Gli autori ringraziano il prof Hendrik Tevaearai e il Dipartimento di Chirurgia Cardiovascolare di sostegno finanziario per la parte iniziale dello studio.

NOTA: Lewis ratti maschi e femmine, 200-220 g sono stati alloggiati in condizioni standard di laboratorio (12 ore di luce e buio ciclo, ad libitum acqua e cibo, IVC gabbia). Tutti gli animali sono stati trattati in conformità con le raccomandazioni della FELASA e la legge svizzera sulla protezione degli animali. 1 Cella Preparazione: isolamento di cellule staminali mesenchimali dal midollo osseo <ol><li> Anestetizzare i ratti con isoflurano 5% O 2 e 5 L / min in una camera di induzione per 5 min. Posizionare muso dell&#39;animale in un cono collegato al sistema di anestesia. Eseguire un pizzico punta o la coda per confermare la sedazione. </li><li> Togliere la pelle dalla gamba con le forbici tagliando la pelle dalla caviglia all&#39;anca. Togliere i muscoli e tagliare l&#39;arteria femorale per dissanguamento degli animali. Esporre l&#39;articolazione dell&#39;anca e dislocare la testa del femore. E &#39;importante non danneggiare la testa del femore. Mettere le ossa in PBS sterile in un tubo di plastica da 50 ml. </li><li> Rimuovere tutti i muscles e legamenti delle ossa senza romperle. NOTA: E &#39;importante che l&#39;osso rimane intatto per evitare il collegamento di soluzione di lavaggio o contaminazione con etanolo. Sciacquare le ossa pelati in etanolo al 70% per 5 min. </li><li> Sotto la cappa a flusso laminare sterile, tagliare entrambe le punte delle ossa con le forbici. Lavare il midollo osseo mediante iniezione di PBS sterile dalla estremità dell&#39;osso. Raccogliere osseo in una provetta da 15 ml. Centrifugare 7 min a 300 x g. </li><li> Rimuovere il surnatante. Sospendere il pellet in 3 ml di globuli rossi Lysis buffer. Incubare la sospensione per 1 min a temperatura ambiente prima della filatura ancora per 7 min a 300 x g. In un pallone di coltura di 150 ml, aggiungere sterile terreno di coltura cellulare (395 ml medio IMDM, 5 penna ml / strep, 100 ml FBS) e seminare le cellule. </li><li> Modificare media il secondo giorno per rimuovere le cellule non aderenti. <ol><li> Eseguire i cambiamenti medi ogni secondo giorno per 2-3 settimane, fino ad ottenere la quantità cella desiderata. </li><li> In alternativa, preparare il biograft per semezione le cellule sulla matrice solida come necessario e descritto altrove 3. </li></ol></li><li> Il giorno di impianto cardiaco, preparare le cellule o raccogliere il biograft poco prima dell&#39;applicazione epicardico. <ol><li> Raccogliere le cellule utilizzando soluzione distacco delle cellule Accutase. Contare le cellule. Riempire una provetta da 1,5 ml per centrifuga con il volume della sospensione cellulare calcolate per ottenere quantità cella desiderata. Centrifugare per 7 minuti a 300 xg e rimuovere il surnatante. </li><li> In alternativa, raccogliere il biograft da terreno di coltura cellulare, sciacquare in PBS sterile, e tenere in terreno di coltura fresco senza siero. </li></ol></li></ol> 2 Prima Toracotomia e KOP legatura <ol><li> Pesare il ratto. Accendere il rilievo di riscaldamento a 37 ° C. Anestetizzare il topo con il 5% isoflurano e 5 L / min 100% O 2 in una camera di induzione per 5-7 min. Eseguire un pizzico punta o la coda per confermare la sedazione. Posizionare il ratto sulla rampa di riscaldamento. </li><li> Intubare l&#39;animale con un 14G catetere iv. Collegare il catetere intubazione di un ventilatore roditore programmato per 2.5 L / min di ossigeno, 2,5% isoflurano, volume corrente di 2 ml, e una frequenza respiratoria di 90 respiri / min. </li><li> Preparare una soluzione di buprenorfina a 0.1 mg / kg. Iniettare per via sottocutanea un terzo della soluzione. Radere la parte sinistra del torace. Disinfettare con una soluzione di Betadine 1%. </li><li> Incidere la pelle perpendicolare allo sterno al quarto spazio intercostale. Separare i 3 strati di muscolatura toracica (pettorale, ascendente pettorale, e pettorale obliqua esterna). Aprire il quarto spazio intercostale (tra nervature 4 e 5). </li><li> Utilizzare un piccolo divaricatore per diffondere le costole e per esporre il cuore. Aprire con cautela il pericardio. Individuare e legare la discendente anteriore dell&#39;arteria coronaria (LAD) con un 7,0 mm 4 sutura sotto l&#39;atrio. </li><li> Chiudere lo spazio intercostale con due punti con 3.0 punti di sutura. Posizionare i due punti di sutura prossimale e distale dallo sterno. In primo luogo, tSerrate la sutura distale. Fissare il tubo di scarico ventilatore per 2 secondi per gonfiare il petto ed evitare qualsiasi pneumotorace. Stringere la seconda sutura. Posizionare i muscoli strati a posto (non sono necessari punti di sutura). Chiudere la pelle con un 5.0 sutura. </li><li> Disinfettare la sutura con una soluzione di Betadine 1%. Iniettare il resto della soluzione buprenorfina. Spegnere il sistema di anestesia. Rimuovere il catetere intubazione. </li><li> Tenere il topo in gabbia sotto una lampada caldo per 1-2 ore. Mantenere un termometro nella gabbia e controllare la distanza tra la luce calda e la gabbia per evitare il surriscaldamento. Restituire il ratto nell&#39;unità IVC. Iniettare per via sottocutanea Buprenorfina 0,05-0,1 mg / kg dopo -operatively ogni 6-12 ore per 48 ore. </li></ol> 3. epicardico Amministrazione della Via trattamento una seconda Toracotomia <ol><li> Ripetere i passaggi 2,1-2,3. </li><li> Incidere la pelle perpendicolare allo sterno al quinto spazio intercostale. Separare il 3 stratis del muscolo toracico (grande pettorale, ascendente pettorale e pettorale obliqua esterna). Aprire il quinto spazio intercostale (fra nervature 5 e 6). </li><li> Utilizzare un piccolo divaricatore per diffondere le costole e per esporre il cuore. Se c&#39;è qualche aderenza, rimuoverli delicatamente con una pinza sottile. </li><li> Individuare la zona infartuata che appare come un&#39;area pallida sotto la legatura. NOTA: Se necessario, utilizzare un 10 centimetri pezzo di sutura (7.0) inserito al vertice per visualizzare meglio il ventricolo sinistro. Estrarre delicatamente la sutura mantenuto liberamente da un morsetto per esporre il cuore. </li><li> Applicare uno dei seguenti trattamenti: <ol><li> Posizionare il biograft alla superficie del cuore. Utilizzare la siringa preriempita di fibrina sigillante preriscaldata a temperatura ambiente per 10 min. Applicare una goccia (50-100 ml) di fibrina sigillante sotto la biograft utilizzando un ago 25 G Luer. Verificare che il biograft sia perfettamente sigillato. </li><li> In alternativa, applicare il pellet di cellule raccolte con un puntale in superficie of cuore sull&#39;area pallido sotto la legatura. Applicare una goccia (50-100 ml) di fibrina sigillante sul pellet di cellule. </li></ol></li><li> Rimuovere il pezzo di sutura dall&#39;apice. </li><li> Ripetere i passaggi 2,6-2,8. </li></ol>

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The authors have nothing to disclose.

La legatura permanente di LAD provoca danno miocardico irreversibile. Il primo modello animale è stato descritto nel 1960 4. Da allora, è stato considerato come un modello standard e adatto a MI cronica. La sua stabilità e riproducibilità consentito valutazione sperimentale delle terapie per MI 5. Miglioramento delle procedure di seguito la descrizione iniziale riportato un tasso di mortalità operatoria del 35-13% 6. Come previsto, LAD legatura indotto f...

Dalla fine del 19 ° secolo, le malattie cardiovascolari è rimasto il serial killer numero uno nei paesi industrializzati. Tra questi, malattia coronarica rappresenta la eziologia principale. I risultati della fase acuta di infarto del miocardio (MI) ed è seguita da rimodellamento disadattivi che si sviluppa progressivamente verso una fase cronica e insufficienza cardiaca grave. Nonostante i recenti significativi progressi tecnologici e terapeutici, la morbilità e la mortalità dovute alla progressione dello scompenso cardiaco è ancora in crescita 1. In questo contesto, la terapia cellulare ha guadagnato crescente interesse come una nuova opzione terapeutica per arrestare la progressione della malattia verso l&#39;insufficienza cardiaca e per stimolare la capacità rigenerativa recentemente identificato del miocardio. Indagini sperimentali e clinici hanno fornito prove convincenti degli effetti benefici ottenuti dopo il trapianto cardiaco di vari tipi di cellule. I principali risultati inclusi migliorata cardifunzione contrattile ac, diminuito rimodellamento ventricolare sinistro, ridotta dimensione infartuale, ed ha aumentato la densità vascolare nella zona infartuata. Tuttavia, il basso numero di cellule di conservazione dopo l&#39;iniezione delle cellule è rimasto un importante svantaggio. Associazione di celle con un biomatrice per migliorare di consegna cella 2 ha recentemente promosso ricercatore e interessi clinici. Legatura della discendente anteriore dell&#39;arteria coronaria (LAD) è un metodo di riferimento per MI in piccolo modello animale che provoca infarto transmurale e una cicatrice maturo. La terapia cellulare applicata nella fase cronica di MI richiede un secondo intervento chirurgico. Un sternotomia mediana è di solito eseguita per consentire l&#39;iniezione intramiocardico delle cellule o impianto epicardico di biografts. Tali procedure chirurgiche invasive aumentare il tasso di mortalità, i tempi di recupero post-operatorio, il dolore, e il rischio di infezione. L&#39;approccio mini-invasivo qui presentato non solo impedisce tale distorsione, ma fornisce anche oaccessibilità ptimal del cuore per l&#39;applicazione del trattamento. MI e impianto epicardico delle cellule associate a un tipo di gel biomatrice vengono eseguite su un cuore che batte con toracotomia intercostale sinistro.

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			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:104px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:239px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">POLYSORB 3-0 suture</td>
			<td style="width:104px;">COVIDIEN</td>
			<td style="width:119px;">UL 204</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">POLYSORB 5-0 suture</td>
			<td style="width:104px;">COVIDIEN</td>
			<td style="width:119px;">UL 202</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SURGIPRO II 7-0 Suture</td>
			<td style="width:104px;">COVIDIEN</td>
			<td style="width:119px;">VP904X</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CATHETER INSYTE 14G</td>
			<td style="width:104px;">BD</td>
			<td style="width:119px;">381267</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">INSPIRA ADVANCED VOLUME CONTROLLED VENTILATOR</td>
			<td style="width:104px;">HARVARD APPARATUS</td>
			<td style="width:119px;">557058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DUMONT #7 Forceps</td>
			<td style="width:104px;">FST Germany</td>
			<td style="width:119px;">11274-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fibrin sealant</td>
			<td style="width:104px;">BAXTER</td>
			<td style="width:119px;">1501441</td>
			<td>TISSEEL Glue, Frozen -20&#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CASTROVIEJO EYE SPECULA</td>
			<td style="width:104px;">HARVARD APPARATUS</td>
			<td style="width:119px;">72-8925</td>
			<td>use as retractor for the ribs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IMDM Glutamax</td>
			<td>Gibco</td>
			<td>31980</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pen/Strep</td>
			<td>Gibco</td>
			<td>15140</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS</td>
			<td>PAA Clone</td>
			<td>&#160;&#160;&#160;&#160;&#160; A15-101</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">bone scissors</td>
			<td style="width:104px;">Fine Science Tols</td>
			<td style="width:119px;">16044-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Red Blood Cell Lysis Solution</td>
			<td>Gentra Systems</td>
			<td>D-50k1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Accutase cell detachment solution</td>
			<td style="width:104px;">Stem cell technology</td>
			<td style="width:119px;">7920</td>
			<td></td>
		</tr>
	</tbody>
</table>

cell-based therapy for heart failure in rat: double thoracotomy for myocardial infarction and epicardial implantation of cells and biomatrix

Terapia cellulare cardiaca ha guadagnato crescente interesse e l&#39;impianto di biomateriali associati con le cellule è diventata una questione importante per ottimizzare la consegna delle cellule del miocardio. Modello di roditore di infarto del miocardio (MI) composto da discendente anteriore sinistra (LAD) legatura è stata comunemente eseguita mediante una toracotomia; un secondo intervento a cuore aperto tramite una sternotomia viene tradizionalmente eseguita per l&#39;applicazione epicardica del trattamento. Dal momento che la descrizione di LAD modello legatura, il tasso di mortalità post-operatoria è sceso 35-13%, comunque il secondo intervento chirurgico è rimasta critica. Al fine di migliorare il recupero post-intervento chirurgico e ridurre il dolore e l&#39;infezione, procedure chirurgiche minimamente invasive sono presentati. Due toracotomie state eseguite, quello iniziale per LAD legatura e la seconda per il trattamento somministrazione epicardico. Biografts costituiti da cellule associate con matrici di tipo solido o gel sono stati applicati sulla ar infartuatoEA. LAD legatura comportato la perdita della funzione cardiaca, come confermato da ecocardiografia eseguita dopo 2 e 6 settimane. Colorazione tricromica Goldner eseguita su sezioni di cuore confermata la formazione della cicatrice transmurale. Ambulatori di primo e secondo portato a meno che il 10% la mortalità post-operatoria.

Tutti gli animali recuperati entro 1 ora dopo toracotomia. La guarigione della ferita è stata rapida. È stata osservata nessuna infezione o edema. La doppia toracotomia sinistra consentito un accesso ottimale al cuore (Figura 1). Il dolore e la mortalità post-operatoria erano bassi. L&#39;animale recuperato rapidamente dalla chirurgia ed ha guadagnato peso (Figura 2). Kaplan Meir sopravvivenza percentuale è stata del 96% per il primo toracotomia. Quattro...

Watch this Scientific Journal Video about Cell-based Therapy for Heart Failure in Rat: Double Thoracotomy for Myocardial Infarction and Epicardial Implantation of Cells and Biomatrix at JoVE.com

Cell-based Therapy for Heart Failure in Rat: Double Thoracotomy for Myocardial Infarction and Epicardial Implantation of Cells and Biomatrix

L&#39;impianto di una biograft per trattare infarto del miocardio indotta da legatura LAD in un modello di roditore è convenzionalmente richiesto due operazioni a cuore aperto. Al fine di ridurre la mortalità e fornire le condizioni ottimali per la fissazione di biomatrici solidi e gelatinosi associati con le cellule, sono state sviluppate procedure minimamente invasive.

Terapia cellulare per insufficienza cardiaca nel ratto: Doppia Toracotomia per infarto miocardico e epicardico impianto di cellule e Biomatrix

Cardiac cell therapy has gained increasing interest and implantation of biomaterials associated with cells has become a major issue to optimize myocardial ...

cell-based-therapy-for-heart-failure-rat-double-thoracotomy-for

Research

JoVE Journal

Bioengineering

11.8K Views.  University of Fribourg.  L'impianto di una biograft per trattare infarto del miocardio indotta da legatura LAD in un modello di roditore è convenzionalmente richiesto due operazioni a cuore aperto. Al fine di ridurre la mortalità e fornire le condizioni ottimali per la fissazione di biomatrici solidi e gelatinosi associati con le cellule, sono state sviluppate procedure minimamente invasive. 

Video: Cell-based Therapy for Heart Failure in Rat: Double Thoracotomy for Myocardial Infarction and Epicardial Implantation of Cells and Biomatrix

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or bioartificial scaffold materials, entrapment of cardiac myocytes in hydrogels such as fibrin or collagen and stacking of myocyte monolayers 1. These concepts aim at restoration of compromised cardiac function (e.g. after myocardial infarction) or as experimental models (e.g. predictive toxicology and substance screening or disease modelling). Precise monitoring of cell survival after implantation of engineered heart tissue (EHT) has now become possible using in-vivo bioluminescence imaging (BLI) techniques 2. Here we describe the generation of fibrin-based EHT from a transgenic rat strain with ubiquitous expression of firefly luciferase (ROSA/luciferase-LEW Tg; 3). Implantation is performed into the greater omentum of different rat strains to assess immune responses of the recipient organism following EHT implantation. Comparison of results generated by BLI and the Enzyme Linked Immuno Spot Technique (ELISPOT) confirm the usability of BLI for the assessment of immune responses.

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT

This video demonstrates the use of in vivo bioluminescence imaging to study immune responses after implantation of Engineered Heart Tissue (EHT) in rats.

Imaging bioluminescenza per la valutazione delle risposte immunitarie A seguito di impianto di tessuto cardiaco Composti (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or ...

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Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Trapianto di progenitori miogenici pluripotenti derivati da cellule staminali indotte simili a mesoangioblasti nei modelli di topo di rigenerazione muscolare

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles

In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. While the outer surface membrane and associated TATS membrane components appear to be continuous, there are substantial differences in lipid and protein content. In ventricular myocytes (VMs), certain TATS components are highly abundant contributing to rectilinear tubule networks and regular branching 3D architectures. It is thought that peripheral TATS components propagate action potentials from the cell surface to thousands of remote intracellular sarcoendoplasmic reticulum (SER) membrane contact domains, thereby activating intracellular Ca2+ release units (CRUs). In contrast to VMs, the organization and functional role of TATS membranes in atrial myocytes (AMs) is significantly different and much less understood. Taken together, quantitative structural characterization of TATS membrane networks in healthy and diseased myocytes is an essential prerequisite towards better understanding of functional plasticity and pathophysiological reorganization. Here, we present a strategic combination of protocols for direct quantitative analysis of TATS membrane networks in living VMs and AMs. For this, we accompany primary cell isolations of mouse VMs and/or AMs with critical quality control steps and direct membrane staining protocols for fluorescence imaging of TATS membranes. Using an optimized workflow for confocal or superresolution TATS image processing, binarized and skeletonized data are generated for quantitative analysis of the TATS network and its components. Unlike previously published indirect regional aggregate image analysis strategies, our protocols enable direct characterization of specific components and derive complex physiological properties of TATS membrane networks in living myocytes with high throughput and open access software tools. In summary, the combined protocol strategy can be readily applied for quantitative TATS network studies during physiological myocyte adaptation or disease changes, comparison of different cardiac or skeletal muscle cell types, phenotyping of transgenic models, and pharmacological or therapeutic interventions.

In cardiac myocytes, tubular membrane structures form intracellular networks. We describe optimized protocols for i) isolation of myocytes from mouse heart including quality control, ii) live cell staining for state-of-the-art fluorescence microscopy, and iii) direct image analysis to quantify the component complexity and the plasticity of intracellular membrane networks.

Analisi della membrana tubolare reti in Cardiac miociti da atri e ventricoli

In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. ...

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional tissue. Tissue engineering holds great promise as a potential solution with its ability to integrate cells and signaling molecules into a 3-dimensional scaffold. Recent work with tissue engineered tracheal grafts (TETGs) has seen some success but their translation has been limited by graft stenosis, graft collapse, and delayed epithelialization. In order to investigate the mechanisms driving these issues, we have developed a mouse model for tissue engineered tracheal graft implantation. TETGs were constructed using electrospun polymers polyethylene terephthalate (PET) and polyurethane (PU) in a mixture of PET and PU (20:80 percent weight). Scaffolds were then seeded using bone marrow mononuclear cells isolated from 6-8 week-old C57BL/6 mice by gradient centrifugation. Ten million cells per graft were seeded onto the lumen of the scaffold and allowed to incubate overnight before implantation between the third and seventh tracheal rings. These grafts were able to recapitulate the findings of stenosis and delayed epithelialization as demonstrated by histological analysis and lack of Keratin 5 and Keratin 14 basal epithelial cells on immunofluorescence. This model will serve as a tool for investigating cellular and molecular mechanisms involved in host remodeling.

Graft stenosis poses a critical obstacle in tissue engineered airway replacement. To investigate cellular mechanisms underlying stenosis, we utilize a murine model of tissue engineered tracheal replacement with seeded bone marrow mononuclear cells (BM-MNC). Here, we detail our protocol, including scaffold manufacturing, BM-MNC isolation, graft seeding, and implantation.

Semina e impianto di un innesto trachea tessutale biosintetico in un modello murino

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. Both forms can be prepared with the same type I collagen. In general, the non-fibrous form promotes cell adhesion and proliferation. The fibril form (gels) provides more physiological conditions in many types of cells; therefore, gel culture is useful for examining physiological behaviors of cells, such as drug efficacy.
Researchers can select the appropriate form according to the purpose of its use. For example, in the case of keratinocytes, on-gel culture has been used as a wound healing model. FEPE1L-8, a keratinocyte cell line cultured on the non-fibrous form of type I collagen, promote cell adhesion. Notably, keratinocyte proliferation is slower on the fibril form than the non-fibrous form. Protocols for the preparation of type I collagen for cell culture are simple and have wide applications depending on the experimental needs.

Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

Valutazione della proliferazione dei cheratinociti su bi - e tri - dimensionale substrati di collagene di tipo I

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...

Magnetic-, Acoustic-, and Optical-Triple-Responsive Microbubbles for Magnetic Hyperthermia and Pothotothermal Combination Cancer Therapy

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has been challenged. Here, we fabricate iron oxide nanoparticle shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform. Iron oxide nanoparticles serve as both magnetic and photothermal agents. Once intravenously injected, NSMs can be magnetically guided to the tumor site. Ultrasound triggers the release of iron oxide nanoparticles, facilitating the penetration of nanoparticles deep into the tumor due to the cavitation effect of microbubbles. Thereafter, magnetic hyperthermia and photothermal therapy can be performed on the tumor for combinational cancer therapy, a solution for cancer resistance due to the tumor heterogeneity. In this protocol, the synthesis and characterization of NSMs including structural, chemical, magnetic and acoustic properties were performed. In addition, the anti-cancer efficacy by thermal therapy was investigated using in vitro cell cultures. The proposed delivery strategy and combination therapy holds great promise in cancer treatment to improve both delivery and anticancer efficacies.

Presented here is a protocol for the fabrication of iron oxide nanoparticle-shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform for magnetic hyperthermia and photothermal combination cancer therapy.

Microbolle magnetiche, acustiche e ottiche a tripla risposta per ipertermia magnetica e terapia del cancro combinata pototermica

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has ...

Preparation of Mesh-Shaped Engineered Cardiac Tissues Derived from Human iPS Cells for In Vivo Myocardial Repair

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from human induced pluripotent stem cells (hiPSCs), which are developed towards the goal of clinical use. HiPSC-derived cardiomyocytes, endothelial cells, and vascular mural cells are mixed with gel matrix and then poured into a polydimethylsiloxane (PDMS) tissue mold with rectangular internal staggered posts. By culture day 14 ECTs mature into a 1.5 cm x 1.5 cm mesh structure with 0.5 mm diameter myofiber bundles. Cardiomyocytes align to the long-axis of each bundle and spontaneously beat synchronously. This approach can be scaled up to a larger (3.0 cm x 3.0 cm) mesh ECT while preserving construct maturation and function. Thus, mesh-shaped ECTs generated from hiPSC-derived cardiac cells may be feasible for cardiac regeneration paradigms.

The present protocol generates mesh-shaped engineered cardiac tissues containing cardiovascular cells derived from human induced pluripotent stem cells to allow the investigation of cell implantation therapy for heart diseases.

Preparazione di tessuti cardiaci ingegnerizzati a forma di mesh derivati da cellule iPS umane per la riparazione miocardia in Vivo

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from ...

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ligation is a challenging procedure and has a high mortality rate due to its nature. We developed a method to consistently transplant bioprinted patches of cells and hydrogels onto the epicardium of an infarcted mouse heart to test their regenerative properties in a robust and feasible way. First, a deeply anesthetized mouse is carefully intubated and ventilated. Following left lateral thoracotomy (surgical opening of the chest), the exposed LAD is permanently ligated and the bioprinted patch transplanted onto the epicardium. The mouse quickly recovers from the procedure after chest closure. The advantages of this robust and quick approach include a predicted 28-day mortality rate of up to 30% (lower than the 44% reported by other studies using a similar model of permanent LAD ligation in mice). Moreover, the approach described in this protocol is versatile and could be adapted to test bioprinted patches using different cell types or hydrogels where high numbers of animals are needed to optimally power studies. Overall, we present this as an advantageous approach which may change preclinical testing in future studies for the field of cardiac regeneration and tissue engineering.

This protocol aims to transplant a 3D bioprinted patch onto the epicardium of infarcted mice modeling heart failure. It includes details regarding anesthesia, the surgical chest opening, permanent ligation of the left anterior descending (LAD) coronary artery and application of a bioprinted patch onto the infarcted area of the heart.

Trapianto di una patch biostampa 3D in un modello Murine di infarto miocardico

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ...

Quantification of Mouse Heart Left Ventricular Function, Myocardial Strain, and Hemodynamic Forces by Cardiovascular Magnetic Resonance Imaging

Mouse models have contributed significantly to understanding genetic and physiological factors involved in healthy cardiac function, how perturbations result in pathology, and how myocardial diseases may be treated. Cardiovascular magnetic resonance imaging (CMR) has become an indispensable tool for a comprehensive in vivo assessment of cardiac anatomy and function. This protocol shows detailed measurements of mouse heart left ventricular function, myocardial strain, and hemodynamic forces using 7-Tesla CMR. First, animal preparation and positioning in the scanner are demonstrated. Survey scans are performed for planning imaging slices in various short- and long-axis views. A series of prospective ECG-triggered short-axis (SA) movies (or CINE images) are acquired covering the heart from apex to base, capturing end-systolic and end-diastolic phases. Subsequently, single-slice, retrospectively gated CINE images are acquired in a midventricular SA view, and in 2-, 3-, and 4-chamber views, to be reconstructed into high-temporal resolution CINE images using custom-built and open-source software. CINE images are subsequently analyzed using dedicated CMR image analysis software.
Delineating endomyocardial and epicardial borders in SA end-systolic and end-diastolic CINE images allows for the calculation of end-systolic and end-diastolic volumes, ejection fraction, and cardiac output. The midventricular SA CINE images are delineated for all cardiac time frames to extract a detailed volume-time curve. Its time derivative allows for the calculation of the diastolic function as the ratio of the early filling and atrial contraction waves. Finally, left ventricular endocardial walls in the 2-, 3-, and 4-chamber views are delineated using feature-tracking, from which longitudinal myocardial strain parameters and left ventricular hemodynamic forces are calculated. In conclusion, this protocol provides detailed in vivo quantification of the mouse cardiac parameters, which can be used to study temporal alterations in cardiac function in various mouse models of heart disease.

This study describes a comprehensive cardiovascular magnetic resonance imaging (CMR) protocol to quantify the left ventricular functional parameters of the mouse heart. The protocol describes the acquisition, post-processing, and analysis of the CMR images as well as assessment of different cardiac functional parameters.

Quantificazione della funzione ventricolare sinistra del cuore di topo, del ceppo miocardico e delle forze emodinamiche mediante risonanza magnetica cardiovascolare

Mouse models have contributed significantly to understanding genetic and physiological factors involved in healthy cardiac function, how perturbations ...

Evaluation of Cardiac Contractility Modulation Therapy in 2D Human Stem Cell-Derived Cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used in regulatory submissions. Here, we extend their use to cardiac medical device safety or performance assessments. We developed a novel method to evaluate cardiac medical device contractile properties in robustly contracting 2D hiPSC-CMs monolayers plated on a flexible extracellular matrix (ECM)-based hydrogel substrate. This tool enables the quantification of the effects of cardiac electrophysiology device signals on human cardiac function (e.g., contractile properties) with standard laboratory equipment. The 2D hiPSC-CM monolayers were cultured for 2-4 days on a flexible hydrogel substrate in a 48-well format.
The hiPSC-CMs were exposed to standard cardiac contractility modulation (CCM) medical device electrical signals and compared to control (i.e., pacing only) hiPSC-CMs. The baseline contractile properties of the 2D hiPSC-CMs were quantified by video-based detection analysis based on pixel displacement. The CCM-stimulated 2D hiPSC-CMs plated on the flexible hydrogel substrate displayed significantly enhanced contractile properties relative to baseline (i.e., before CCM stimulation), including an increased peak contraction amplitude and accelerated contraction and relaxation kinetics. Furthermore, the utilization of the flexible hydrogel substrate enables the multiplexing of the video-based cardiac-excitation contraction coupling readouts (i.e., electrophysiology, calcium handling, and contraction) in healthy and diseased hiPSC-CMs. The accurate detection and quantification of the effects of cardiac electrophysiological signals on human cardiac contraction is vital for cardiac medical device development, optimization, and de-risking. This method enables the robust visualization and quantification of the contractile properties of the cardiac syncytium, which should be valuable for nonclinical cardiac medical device safety or effectiveness testing. This paper describes, in detail, the methodology to generate 2D hiPSC-CM hydrogel substrate monolayers.

Here, we demonstrate a non-invasive cardiac medical device contractility evaluation method using 2D human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) monolayers, plated on a flexible substrate, coupled with video-based microscopy. This tool will be useful for the in vitro evaluation of the contractile properties of cardiac electrophysiology devices.

Valutazione della terapia di modulazione della contrattilità cardiaca in cardiomiociti 2D derivati da cellule staminali umane

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used ...