mice, and offers a novel system for the in vivo analysis of drug action in the skeletal system.

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Small laboratory fish have become popular models for bone research on the mechanisms underlying human bone disorders and for the screening of bone-modulating drugs. In this report, we describe a protocol to assess the effect of alendronate on bone cells in medaka larvae with osteoporotic lesions.

Il trattamento di droga e<em&gt; In Vivo</em&gt; Imaging di osteoblasti-osteoclasti interazioni in un modello di Medaka pesce osteoporosi

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka ...

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.

Isolamento rapido di BMPR-IB + derivate da tessuto adiposo cellule stromali per l&#39;uso in un cranica difetto di guarigione Modello

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

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor metastasis. In this assay, cells are grown to form a confluent monolayer and a mechanical wound is created by scratching with a device. Then the migration rate of the cells towards the denuded area can be monitored by imaging. Our 8-channel mechanical wounder is designed to tackle most of the problems associated with the cell migration assay. Firstly, our wounder can be easily sterilized by autoclaving or with common disinfectants. Secondly, the individual adjustable pins allow even contact with the cell culture plate so that sharp and reproducible wounds can be created. Thirdly, the guiding bars on both sides of the wounder ensure consistent wounding position in each well. The use of disposable plastic pipette tips for wounding can further provide better handling of the wounder as well as to minimize cross-contamination. In conclusion, our cell wounder can provide researchers with a user friendly and reproducible device for performing the cell migration assay using the standard 96-well culture plate.

Here, we present a protocol to perform a semi-high throughput cell migration assay on a 96-well cell culture plate. This protocol is a fast, simple and economical method to create consistent scratch wounds on a cell monolayer.

Un dispositivo per realizzare la migrazione delle cellule / Wound Healing in una piastra a 96 pozzetti

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

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding and invasion. Current animal models for the study of T. cruzi allow limited observation of parasites in vivo, representing a challenge for understanding parasite behavior during the initial stages of infection in humans. This protozoan has a flagellar stage in both vector and mammalian hosts, but there are no studies describing its motility in vivo.The objective of this project was to establish a live vertebrate zebrafish model to evaluate T. cruzi motility in the vascular system. Transparent zebrafish larvae were injected with fluorescently labeled trypomastigotes and observed using light sheet fluorescence microscopy (LSFM), a noninvasive method to visualize live organisms with high optical resolution. The parasites could be visualized for extended periods of time due to this technique&#39;s relatively low risk of photodamage compared to confocal or epifluorescence microscopy. T. cruzi parasites were observed traveling in the circulatory system of live zebrafish in different-sized blood vessels and the yolk. They could also be seen attached to the yolk sac wall and to the atrioventricular valve despite the strong forces associated with heart contractions. LSFM of T. cruzi-inoculated zebrafish larvae is a valuable method that can be used to visualize circulating parasites and evaluate their tropism, migration patterns, and motility in the dynamic environment of the cardiovascular system of a live animal.

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

In this protocol, fluorescently labeled T. cruzi&#160;were injected into transparent zebrafish larvae, and parasite motility was observed in vivo using light sheet fluorescence microscopy.

Stabilimento di larvale Zebrafish come modello animale per indagare Trypanosoma cruzi motilità In Vivo

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding ...

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological questions in cell and developmental biology. In addition, organoids together with recent technical advances in gene editing and viral gene delivery promises to advance medical research and development of new drugs for treatment of diseases. Organoids grown in vitro in basement matrix provide powerful model systems for studying the behavior and function of various proteins and are well suited for live-imaging of fluorescent-tagged proteins. However, establishing the expression and localization of the endogenous proteins in ex vivo tissue and in in vitro organoids is important to verify the behavior of the tagged proteins. To this end we have developed and modified tissue isolation, fixation, and immuno-labeling protocols for localization of microtubules, centrosomal, and associated proteins in ex vivo intestinal tissue and in in vitro intestinal organoids. The aim was for the fixative to preserve the 3D architecture of the organoids/tissue while also preserving antibody antigenicity and enabling good penetration and clearance of fixative and antibodies. Exposure to cold depolymerizes all but stable microtubules and this was a key factor when modifying the various protocols. We found that increasing the ethylenediaminetetraacetic acid (EDTA) concentration from 3 mM to 30 mM gave efficient detachment of villi and crypts in the small intestine while 3 mM EDTA was sufficient for colonic crypts. The developed formaldehyde/methanol fixation protocol gave very good structural preservation while also preserving antigenicity for effective labeling of microtubules, actin, and the end-binding (EB) proteins. It also worked for the centrosomal protein ninein although the methanol protocol worked more consistently. We further established that fixation and immuno-labeling of microtubules and associated proteins could be achieved with organoids isolated from or remaining within the basement matrix.

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

We present protocols for isolation of intestinal 3D structures from in vivo tissue and in vitro basement matrix embedded organoids, and detail different fixation and staining protocols optimized for immuno-labeling of microtubule, centrosomal, and junctional proteins as well as cell markers including the stem cell protein Lgr5.

Immuno-fluorescenza etichettatura dei microtubuli e proteine centrosomali in tessuto intestinale Ex Vivo e 3D In Vitro intestinale Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological ...

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic age-related macular degeneration (AMD) which represents 90% of the cases. Therapies are needed to slow or retard the development of geographic atrophy (GA), and understanding Bruch&#39;s membrane pathology is part of this process. Alterations in human Bruch&#39;s membrane precede the progression of AMD by contributing to the damage of retinal pigment epithelial (RPE) cells. Given the lack of sufficient animal models to study AMD, ex vivo models of aged human Bruch&#39;s membrane serve as a useful tool to study the behavior of RPE cells from immortalized and primary cell lines as well as RPE lines derived from induced pluripotent stem cells (iPSCs). Here, we present a detailed method that allows one to determine the effects of RPE cell behavior seeded on harvested human Bruch&#39;s membrane explants from human donors, including attachment, apoptosis and proliferation, ability to phagocytize photoreceptor outer segments, establishment of polarity, and gene expression. This assay provides an ex vivo model of aged Bruch&#39;s membrane to assess the functional characteristics of RPE cells when seeded on aged/compromised extracellular matrix.

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

The goal of this protocol is to demonstrate the culturing of retinal pigment epithelial (RPE) cells on aged and/or diseased human Bruch&#39;s membrane. This method is suitable to study RPE cell behavior on a compromised extracellular matrix.

Coltura delle cellule epiteliali del pigmento retinico su un modello Ex Vivo della membrana di Bruch umano invecchiato

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic ...

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Coltura delle cellule epiteliali del pigmento retinico su un modello Ex Vivo della membrana di Bruch umano invecchiato