Questo lavoro è stato sostenuto da finanziamenti del SNF [31003A-138528], la Octav e la Botnar Fondazione Marcella, la Fondazione Novartis e la Muschamp Fondazione Emma. Ringraziamo Martine Lambelet, e Jean-Christophe Stehle per la loro assistenza tecnica eccellente.

Il Comitato Etico dell&#39;Università di Losanna ha approvato gli esperimenti, che sono in conformità con i principi enunciati nella Dichiarazione di Helsinki del 1975, riveduta nel 1983 per l&#39;uso di tessuti umani. 1. umano Grande vena safena Harvest <ol><li> Ottenere segmenti eccedenti di non varicose safene umane da pazienti sottoposti a chirurgia degli arti inferiori di bypass per ischemia. In sala operatoria, disinfettare tutta la gamba con una soluzione di iodio e drappeggio al paziente di esporre la gamba dall&#39;inguine al piede. </li><li> Eseguire un&#39;incisione mediana dall&#39;inguine al ginocchio (lasciando la parte della pelle interrotto). </li><li> Raccogliere la vena grande safena con un peduncolo di circostante (tecnica no-touch) del tessuto. Sicuro rami laterali delle vene con 4-0 cravatte di seta. Memorizzare immediatamente un minimo di 9 cm segmento avanzo lungo della grande safena, con un diametro esterno di 2,5-4 mm a 4 ° C in RPMI-1640 Glutamax medio, integrato con siero di vitello fetale 12,5% e portarlo al laboratorio. </li></ol> 2 EVPS design <ol><li> Montare l&#39;apparecchiatura generale mostrato in Figura 1. Autoclave tutte le apparecchiature e mantenere tutti i componenti in condizioni sterili. Inoltre, assicurarsi che il sistema è impermeabile e non perde sostanze chimiche nel mezzo. Utilizzare polimetacrilato di metile (PMMA-GS) per la copertina. Acciaio (X5 Cr Ni 18 10) e plastica poliossimetilene (POM) come supporto vena. </li><li> Progettare la camera di perfusione alla geometria desiderata per consentire il posizionamento della vena e la sua connessione. Assicurarsi che la profondità (o raggio se si utilizza la costruzione cilindrica) è almeno 2,5 cm in modo che permette la flessione minima e dilatazione del vaso con una copertura costante da parte del terreno di coltura (Figura 1). Tenuta è una questione importante ed è il motivo rettangolare costruzione PMMA-GS viene utilizzato. </li><li> Progettare il sostegno vena al geometr desideratoy. Per evitare piegatura vena o sopra distensione, consentire la regolazione della lunghezza spingendo o tirando (vite non può essere usato per questo scopo, come la vena potrebbe essere attorcigliato insieme con la vite). NOTA: Un tondino di acciaio pieno collegati da 2 pezzi scorrevoli a forma di L che supportano i cilindri 2 vena (5 mm di diametro per adattarsi nave) e la vena (Figura 1B e Figura 2) è qui utilizzato. </li><li> Progettare colonna di pressione, in modo tale che la &quot;pressione di riposo&quot; applicato al sistema è: p = 0-10 = hx ρ xg, dove p = pressione (N / m 2, Pa) h = altezza della colonna di liquido (m) ρ = densità del liquido (kg / m 3) e g = costante gravitazionale (9,81 m / sec 2). Condotti di collegamento Progettazione quattro, dall&#39;alto verso il basso: per applicare la pressione, per il deflusso (dalla vena), l&#39;afflusso (alla vena) e per consentire medie cambiamento. </li><li> Preparare il terreno. Sulla base di studi precedenti 5,11-14, scegliere RPMI-1640, integrato con Glutamax, 12,5Siero di vitello fetale%, e soluzione antibiotica-antimicotica 1% (10.000 U / ml di penicillina G, oltre a 10 mg / ml di streptomicina solfato, più 25 mg / ml di amfotericina B, oltre a 0,5 mg / ml: gentamicina). Lo sforzo di taglio (SS) è data da SS = 4 μQ / π * r 3 Q è la portata (ml / sec), r il raggio (cm) del segmento di vena, e μ è la viscosità del mezzo di perfusione. <ol><li> Modulare SS regolando la viscosità mediante aggiunta di 70 kDa destrano. Misurare la viscosità con un viscosimetro. Qui, aggiungere 8% 70 kDa destrano per impostare SS a 9-15 dyn / cm 2. </li></ol></li><li> Impostare la pompa ingranaggi per indurre un segnale cardioide pulsatile di 60 impulsi / min e ampiezza costante generando un flusso unidirezionale di 150 ± 15 ml / min, indipendentemente dalla pressione applicata nel sistema e controllato da un computer. Assicurarsi che il software di guida integra l&#39;acquisizione costante e il monitoraggio delle pressioni, velocità di flusso, frequenza del polso, e il segnale. Se lo si desidera, utilizzare una seconda pompa (non synchronized) per produrre un non-laminare, turbolento. </li></ol> 3. EVPS Assembly (Figura 1) <ol><li> Prima di iniziare, assicurarsi che tutta l&#39;attrezzatura è sterile. Eseguire le seguenti operazioni sotto asepsi in una cappa a flusso laminare. </li><li> Posizionare la vena in una capsula di Petri riempite con terreno. Utilizzare una lama chirurgica e dividere la vena in 3 segmenti uguali. </li><li> Sciacquare immediatamente un segmento in PBS. Dividere il segmento in 3 parti, fissare uno in formalina per morfometria. Congelare gli altri due per la trascrizione quantitativa (RT-PCR) e di proteine ​​(Western Blot) analisi. Considerare questi segmenti come controllo, vena non perfuso. </li><li> Utilizzare i 2 segmenti rimanenti per perfusione. <ol><li> Iniettare molto delicatamente medio nella vena e determinare la normale direzione del flusso; in presenza di valvole vena è invertita. </li><li> Tenuta le vene è della massima importanza per il successo sperimentale. Controllare eventuali perdite attraverso collaterali. Fissare eventuali perdite con 6-0 punti di sutura in seta. </li> &lt;/ Ol&gt; </li><li> Collegare il segmento vena tra i due cilindri metallici, un&#39;estremità alla volta (2.3, figura 1). Fissare i cilindri con Ethibon 3-0 intorno alle rientranze (Figure 1A e B). <ol><li> Posizionare l&#39;intero segmento venoso nella camera di perfusione in precedenza occupato dal mezzo. Ripetere la stessa procedura per il secondo segmento. NOTA: La mancata sigillare adeguatamente la vena al cilindro sarà una fonte di perdita, richiede reintervento, e aumentare significativamente il rischio di infezione e fallimento sperimentale. </li></ol></li><li> Per rinforzare (mesh) il secondo segmento, rilasciare i due cilindri (con la vena allegato) dai pezzi a forma di L (2.3 e Figura 1). <ol><li> Sii gentile e non toccare la vena con qualsiasi strumento. Far scorrere la maglia prima sul cilindro poi sulla vena. A spintoni push / pull otterrà la maglia sulla vena. </li><li> Una volta che la rete copre l&#39;intera superficie della vena fissare il jvena acketed ai cilindri con Ethibon 3-0. </li><li> Rimontare il composto vena / cilindro al supporto a forma di L e trasferirlo alla camera di perfusione, precedentemente occupato dal mezzo. </li></ol></li><li> Collegare ogni cilindro metallico (in-e deflusso) di un Y-splitter utilizzando tubi al silicone perossido-trattato con un diametro interno di 3,2 mm. </li><li> Collegare lo splitter deflusso di un secondo partitore a Y utilizzando lo stesso tipo di tubazione. Da questo Y-splitter, utilizzare un tubo per misurare la pressione di perfusione attraverso entrambe le navi. Collegare l&#39;altra posteriore alla colonna per formare un sistema ad anello chiuso (Figura 2). </li><li> All&#39;interno l&#39;incubatore, utilizzare un tubo lungo (lunghezza di un metro) per collegare la colonna di pressione alla testa della pompa. </li><li> Completa il set up collegando la testa della pompa per l&#39;afflusso Y-splitter con un altro tubo lunga durata (Figura 1). </li></ol> 4. Vene Perfusione <ol><li> Dopo l&#39;assemblea EVPS è stato completed, riempire la colonna con supporto (rimanere al di sotto del condotto di scarico vena per consentire la ricarica). Aggiungere più media nella colonna fino a quando il sistema è pieno. Spostare tutto il sistema in incubatore mantenuto a 37 ± 0,1 ° C con un pH mantenuto costante a 7.40 ± 0.01 (utilizzando un algoritmo di CO 2 / pH sulla base dell&#39;equazione di Henderson-Hasselbach). </li><li> Portare la testa della pompa ad ingranaggi all&#39;esterno dell&#39;incubatrice e collegarlo all&#39;unità pompa ad ingranaggi. Avvitare le aste per fissare il gruppo. </li><li> Accendere la pompa di alimentazione, assicurarsi che sia attivato il software di guida e lasciare 5 minuti a media e devono essere distribuiti equamente in ogni vano. </li><li> Per monitorare la pressione, utilizzare un monitoraggio linea arteriosa. Collegate l&#39;uscita di pressione EVPS (corrisponde al catetere arterioso) al trasduttore di pressione collegato al computer. <ol><li> Assicurarsi che il tubo è interamente occupata dal mezzo e non contiene bolle. De-bolla il sistema cultura attraverso la &quot;li arteriosane tubo &quot;(Figura 2). Prestare attenzione al display e cercare un segnale cardioide pulsatile di 60 impulsi / min di ampiezza costante. A questo punto, la pressione media è compresa tra 0-10 mm Hg. Se la pressione è &lt;0 e la colonna si svuota progressivamente cercare una perdita (collaterale della vena o tenuta inadeguata tra la vena e il tubo). </li></ol></li><li> Impostare la pressione minima a 6 mm Hg per un test venoso o 90 mm Hg per un test arteriosa. In queste condizioni, un iniettore aria si applica la pressione necessaria alla colonna e sistema. </li><li> Cambiare il mezzo ogni 2 giorni utilizzando il tubo collegato alla colonna di pressione. Per evitare danni cambiamento di pressione, aprire il tappo della colonna prima. </li></ol> 5 Completamento della perfusione <ol><li> Dopo 3 o 7 giorni di perfusione: prendere i EVPS fuori dell&#39;incubatore e smontare le vene. Eliminare il prossimale 5 mm e vena distale estremità collegata alle apparecchiature. Tagliare un centrale, rin spessore di 5 mmgs del segmento rimanente e fissare in formalina (morfometria). Congelare i frammenti rimanenti e ridurre in polvere per ulteriori analisi molecolari. </li></ol>

<ol>
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Questo studio rivela un ex vivo sistema di perfusione venosa (EVPS) per eseguire studi approfonditi emodinamiche nelle vene umane. Questo sistema permette safene perfusione sotto parametri emodinamici definiti in assenza di fattori infiammatori e di crescita aggravanti rilasciati dalle cellule circolanti in vivo. Pertanto, esso fornisce una migliore comprensione dei meccanismi sottostanti coinvolti nel controllo della IH in vene umane innesti 5,11,12,15. Perturba...

Le malattie cardiovascolari sono la principale causa di morbilità e mortalità nei paesi occidentali 1. Nonostante i progressi compiuti nei trattamenti endovascolari, la chirurgia di bypass rimane il fondamento delle terapie contemporanee, così oltre mezzo milione di innesti di vena vengono eseguite ogni anno negli Stati Uniti. Tuttavia, nonostante decenni di ricerca, 30-60% dei minori innesti estremità vena falliscono entro i primi anni a causa di iperplasia intimale (IH) 2. Forze meccaniche, particolarmente basso sforzo di taglio (SS) e la tensione alto muro, sono fondamentali nel l&#39;avvio e lo sviluppo di questa risposta iperplastica 3,4. Per risolvere questo problema, un ex vivo vene sistema di perfusione (EVPS) è stato generato per studiare, in condizioni strettamente controllate emodinamiche (pressione e lo stress di taglio), il comportamento di safene umane. In questo studio, dopo l&#39;inserimento nella circolazione arteriosa simile, ad alta pressione (media = 100 mm Hg) era sufficiente per stimolare prolifrazione e la migrazione delle cellule muscolari lisce nello strato intimale (IH) 5. Studi mammiferi hanno suggerito l&#39;uso del rinforzo esterno come un metodo efficace per sostenere la &quot;vena arterializzato&quot; e contrastare l&#39;emodinamica acuta cambia la vena facce, una volta impiantato in un ambiente arteriosa. La maglia ha impedito sovra-distensione, aumento dello stress di taglio, e ridotta tensione di parete e di conseguenza IH 6-10. Tuttavia, i meccanismi alla base e la sua applicabilità alle vene umane per migliorare bypass pervietà non sono stati completamente caratterizzati. I nostri EVPS è stato utilizzato per confrontare, in condizioni che mimano le alterazioni vena volti, una volta inserito in un regime arteriosa (stress e pressione alta di taglio), il comportamento di safene umane in assenza e presenza di una rete tubolare in poliestere macroporosa esterno. Impedendo rimodellamento patologico e IH, la rete ha fornito prove della sua potenziale efficacia clinica 11 </sup&gt;. Questo studio 1) introduce un modello di ex vivo safene umane perfusione sotto pressione controllata e sforzo di taglio 2) dimostra che in poliestere maglia macro-poroso esterno riduce IH e fornisce informazioni fondamentali per la sua potenziale applicazione clinica.

<table border="0" cellpadding="0" cellspacing="0" width="1113">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Name</td>
			<td style="width:371px;">Company</td>
			<td style="width:145px;">Catalog Number</td>
			<td style="width:144px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640 - Glutamax</td>
			<td>Life Technologies</td>
			<td>61870-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"><a name="RANGE!A3">Penicilline/Streptomycine/Fungizone</a></td>
			<td>Bioconcept</td>
			<td>4-02F00-H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dextran from Leuconostoc spp. 500 gr.</td>
			<td>Sigma-Aldrich</td>
			<td>31390</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tampon PBS CHUV pH 7.1-7.3 1 lt.</td>
			<td>Laboratorium und Grosse Apotheke Dr. G. Bichsel AG</td>
			<td>100 0 324 00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryosectionning embedding medium - Tissue-Tek OCT Compound</td>
			<td>Fisher Scientific</td>
			<td>14-373-65</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silicon Tubing (Peroxide) L/S 16 (96400-16 ) - 7.5m</td>
			<td>Idex Health &#38; Science GMBH</td>
			<td>MF0037ST</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Y-splitter&#160;</td>
			<td>Idex Health &#38; Science GMBH</td>
			<td>Y-connector</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35 mm Culture dish</td>
			<td>Sigma-Aldrich</td>
			<td>CLS430165-100EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 ml Falcon tube</td>
			<td>BD Bioscence</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 ml Falcon tube</td>
			<td>BD Bioscence</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gearing pump - Reglo-Z</td>
			<td>Idex Health &#38; Science GMBH</td>
			<td>SM 895&#160;</td>
			<td>&#160;App-Nr 03736-00194</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pump Head</td>
			<td>Idex Health &#38; Science GMBH</td>
			<td>MI0008&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Monitoring Kit TRANSPAC IV</td>
			<td>icumedical</td>
			<td>011-0J736-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20 mL Syringes</td>
			<td>B. Braun Medical SA</td>
			<td>4612041-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Etibon 3-0 FS-2</td>
			<td>Ethicon- Johnson&#38;Johnson</td>
			<td>EH7346H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mesh ProVena 6-8mm</td>
			<td>B. Braun Medical SA</td>
			<td>1105012-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl: Sodium Chlorure Solution perfusion 0.9% (100 ml)</td>
			<td>B. Braun Medical SA</td>
			<td>534534</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Masterflex L/S Standard Drive</td>
			<td>Cole-Parmer Instrument Co</td>
			<td>7521-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acquisition card</td>
			<td>National Instruments</td>
			<td>PCI-6024 E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flowmeter module</td>
			<td>Transonic Systems Inc.</td>
			<td>TS410 and T402</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stopcock with 3-ways</td>
			<td>BD Connexta Luerlock</td>
			<td>394600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Millex Filter</td>
			<td>Milian</td>
			<td>SE2M229I04</td>
			<td></td>
		</tr>
	</tbody>
</table>

procedure for human saphenous veins ex vivo perfusion and external reinforcement

Il pilastro di terapie contemporanee per esteso arteriopatia obliterante è venosa bypass. Tuttavia, la sua durata è minacciato da iperplasia intimale (IH), che alla fine porta a occlusione nave e fallimento del trapianto. Forze meccaniche, particolarmente basso shear stress e la tensione alto muro, si pensa di avviare e sostenere questi cambiamenti molecolari e cellulari, ma il loro contributo esatto resta da dipanare. Per valutare in modo selettivo il ruolo di pressione e sollecitazione di taglio sulla biologia di IH, un ex vivo sistema di perfusione (EVPS) è stato creato per perfusione segmenti di vene safene umani sotto il regime arteriosa (stress elevato shear e alta pressione). Ulteriori innovazioni tecniche hanno permesso di perfusione simultanea di due segmenti della stessa vena, uno armato con una rete esterna. Le vene sono state raccolte utilizzando una tecnica no-touch e immediatamente trasferiti al laboratorio per il montaggio in EVPS. Un segmento del isol appenaated vena non è stato perfuso (controllo, giorno 0). Gli altri due segmenti sono stati perfusi per un massimo di 7 giorni, un essere completamente al riparo con un 4 mm (diametro) della maglia esterno. La pressione, velocità del flusso, e la frequenza cardiaca sono stati costantemente monitorati e adeguati per simulare le condizioni emodinamiche prevalenti nell&#39;arteria femorale. Al termine della perfusione, vene sono stati smontati e utilizzati per l&#39;analisi istologica e molecolare. In ex vivo, condizioni di perfusione ad alta pressione (arteriosa, medio = 100 mm Hg) è sufficiente a generare IH e il rimodellamento delle vene umane. Queste alterazioni sono ridotti in presenza di una rete di poliestere esterno.

Il EVPS fornisce un prezioso strumento per valutare autonomamente le forze emodinamiche sulla vena safena umana innesta rimodellamento e IH. La Figura 1 mostra la camera di perfusione e il supporto vena. Nelle figure 1A e B, il supporto vena prima (Figura 1A) e dopo (Figura 1B) assemblaggio, rispettivamente è raffigurato. È composto (dalla parte superiore alla parte inferiore) di 1 tubo di acciaio inox pia...

Watch this Scientific Journal Video about Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement at JoVE.com

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

I meccanismi che portano allo sviluppo di iperplasia intimale (IH) e il fallimento del trapianto vena sono ancora poco conosciuti. Questo studio descrive un sistema ex vivo di perfusione vene umane sotto flusso e pressione controllata. Inoltre l&#39;efficienza di rinforzo maglia esterna per limitare lo sviluppo di IH è stata valutata.

Procedura per la safena umane Veins<em&gt; Ex Vivo</em&gt; Perfusione e rinforzo esterno

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal ...

procedure-for-human-saphenous-veins-ex-vivo-perfusion-external

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

11.1K Views.  Brigham and Women's Hospital/Harvard Medical School.  I meccanismi che portano allo sviluppo di iperplasia intimale (IH) e il fallimento del trapianto vena sono ancora poco conosciuti. Questo studio descrive un sistema ex vivo di perfusione vene umane sotto flusso e pressione controllata. Inoltre l'efficienza di rinforzo maglia esterna per limitare lo sviluppo di IH è stata valutata. 

Video: Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

The success of liver transplantation has resulted in a dramatic organ shortage. In most transplant regions 20-30% of patients on the waiting list for liver transplantation die without receiving an organ transplant or are delisted for disease progression. One strategy to increase the donor pool is the utilization of marginal grafts, such as fatty livers, grafts from older donors, or donation after cardiac death (DCD). The current preservation technique of cold static storage is only poorly tolerated by marginal livers resulting in significant organ damage. In addition, cold static organ storage does not allow graft assessment or repair prior to transplantation.
These shortcomings of cold static preservation have triggered an interest in warm perfused organ preservation to reduce cold ischemic injury, assess liver grafts during preservation, and explore the opportunity to repair marginal livers prior to transplantation. The optimal pressure and flow conditions, perfusion temperature, composition of the perfusion solution and the need for an oxygen carrier has been controversial in the past.
In spite of promising results in several animal studies, the complexity and the costs have prevented a broader clinical application so far. Recently, with enhanced technology and a better understanding of liver physiology during ex vivo perfusion the outcome of warm liver perfusion has improved and consistently good results can be achieved.
This paper will provide information about liver retrieval, storage techniques, and isolated liver perfusion in pigs. We will illustrate a) the requirements to ensure sufficient oxygen supply to the organ, b) technical considerations about the perfusion machine and the perfusion solution, and c) biochemical aspects of isolated organs.

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

Marginal grafts, such as fatty livers, grafts from older donors, or livers retrieved after cardiac death (DCD) tolerate conventional, cold static storage only poorly. We developed a novel model of subnormothermic ex vivo liver perfusion for preservation, assessment, and repair of marginal liver grafts prior to transplantation.

Tecnica di Subnormothermic<em&gt; Ex Vivo</em&gt; Fegato perfusione per la Conservazione, valutazione e riparazione dei marginali fegato innesti

The success of liver transplantation has resulted in a dramatic organ shortage. In most transplant regions 20-30% of patients on the waiting list for ...

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Medicina

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and &#8220;pearls of wisdom&#8221;/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

Metodo di isolato<em&gt; Ex Vivo</em&gt; Lung Perfusion in un modello di ratto: Lezioni apprese da Sviluppo di un programma EVLP Rat

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

Un<em&gt; Ex vivo</em&gt; Modello per studiare Hormone azione in seno umano

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from cirrhosis to cardiovascular diseases). There is no effective treatment and the in vitro tools available do not mimic the in vivo situation rendering the progress of the out of control wound healing process still enigmatic.
To date, 2D and 3D cultures of fibroblasts derived from DD patients are the main experimental models available. Primary cell cultures have many limitations; the fibroblasts derived from DD are altered by the culture conditions, lack cellular context and interactions, which are crucial for the development of fibrosis and weakly represent the derived tissue. Real-time PCR analysis of fibroblasts derived from control and DD samples show that little difference is detectable. 3D cultures of fibroblasts include addition of extracellular matrix that alters the native conditions of these cells. 
As a way to characterize the fibrotic, proliferative properties of these resection specimens we have developed a 3D culture system, using intact human resections of the nodule part of the cord. The system is based on transwell plates with an attached nitrocellulose membrane that allows contact of the tissue with the medium but not with the plastic, thus, preventing the alteration of the tissue. No collagen gel or other extracellular matrix protein substrate is required. The tissue resection specimens maintain their viability and proliferative properties for 7 days. This is the first &#8220;organ&#8221; culture system that allows human resection specimens from DD patients to be grown ex vivo and functionally tested, recapitulating the in vivo situation.

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Dupuytren&#8217;s disease (DD) is a fibroproliferative disease of the palm of the hand. Here, we present a protocol to culture resection specimens from DD in a three-dimensional (3D) culture system. Such short-term culture system allows preservation of the 3D structure and molecular properties of the fibrotic tissue.

Umana Dupuytren di<em&gt; Ex Vivo</em&gt; Cultura per lo Studio dei miofibroblasti e matrice extracellulare Interazioni

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from ...

Ex Situ Normothermic Machine Perfusion of Donor Livers

In contrast to conventional static cold preservation (0-4 &#176;C), ex situ machine perfusion may provide better preservation of donor livers. Continuous perfusion of organs provides the opportunity to improve organ quality and allows ex situ viability assessment of donor livers prior to transplantation. This video article provides a step by step protocol for ex situ normothermic machine perfusion (37 &#176;C) of human donor livers using a device that provides a pressure and temperature controlled pulsatile perfusion of the hepatic artery and continuous perfusion of the portal vein. The perfusion fluid is oxygenated by two hollow fiber membrane oxygenators and the temperature can be regulated between 10 &#176;C and 37 &#176;C. During perfusion, the metabolic activity of the liver as well as the degree of injury can be assessed by biochemical analysis of samples taken from the perfusion fluid. Machine perfusion is a very promising tool to increase the number of livers that are suitable for transplantation.

Ex Situ Normothermic Machine Perfusion of Donor Livers

Here we present a protocol describing oxygenated ex situ machine perfusion of donor liver grafts. This article contains a step by step protocol to procure and prepare the liver graft for machine perfusion, prepare the perfusion fluid, prime the perfusion machine and perform oxygenated normothermic machine perfusion of the liver graft.

Ex Situ normotermica macchina di perfusione di Vivi donatori

In contrast to conventional static cold preservation (0-4 &#176;C), ex situ machine perfusion may provide better preservation of donor livers. Continuous ...

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a serious problem. Therefore, the acceptance criteria for organ donors have been extended leading to the usage of marginal kidney grafts. These marginal organs tolerate cold storage poorly resulting in increased preservation injury and higher rates of delayed graft function. To overcome the limitations of cold storage, extensive research is focused on alternative normothermic preservation methods.
Ex vivo normothermic organ perfusion is an innovative preservation technique. The first experimental and clinical trials for ex vivo lung, liver, and kidney perfusions demonstrated favorable outcomes.
In addition to the reduction of cold ischemic injury, the method of normothermic kidney storage offers the opportunity for organ assessment and repair. This manuscript provides information about kidney retrieval, organ preservation techniques, and isolated ex vivo normothermic kidney perfusion (NEVKP) in a porcine model. Surgical techniques, set up for the perfusion solution and the circuit, potential assessment options, and representative results are demonstrated.

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

The severe organ shortage has resulted in increased use of marginal kidney grafts for transplantation. This has triggered interest in alternative storage methods, since marginal grafts especially tolerate cold storage poorly. The technique of normothermic ex vivo kidney perfusion (NEVKP) represents a novel preservation method for kidney grafts prior to &#160;transplantation.

Normotermica<em&gt; Ex Vivo</em&gt; Rene perfusione per la Preservazione della Kidney Innesti prima del trapianto

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

Generazione di Microtumors Uso 3D Biogel Umano Sistema Cultura e cellule di glioblastoma paziente-derivato per Kinomic Profiling and Drug Testing risposta

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, normothermic ex vivo liver perfusion (NEVLP) has been introduced as a method to evaluate and modify organ function. NEVLP has many advantages in comparison to hypothermic and subnormothermic perfusion including reduced preservation injury, restoration of normal organ function under physiologic conditions, assessment of organ performance, and as a platform for organ repair, remodeling, and modification. Both murine and porcine NEVLP models have been described. We demonstrate a rat model of NEVLP and use this model to show one of its important applications &#8212; the use of a therapeutic molecule added to liver perfusate. Catalase is an endogenous reactive oxygen species (ROS) scavenger and has been demonstrated to decrease ischemia-reperfusion in the eye, brain, and lung. Pegylation has been shown to target catalase to the endothelium. Here, we added pegylated-catalase (PEG-CAT) to the base perfusate and demonstrated its ability to mitigate liver preservation injury. An advantage of our rodent NEVLP model is that it is inexpensive in comparison to larger animal models. A limitation of this study is that it does not currently include post-perfusion liver transplantation. Therefore, prediction of the function of the organ post-transplantation cannot be made with certainty. However, the rat liver transplant model is well established and certainly could be used in conjunction with this model. In conclusion, we have demonstrated an inexpensive, simple, easily replicable NEVLP model using rats. Applications of this model can include testing novel perfusates and perfusate additives, testing software designed for organ evaluation, and experiments designed to repair organs.

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant liver donor shortage, and criteria for liver donors have been expanded. Normothermic ex vivo liver perfusion (NEVLP) has been developed to evaluate and modify organ function. This study demonstrates a rat model of NEVLP and tests the ability of pegylated-catalase, to mitigate liver preservation injury.

Un piccolo modello animale di aspersione epatica normotermica Ex Vivo

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, ...

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to larger animals. Yet, larger mammals are better suited for translational research questions related to normal cardiac physiology, pathophysiology, and preclinical testing of therapeutic agents. To overcome the technical barriers associated with employing a larger animal model in cardiac research, we describe an approach to measure physiological parameters in an isolated, Langendorff-perfused piglet heart. This approach combines two powerful experimental tools to evaluate the state of the heart: electrophysiology (EP) study and simultaneous optical mapping of transmembrane voltage and intracellular calcium using parameter sensitive dyes (RH237, Rhod2-AM). The described methodologies are well suited for translational studies investigating the cardiac conduction system, alterations in action potential morphology, calcium handling, excitation-contraction coupling and the incidence of cardiac alternans or arrhythmias.

The objective of this study was to establish a method for investigating cardiac dynamics using a translational animal model. The described experimental approach incorporates dual-emission optocardiography in conjunction with an electrophysiological study to assess electrical activity in an isolated, intact porcine heart model.

Optocardiografia ed elettrofisiologia Studi di Ex Vivo Langendorff-perfusi

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to ...

Determining and Controlling External Power Output During Regular Handrim Wheelchair Propulsion

The use of a manual wheelchair is critical to 1% of the world&#39;s population. Human powered wheeled mobility research has considerably matured, which has led to improved research techniques becoming available over the last decades. To increase the understanding of wheeled mobility performance, monitoring, training, skill acquisition, and optimization of the wheelchair-user interface in rehabilitation, daily life, and sports, further standardization of measurement set-ups and analyses is required. A crucial stepping-stone is the accurate measurement and standardization of external power output (measured in Watts), which is pivotal for the interpretation and comparison of experiments aiming to improve rehabilitation practice, activities of daily living, and adaptive sports. The different methodologies and advantages of accurate power output determination during overground, treadmill, and ergometer-based testing are presented and discussed in detail. Overground propulsion provides the most externally valid mode for testing, but standardization can be troublesome. Treadmill propulsion is mechanically similar to overground propulsion, but turning and accelerating is not possible. An ergometer is the most constrained and standardization is relatively easy. The goal is to stimulate good practice and standardization to facilitate the further development of theory and its application among research facilities and applied clinical and sports sciences around the world.

Accurate and standardized assessment of external power output is crucial in the evaluation of physiological, biomechanical, and perceived stress, strain, and capacity in manual wheelchair propulsion. The current article presents various methods to determine and control power output during wheelchair propulsion studies in the laboratory and beyond.

Determinazione e controllo della potenza esterna durante la propulsione regolare della sedia a rotelle Handrim

The use of a manual wheelchair is critical to 1% of the world&#39;s population. Human powered wheeled mobility research has considerably matured, which ...

Procedura per la safena umane Veins

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Tecnica di Subnormothermic

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Ex Situ normotermica macchina di perfusione di Vivi donatori

Normotermica

Generazione di Microtumors Uso 3D Biogel Umano Sistema Cultura e cellule di glioblastoma paziente-derivato per Kinomic Profiling and Drug Testing risposta

Un piccolo modello animale di aspersione epatica normotermica Ex Vivo

Optocardiografia ed elettrofisiologia Studi di Ex Vivo Langendorff-perfusi

Determinazione e controllo della potenza esterna durante la propulsione regolare della sedia a rotelle Handrim