Gli autori desiderano ringraziare i membri passati della Fotografico laboratorio: Ming-Min Lee, Kathryn Plichta, Victoria Miranda-Thompson e Sam-Kyu Kim per il loro contributo al miglioramento di molti di questi protocolli. Questo lavoro è stato finanziato in parte mediante la concessione National Science Foundation IOS e IOS-0840932-0724978 SP Archivio

1 In vivo Allevamento di Nematodi entomopatogeni con i loro batteri Symboitic <ol><li> Invertire una capsula di Petri in plastica 100 x 15 mm e collocare due dischi di carta da filtro (90 mm) nel coperchio del piatto. </li><li> Distribuire uniformemente 1 ml della sospensione acqua IJ (larve infettive) (ad una concentrazione di 1.000-2.000 IJ / ml) sulla carta da filtro. NOTA: IJs non devono essere sterilizzati in superficie. </li><li> Aggiungere 10 ultimo instar larve della maggior waxmoth Galleria mellonella al piatto. L&#39;obiettivo è quello di fornire circa 100-200 IJs / larva. </li><li> Coprire il coperchio con la parte inferiore della scatola di Petri (Figura 1) ed etichettare la piastra di Petri. Indicare le seguenti informazioni: nematode nome della specie (se noto); isolare il codice / designazione; trappola infezione data è stata impostata. </li><li> Porre la capsula all&#39;interno di un sacchetto di plastica sigillato liberamente (per conservare l&#39;umidità) e tenerlo al buio sia a temperatura ambiente o in un incubatore tra i 20-25 ° C. Controllare i piatti tutti i giorni NOTA: L&#39;oscurità facilita l&#39;infezione da nematodi, in quanto imita le condizioni naturali di infezione nel terreno. </li><li> Dopo 3-5 giorni, rimuovere i cadaveri con segni di infezione da nematodi ad una modifica trappola Bianco 7. NOTA: Se i cadaveri odore putrido, questo può essere un&#39;indicazione che la coltura non ha avuto successo e / o c&#39;era contaminazione. Impostare una nuova camera di infezione con un nuovo lotto di nematodi e insetti. </li></ol> 2 in vitro Allevamento di Nematodi entomopatogeni con i loro batteri simbionti <ol><li> Fegato-rene Agar Metodo di 9,19 <ol><li> Tritate il fegato e reni in piccoli pezzi (da 2 cm 3 o più piccoli) e mettere in un frullatore con NaCl, agar e 300 ml di acqua e mescolare fino a quando la carne diventa una poltiglia liquida, pasta spessa o purea. Poi, il trasferimento miscela di un pallone da 1 L Erlenmeyer. </li><li> Aggiungere gli ultimi 200 ml di acqua al vaso frullatore e decantare eventuali supporti rimasti nelBeuta. Autoclave per 15 minuti a 121 ° C. Dopo la sterilizzazione in autoclave agar lasciare raffreddare a toccare prima di versare. NOTA: dispensazione dei media non è raccomandato perché questo mezzo tende ad avere pezzi di carne. </li><li> Versare ~ 20 ml di agar su 5 (o 6) cm piastre di Petri agitando la mano o agitando la beuta tra versa per un supporto più omogeneo. Lasciare agar per solidificare e piatti conservare a 4 ° C fino al momento NOTA: Utilizzare una spatola di metallo-sterilizzati fiamma per rompere tutti i grandi pezzi di carne versato nella piastra di Petri. </li></ol></li><li> Lipid Agar Metodo 9,19 <ol><li> Preparare mezzo lipidico agar mescolando brodo nutriente, estratto di agar e il lievito e mescolare versare in un pallone da 2 L Erlenmeyer. Aggiungere acqua distillata H 2 O e MgCl 2 • 6H 2 O e autoclave per 15 minuti a 121 ° C. </li><li> Aggiungere miscela sterile di olio e di sciroppo di mais mais mix e mescolare o agitare energicamente e spesso per disperdere goccioline di olio in modo uniforme. <br/&gt; NOTA: Sterilizzare olio di mais e lo sciroppo mettendo il volume necessario in un UV cross-linker. L&#39;olio non si dissolvono nel liquido, ma assicurarsi che sia in minuscole goccioline. </li><li> Versare ~ 20 ml di agar su un 5 (o 6) cm piastre di Petri e lasciare solidificare l&#39;agar e piatti conservare a 4 ° C fino al momento dell&#39;uso. </li><li> Streak il batteri simbionti desiderato sulla piastra di agar lipidi e incubare le piastre a 28 ° C per 24-48 ore. NOTA: Stendere 100-200 ml di una notte (12-16 ore) LB sottocultura. </li><li> Il giorno dopo, aggiungere circa 0,4 ml di sospensione sterile superficie nematode (uova o larve infettive) e incubare le piastre a temperatura ambiente al buio o in un incubatore a 22 ± 3 ° C. NOTA: Non aggiungere più di 0,4 ml di sospensione nematode come si può creare un ambiente eccessivamente umido che è sfavorevole alla crescita nematode. </li><li> Monitorare culture quotidiana. Le culture dovrebbero produrre una nuova generazione di IJs in circa 12 giorni (Figura 2).</li><li> Trasferire piatto fondo con agar quando nematodi sono visti strisciare i lati del piatto (Figura 3A) ad una trappola Bianco modificata (vedi Orozco et al. 12) per raccogliere IJs (Figura 3B). Eseguire questo passaggio sotto una cappa a flusso laminare per ridurre la contaminazione dei media. </li><li> Raccogliere IJs dalla trappola Bianco modificato sulla nascita e risciacquare sospensione IJ per rimuovere eventuali detriti mezzo. Conservare IJ in acqua distillata sterilizzata a 10 ° C (in un incubatore a freddo-temperatura o cella frigorifera) o utilizzare immediatamente, se necessario. NOTA: A seconda l&#39;obiettivo dello studio, il seme piatti sia con nematodi simbionti-colonizzati o aposymbiotic. </li></ol></li></ol> 3. in vitro allevamento di Aposymbiotic (Symbiont-gratuito) Nematodi entomopatogeni <ol><li> Infettare 10-20 G. mellonella larve con IJs delle specie di nematodi desiderati (vedi capitolo 1). Dopo 3 o 4 giorni (in questo momento IJs avrebbe maturatoagli adulti di prima generazione) raccogliere cadaveri. NOTA: Il tempo a scadenza e numero di femmine necessari per ottenere un adeguato numero di uova può variare da specie. Infettare più G. mellonella larve se necessario e iniziare dissezioni già alla 48 ore dopo l&#39;infezione per valutare se sono in fase di sviluppo destra. </li><li> Mettete ogni cadavere singolarmente in una capsula di Petri con una soluzione salina (tampone M9 18). Sezionare un cadavere tirando la sua testa con una pinza sottile punta. </li><li> Raccogliere almeno 20 gravido (con le uova all&#39;interno) femmine con un ago sottile (L-forma preferibilmente) e metterli in un vetro da orologio contenente 10 ml di tampone M9 (Figura 4A). </li><li> Preparare una soluzione axenizing considerando i seguenti ingredienti: 6,75 ml di acqua distillata, 1,25 ml 5 N NaOH, e 2,25 ml di soluzione di candeggina disponibile in commercio diluito al 3% di ipoclorito. NOTA: NaOH è una soluzione di base-guanti, occhiali e altro adeguato di protezioneabbigliamento deve essere indossato. NOTA: Preparare soluzione axenizing fresco ogni volta, come candeggina degrada alla luce. Preparare diversi lotti di soluzione axenizing ed eseguire la sterilizzazione di superficie e la raccolta delle uova in più vetri da orologio. </li><li> Risciacquare femmine almeno tre volte con riempimento del buffer M9 vetro d&#39;orologio con soluzione tampone e succhiare buffer con una pipetta. Assicurarsi che le femmine rimangono nel vetro d&#39;orologio. Ripetere questa operazione altre due volte. </li><li> Aggiungere immediatamente la soluzione axenizing e permettono le femmine di rimanere in questa soluzione per non più di 10-15 minuti. Osservare i tessuti nematode cominciano a disintegrarsi, mentre le uova (che sono resistenti alla soluzione axenizing) rimangono intatti. </li><li> Interrompere manualmente corpi femminili per accelerare il processo, tagliandoli con un ago o un bisturi (Figura 4B, C). Rimuovere le uova da loro pipettando in provette da 1,5 ml microcentrifuga, evitando l&#39;aggiunta di qualsiasi tessuto non disciolto. NOTA: Utilizzare come meventuali microprovette se necessario per raccogliere le uova e lavorare rapidamente, come la vitalità delle uova diminuisce per un periodo prolungato di esposizione alla soluzione di axenizing. </li><li> Tubi vortex per 15 secondi utilizzando l&#39;impostazione &quot;alta velocità&quot; per rimuovere ulteriormente i tessuti nematodi attaccati alle uova. In alternativa, se un vortice non è disponibile, pipettare su e giù per le soluzioni più volte. </li><li> Uova pellet di centrifugazione a 18.000 g per circa 2 min. Aumentare la velocità se le uova non fanno sufficientemente pellet. Rimuovere la soluzione axenizing e risciacquare due volte riempiendo la provetta con acqua distillata sterile e centrifugare ancora una volta a 900 xg per 1 min. </li><li> Dopo l&#39;ultimo lavaggio, risospendere le uova in 300-500 ml di acqua distillata sterile e metterli in una sterile 3 centimetri scatola di Petri o vetro da orologio sterilizzati. Ricordate che le uova sono ormai sterilizzato superficie, in modo da utilizzare pipette sterili e / o suggerimenti pippetter e acqua per mantenere la pulizia delle uova. </li><li>Esaminare le uova sotto un microscopio da dissezione (30-50X ingrandimento) per confermare che sono intatti (Figura 4D) e trasferirli in una piastra 5 cm e agar fegato-rene (vedere paragrafo 2.1) NOTA: Non aggiungere più di 400 microlitri della sospensione uovo sulle piastre come farà l&#39;agar eccessivamente bagnato. </li><li> Le targhette di identificazione con le informazioni appropriate e memorizzarli in posizione verticale al buio a 28 ° C. Schiusa delle uova dovrebbe essere evidente durante la notte. NOTA: L&#39;acqua può condensare sul coperchio del piatto ma evapora dopo 1 o 2 giorni. A questo punto, porre il recipiente in sacchetto di plastica per trattenere l&#39;umidità del agar ed evitare la sua essiccazione. </li><li> Osservare piatti periodicamente e scartare tutti i piatti che mostrano segni di contaminazione batterica o fungina. Una volta IJs sono visti migrare il muro della capsula di Petri, togliere il coperchio e trasferire il piatto fondo con agar ad una modifica trappola Bianco 12. Considerare condizioni sterili quando si trasferisce il piatto nel Modified trappola bianco per evitare la contaminazione dei mezzi esposti. </li><li> Continua il monitoraggio delle trappole bianche e raccolto IJ, se necessario. NOTA: nematodi Aposymbiotic continueranno a migrare in acqua della trappola Bianca per molti giorni e addirittura settimane. </li></ol>

<ol>
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Nessun conflitto di interessi dichiarati.

Utilizzando un ospite adatto è un fattore chiave per il successo in allevamento vivo di EPN. Di solito, sia steinernematids e heterorhabditids possono riprodursi e completare il loro ciclo di vita in larve della maggiore tarma della cera, Galleria mellonella (Lepidoptera: Pyralidae). Tuttavia, altre specie di insetti provenienti da diverse famiglie e / o gli ordini possono essere considerati. Alcune delle specie di nematodi attualmente descritte sono noti per avere specificità per un partico...

Nematodi entomopatogeni (EPN) Steinernema e Heterorhabditis spp. (Steinernematidae, Heterorhabditidae) e dei loro simbionti batterici, Xenorhabdus e Photorhabdus spp (Enterobacteriaceae) sono considerati un modello emergente di animali terrestri-microbici relazioni simbiotiche 2-4,6,10,19. Xenorhabdus e Photorhabdus spp. sono nutrito come simbionti nell&#39;intestino della sola fase di vita libera dei nematodi, noto anche come il giovanile infettiva (IJ) o stadio 3 ° infettivo giovanile 8,10,13. La coppia batterio-nematode è patogeno per una vasta gamma di insetti ed è stato implementato con successo in programmi di lotta biologica e lotta integrata in tutto il mondo 6,8. Qui mostriamo una selezione di in vivo e in vitro tecniche che sono spesso esercitati per l&#39;allevamento di EPN in condizioni di laboratorio. In metodi in vivo contemplano un host insetto per l&#39;allevamento dei nematodi. Di solito, stadi immaturi di vari ordini di insetti (ad esempio Lepidotteri, Coleotteri, Ditteri, ecc.) Sono considerati host adatti. In vivo i metodi sono di solito presi in considerazione per la manutenzione delle culture nematodi in laboratorio. Questo metodo potrebbe non essere adatto se si considera la produzione di massa dei nematodi. Grandi quantità di insetti ospiti possono essere richiesti a tale scopo chiedendo più tempo e costi supplementari connesse con l&#39;allevamento degli insetti. Nematodi entomopatogeni possono anche essere coltivate in vitro su diversi supporti. A seconda l&#39;obiettivo dello studio, metodi in vitro può o prendere in considerazione l&#39;incorporazione dei batteri simbiotici nei media. In questa presentazione, descriviamo due metodi comunemente utilizzati per la propagazione della EPN. Gli ingredienti dei media forniscono una fonte di nutrienti per il batterio simbiotico unMetodi in vitro trovare una fonte di steroli per i nematodi. offrono il vantaggio l&#39;allevamento di EPN senza un host insetto. In origine, molti dei media in vitro sviluppati sono stati utilizzati per la moltiplicazione di EPN quando adatti insetti ospiti non sono disponibili. Tuttavia, negli ultimi anni, in vitro metodi di allevamento sono diventati largamente impiegato nel campo della ricerca al fine di comprendere il rapporto mutualistico tra EPN ed i loro batteri simbionti 17,19. Le tecniche descritti in questa presentazione corrispondono a quelli descritti da vari autori e raffinato dal laboratorio Stock, University of Arizona (Tucson, AZ, USA). Queste tecniche sono distinti dal corpo di tecniche che vengono utilizzati nella produzione di massa di tali organismi a fini di gestione nocivo.

<table border="0" cellpadding="0" cellspacing="0" width="1827">
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			<td height="42" style="height:42px;width:485px;">Material&#160;</td>
			<td style="width:260px;">Company</td>
			<td style="width:488px;">Catalog Number</td>
			<td style="width:595px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;"></td>
			<td style="width:260px;"></td>
			<td style="width:488px;"></td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:485px;">For in vivo Infections</td>
			<td style="width:260px;"></td>
			<td style="width:488px;"></td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:485px;">100 x 15 Petri Dishes</td>
			<td style="width:260px;">VWR</td>
			<td style="width:488px;">25384-088</td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:485px;">Filter Paper, 9 cm Grade 1 Cellulose</td>
			<td style="width:260px;">Whatman/VWR</td>
			<td style="width:488px;">28450-081</td>
			<td style="width:595px;">Grade 1 filter paper recommended</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:485px;">Insect hosts</td>
			<td style="width:260px;">Timberline</td>
			<td style="width:488px;">http://www.timberlinefisheries.com/ProductDetails.asp?ProductCode=WAXLG</td>
			<td style="width:595px;">Galleria mellonella are recommended</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;"></td>
			<td style="width:260px;"></td>
			<td style="width:488px;"></td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:485px;">For Liver-Kidney Agar (for 500 ml)</td>
			<td style="width:260px;"></td>
			<td style="width:488px;"></td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;width:485px;">60 x 15 mm Petri Dishes</td>
			<td style="width:260px;">VWR</td>
			<td>25384-092</td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:485px;">Beef Liver, 50 g</td>
			<td style="width:260px;">Locally sourced; butcher or supermarket</td>
			<td style="width:488px;"></td>
			<td style="width:595px;">Remaining may be stored frozen and thawed for future use</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:485px;">Beef Kidney, 50 g</td>
			<td style="width:260px;">Locally sourced; butcher or supermarket</td>
			<td style="width:488px;"></td>
			<td style="width:595px;">Remaining may be stored frozen and thawed for future use</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:485px;">Sodium Chloride 2.5g ( 0.5% final concentration )</td>
			<td style="width:260px;">Acros/VWR</td>
			<td>200002-434</td>
			<td style="width:595px;">any research grade NaCl can be used</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:485px;">Agar, 7.5 g (1.5% agar, final concentration)</td>
			<td style="width:260px;">HiMedia/VWR</td>
			<td>95026-642</td>
			<td style="width:595px;">any media grade agar can be used</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">500 ml distilled H20&#160;</td>
			<td style="width:260px;"></td>
			<td style="width:488px;"></td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:260px;"></td>
			<td style="width:488px;"></td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:485px;">For Lipid Agar (for 1 L)</td>
			<td style="width:260px;"></td>
			<td style="width:488px;"></td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:485px;">100 x 15 Petri Dishes</td>
			<td style="width:260px;">VWR</td>
			<td>25384-088</td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:485px;">Nutrient Broth, 8 g</td>
			<td style="width:260px;">BD/VWR</td>
			<td>90002-660</td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:485px;">Yeast Extract, 5g</td>
			<td style="width:260px;">EMD/VWR</td>
			<td>EM1.03753.0500</td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:485px;">Magnesium Chloride Hexahydrate,&#160; 10 ml (0.2g/ml)</td>
			<td style="width:260px;">EMD/VWR</td>
			<td>EM-MX0045-1</td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:485px;">Corn Oil, 4 ml</td>
			<td style="width:260px;">Any brand</td>
			<td style="width:488px;">Locally source; supermarket</td>
			<td style="width:595px;">Any brand of corn oil can be used</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:485px;">Corn Syrup, 96 ml combine 7 ml corn syrup in 89 ml heated H20 and swirl for homogeneity</td>
			<td style="width:260px;">Karo</td>
			<td style="width:488px;">Locally sourced; supermarket</td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agar, 15 g</td>
			<td style="width:260px;">HiMedia/VWR</td>
			<td>95026-642</td>
			<td style="width:595px;">any media grade agar can be used</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Distilled H20,890 ml</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo and in vitro rearing of entomopathogenic nematodes (steinernematidae and heterorhabditidae)

Nematodi entomopatogeni (EPN) (Steinernematidae e Heterorhabditidae) hanno una partnership mutualistico con Gram-negativi Gamma-Proteobacteria della famiglia Enterobacteriaceae batteri. Xenorhabdus sono associati con steinernematids nematodi, mentre Photorhabdus sono simbionti di heterorhabditids. Insieme nematodi e batteri formano un complesso potente insetticida che uccide una vasta gamma di specie di insetti in una intima e specifico. Qui, dimostriamo in vivo e in vitro tecniche comunemente utilizzate nell&#39;allevamento di questi nematodi in condizioni di laboratorio. Inoltre, queste tecniche rappresentano passaggi fondamentali per la creazione di successo di culture EPN e costituiscono anche la base per altri test biologici che utilizzano questi organismi per la ricerca. La produzione di aposymbiotic nematodi (senza simbionti) è spesso fondamentale per un approfondito e approccio multiforme allo studio della simbiosi. Questoprotocollo non richiede l&#39;aggiunta di antibiotici e può essere realizzato in un breve lasso di tempo con attrezzature di laboratorio standard. Nematodi prodotti in questo modo sono relativamente robusti, anche se la loro sopravvivenza in deposito possono variare a seconda delle specie utilizzate. Le tecniche descritti in questa presentazione corrispondono a quelli descritti da vari autori e raffinato dal Laboratorio di P. Archivio, University of Arizona (Tucson, AZ, USA). Queste tecniche sono distinti dal corpo di tecniche che vengono utilizzati nella produzione di massa di tali organismi a fini di gestione nocivo.

Il metodo di allevamento in vivo utilizza insetti vivi come host per la crescita e la riproduzione dei nematodi. Camere di infezione sono un metodo efficiente per esporre insetti IJs. Questa è l&#39;unica fase del ciclo di vita dei nematodi &#39;che i vettori simbionti batterici da un insetto host all&#39;altro. Figura 1 mostra il set up per una camera un&#39;infezione così come i materiali necessari per costruire questa camera. In vitro metodi di allevamento consentono EPN a crescer...

Watch this Scientific Journal Video about In vivo and In vitro Rearing of Entomopathogenic Nematodes Steinernematidae and Heterorhabditidae at JoVE.com

In vivo and In vitro Rearing of Entomopathogenic Nematodes Steinernematidae and Heterorhabditidae

L&#39;obiettivo di questa presentazione è quello di dimostrare in vivo e in vitro tecniche per l&#39;allevamento di nematodi entomopatogeni. Metodi in vivo considerano l&#39;allevamento di questi nematodi con un host insetto, mentre i metodi in vitro utilizzano rich media agar.

In vivo e in vitro Allevamento di Nematodi entomopatogeni (Steinernematidae e Heterorhabditidae)

Entomopathogenic nematodes (EPN) (Steinernematidae and Heterorhabditidae) have a mutualistic partnership with Gram-negative Gamma-Proteobacteria in the ...

in-vivo-vitro-rearing-entomopathogenic-nematodes-steinernematidae

Research

JoVE Journal

Bioengineering

In vivo and In vitro Rearing of Entomopathogenic Nematodes (Steinernematidae and Heterorhabditidae)

20.4K Views.  University of Arizona.  L'obiettivo di questa presentazione è quello di dimostrare in vivo e in vitro tecniche per l'allevamento di nematodi entomopatogeni. Metodi in vivo considerano l'allevamento di questi nematodi con un host insetto, mentre i metodi in vitro utilizzano rich media agar. 

Video: In vivo and In vitro Rearing of Entomopathogenic Nematodes Steinernematidae and Heterorhabditidae

Decellularization and Recellularization of Whole Livers

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to recreate or mimic the liver microstructure with grounds up approach using tissue engineering and microfabrication techniques have not been successful so far due to this design challenge. In addition, synthetic biomaterials used to create scaffolds for liver tissue engineering applications have been limited in inducing tissue regeneration and repair in large part due to the lack of specific cell binding motifs that would induce the proper cell functions2. Decellularized native tissues such blood vessels3and skin4on the other hand have found many applications in tissue engineering, and have provided a practical solution to some of the challenges. The advantage of decellularized native matrix is that it retains, to an extent, the original composition, and the microstructure, hence enhancing cell attachment and reorganization5.
In this work we describe the methods to perform perfusion-decellularization of the liver, such that an intact liver bioscaffold that retains the structure of major blood vessels is obtained. Further, we describe methods to recellularize these bioscaffolds with adult primary hepatocytes, creating a liver graft that is functional in vitro, and has the vessel access necessary for transplantation in vivo.

Perfusion decellularization is a novel technique to produce whole liver scaffolds that retains the organ's extracellular matrix composition and microarchitecture. Herein, the method of preparing whole organ scaffolds using perfusion decellularization and subsequent repopulation with hepatocytes is described. Functional and transplantable liver grafts can be generated using this technique.

Decellularization e Recellularization di fegati interi

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to ...

Ricerca

Bioingegneria

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

La creazione di substrati Patterned bidimensionali per confinamento di proteine ​​e cellule

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

Mappatura ottico dei potenziali d&#39;azione e transitori Calcio nel Cuore mouse

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

Quantificazione di Global Funzione diastolica da Analisi basata su modellazione cinematica del flusso transmitralico tramite il formalismo di riempimento parametrizzate diastolica

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

Oculare Test (EIT) per identificazione del pericolo di sostanze chimiche irritanti oculari utilizzando Ricostruito umana Cornea come epiteliale (RHCE) Tessuto Modello

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

Un<em&gt; In Vitro</em&gt; Organ Culture Modello del disco intervertebrale Murine

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

Migliorata 3D idrogel culture di cellule gliali primari In Vitro modellazione del Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

Rigida incorporamento di fisso e macchiato, complesso, scala millimetrata esemplari per l'istologia privo di sezione 3D da Micro-tomografia computerizzata

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

Modelli avanzati di fegato 3D per test di genotossicità in vitro a seguito dell'esposizione a lungo termine ai nanomateriali

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

An In vitro System to Gauge the Thrombolytic Efficacy of Histotripsy and a Lytic Drug

Deep vein thrombosis (DVT) is a global health concern. The primary approach to achieve vessel recanalization for critical obstructions is catheter-directed thrombolytics (CDT). To mitigate caustic side effects and the long treatment time associated with CDT, adjuvant and alternative approaches are under development. One such approach is histotripsy, a focused ultrasound therapy to ablate tissue via bubble cloud nucleation. Pre-clinical studies have demonstrated strong synergy between histotripsy and thrombolytics for clot degradation. This report outlines a benchtop method to assess the efficacy of histotripsy-aided thrombolytic therapy, or lysotripsy.
Clots manufactured from fresh human venous blood were introduced into a flow channel whose dimensions and acousto-mechanical properties mimic an iliofemoral vein. The channel was perfused with plasma and the lytic&#160;recombinant tissue-type plasminogen activator. Bubble clouds were generated in the clot with a focused ultrasound source designed for the treatment of femoral venous clots. Motorized positioners were used to translate the source focus along the clot length. At each insonation location, acoustic emissions from the bubble cloud were passively recorded, and beamformed to generate passive cavitation images. Metrics to gauge treatment efficacy included clot mass loss (overall treatment efficacy), and the concentrations of D-dimer (fibrinolysis) and hemoglobin (hemolysis) in the perfusate. There are limitations to this in vitro design, including lack of means to assess in vivo side effects or dynamic changes in flow rate as the clot lyses. Overall, the setup provides an effective method to assess the efficacy of histotripsy-based strategies to treat DVT.

Histotripsy-aided lytic delivery or lysotripsy is under development for the treatment of deep vein thrombosis. An in vitro procedure is presented here to assess the efficacy of this combination therapy. Key protocols for the clot model, image guidance, and assessment of treatment efficacy are discussed.