1. Modifica polimerici Superfici con recCD47 NOTA:. Il protocollo è riassunta schematicamente in Figura 1 Figura 1A illustra generazione di superfici polimeriche tiolo reattivo Figura 1B illustra generazione di tiolo reattivo recCD47.. <ol><li> Giorno 1 <ol><li> Preparare una soluzione di PDT-BzPh (1 mg / ml) e bicarbonato di potassio (KHCO 3) (0,7 mg / ml) in acqua sterile. Mescolare notte a 4 ° C (riparo dalla luce). </li></ol></li><li> Giorno 2 <ol><li> Tagliare il tubo polimerico a 40 centimetri pezzi lunghi (abbastanza a lungo per adattarsi intorno ruote girevoli). </li><li> Bagnare i tubi in una soluzione acquosa 0,1% di hexacylpyridinium per 90 minuti a temperatura ambiente su un agitatore o su Chandler Loop Apparecchiatura. Sciacquare le provette con 3x acqua sterile dopo l&#39;90 min ammollo. </li><li> Acidificare la soluzione di PDT-BzPh dal 1 ° giorno aggiungendo il 15% di potassio fosfato monobasico acquosa (KH 2 </sub&gt; PO 4). Aggiungere 50 ml KH 2 PO 4 per ml di PDT-BzPh, formando una soluzione micellare nuvoloso. </li><li> Bagnare i tubi nella soluzione PDT-BzPh acidificato per 40 minuti a temperatura ambiente su un agitatore o apparato. </li><li> Sciacquare le provette con acido acetico diluito (1: 1.000) una volta. </li><li> Esporre i tubi per irradiazione UV per una durata complessiva di 60 min. Ruotare i tubi ¼ di giro ogni 15 minuti per irradiare l&#39;intera superficie. </li><li> Bagnare i tubi in una soluzione di bicarbonato di potassio / ml 20 mg (KHCO 3) per 20 minuti a temperatura ambiente su un agitatore o apparato. </li><li> Sciacquare le provette con 3x acqua sterile e conservare a 4 ° C in acqua sterile. </li></ol></li><li> Giorno 3 <ol><li> Disciogliere 5 mg solfo-SMCC in 200 microlitri dimetilformammide (DMF) (Attenzione: Eseguire in cappa). </li><li> Aggiungere 50 ml della soluzione di solfo-SMCC preparata al punto 1 a 0,1 mg / ml di soluzione di poli-lisina recCD47, e mescolare per 60 min a rotemperatura om. </li><li> Durante 60 min mescolare, degas sterilizzato Dulbecco Phosphate Buffered Saline (DPBS) e mettere da parte. </li><li> Purificare solfo-SMCC reagito recCD47 utilizzando un 7K peso molecolare di cut-off di spin dissalazione colonna secondo le istruzioni del produttore. Raccogliere finale a flusso continuo (alta qualità tiolo reattivo recCD47) e portare a volume necessario per rivestire l&#39;interno del tubo con DPBS. </li><li> Sciacquare i tubi dal giorno 2 con sterile, degassata DPBS 3x. </li><li> React superficie modificata dei tubi con una soluzione di 20 mg / ml di tris (2-carbossietil) fosfina cloridrato (TCEP) in DPBS degassati per meno di 2 min. Sciacquare le provette con sterili degassificato DPBS 4x. </li><li> Aggiungere il recCD47 Sulfo-SMCC-reagito ai tubi e incubare una notte a 4 ° C su un agitatore o apparati. </li></ol></li></ol> 2 Immunoassay Quantificazione dei recCD47 su superfici modificate <ol><li> Sciacquare i tubi modificati per essere quantificato dal Day 3 with DPBS 3x. Conservare le provette non utilizzate per la quantificazione in DPBS a 4 ° C. </li><li> Utilizzare una biopsia 4 mm e fare i campioni del tubo duplicati e inserire pugni biopsia in pozzetti di una piastra a 96 pozzetti. </li><li> Preparare un controllo negativo costituito da un tubo campione non modificato non trattato con l&#39;anticorpo di rilevazione. Preparare un controllo dell&#39;anticorpo costituito da un tubo campione non modificato trattati con l&#39;anticorpo di rilevamento. Preparare i campioni di prova dei tubi modificati trattati con l&#39;anticorpo di rilevazione. </li><li> Campioni di blocco con albumina sierica bovina 0,4% (BSA) in DPBS per 60 minuti a temperatura ambiente su un agitatore. </li><li> Dopo il blocco, aspirare BSA e sciacquare con tris-buffered saline più l&#39;1% Tween-20 (TBST) 3x, 10 minuti ciascuna su un agitatore. </li><li> Preparare una diluizione di lavoro degli anticorpi nel 0,4% di BSA secondo la diluizione raccomandata dal produttore per saggi immunologici. Diluire CD47 anticorpi umani B6H12-FITC 1: 100 in 0,4% di BSA. </li><li> Aggiungere 200 ml di diluizione anticorpo per pozzetti. Incubate controlli negativi con il 0,4% solo BSA. Incubare a temperatura ambiente per 60 minuti su un agitatore (riparo dalla luce). </li><li> Risciacquare con TBST 3x, 10 minuti ciascuna su un agitatore. Aspirare l&#39;ultimo risciacquo TBST e aggiungere 200 microlitri di DPBS pozzetti del campione del tubo. </li><li> Raccogliere il flow-through finale, che è di alta qualità tiolo-reattiva CD47 ricombinante e portare a un volume necessario per rivestire l&#39;interno dei tubi con DPBS. </li><li> Leggi intensità del segnale FITC utilizzando un lettore di micropiastre con FITC di eccitazione (485 nm) e di emissione impostazioni (538 nm). </li><li> Calcola recCD47 destinato a superficie polimerica sulla base di valori standard, che rappresentano per auto-fluorescenza e non specifico vincolante dal campione di controllo dell&#39;anticorpo. </li></ol> 3. Chandler Loop Apparato protocollo Cercate Institutional Review Board (IRB) approvazione del protocollo di raccolta del sangue e informato consenso scartoffie prima di iniziare la raccolta dei campioni di sangue umano. Ottenere informazionirmed consenso da un donatore di sangue umano. NOTA: Un diagramma raffigurante l&#39;apparato è mostrato nella Figura 2. <ol><li> Riempire bagnomaria con acqua distillata fino a circa mezzo delle ruote di diametro sono sommerso. Aggiungere abbastanza candeggina al bagnomaria per ottenere una soluzione di candeggina al 10%. Set bagno d&#39;acqua a 37 ° C e che la temperatura equilibrare. </li><li> Raccogliere adattatori in metallo (indicati nella Figura 1B), tubi senza modifiche e tubi modificati, e assemblare attorno alle ruote apparecchi, come mostrato nella Figura 1C. Assicurarsi che il tubo si adatta snuggly intorno alla ruota con l&#39;adattatore in metallo posto. </li><li> Ottenere 30 ml di campione di sangue usando un protocollo IRB-approvato, in un flaconcino preriempita con 2 ml di citrato o altro anticoagulante, e mescolare per inversione per prevenire la coagulazione una volta che il campione è stato raccolto. </li><li> Aggiungere circa 10 ml di sangue per ciascun tubo con la valvola di metallo e la siringa, lasciando un po &#39;d&#39;aria nel tubo. Fissare il tappo della valvola e rotate per 3 ore. </li><li> Drenare il sangue in un bicchiere reflue trattate con una soluzione di candeggina al 10% (o come richiesto dalla politica istituzionale). </li><li> Lavare delicatamente il tubo interno con DPBS per rimuovere tutte le tracce di sangue. Raccogliere flusso continuo nel becher rifiuti. Smaltire sangue in conformità con la politica istituzionale. </li><li> Disinfettare l&#39;apparato e il sangue di contattare superfici utilizzando una soluzione di candeggina al 10% o in base alla politica istituzionale. </li><li> Campioni di processo per microscopia a fluorescenza o la scansione microscopia elettronica come descritto di seguito. </li></ol> 4. fluorescente Microscopia e conteggio delle cellule <ol><li> Preparare una soluzione al 4% paraformaldeide (PFA) (Attenzione: Eseguire in cappa). </li><li> Incubare il tubo in una soluzione PFA 4% per una notte a 4 ° C. </li><li> Dopo incubazione overnight, rimuovere 4% PFA e risciacquare film molto delicatamente con DPBS. </li><li> Tagliare il tubo in sezioni per esporre la superficie del lume per la colorazione. </li><li> Stainuna sezione del tubo con alcune gocce di mezzo di montaggio con 4 &#39;, 6-diamino-2-fenilindolo (DAPI) per 30 minuti a temperatura ambiente (proteggere dalla luce). Dopo la colorazione, sciacquare il tubo molto delicatamente con DPBS per ridurre il segnale DAPI sfondo. </li><li> Immagine utilizzando un microscopio a fluorescenza dotato di un filtro DAPI e fotocamera digitale per contare il numero di cellule in 9 settori ciecamente selezionati di vista sotto 200 ingrandimenti. </li><li> Cella Record conta per campo visivo ed eseguire appropriate analisi statistiche per determinare la significatività statistica dei risultati. </li></ol> 5. Microscopia Elettronica a Scansione <ol><li> Preparare una soluzione di glutaraldeide al 2% (Attenzione: Eseguire in cappa). </li><li> Incubare il tubo in una soluzione di glutaraldeide al 2% notte a 4 ° C. </li><li> Dopo incubazione overnight, lavare delicatamente la 3x tubi con DPBS. </li><li> Preparare una soluzione all&#39;1% di tetrossido di osmio (Attenzione: pericolo di inalazione grave! Utilizzare in cappa). </li><li> Incubare il tubo in una soluzione all&#39;1% di tetrossido di osmio per 15 min a temperatura ambiente. </li><li> Lavare delicatamente il tubo 3x con DPBS. </li><li> Preparare una serie di concentrazioni di etanolo (25%, 50%, 75%, 95% e 100% etanolo). </li><li> Disidratare il tubo mediante incubazione nella serie di concentrazioni di etanolo. Incubare a 25%, 50%, 75% e 95% di etanolo per 20 minuti ciascuno. Incubare a 100% di etanolo per 30 min. </li><li> Punto supercritico asciugare il tubo con CO 2 per 45 minuti per rimuovere tutta l&#39;umidità. </li><li> Sezioni del tubo Montare su un stub campione con pasta d&#39;argento o grafite. </li><li> Sezioni del tubo cappotto con 12,5 nm di oro-palladio. </li><li> Esaminate in un microscopio elettronico a scansione. </li></ol>

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La ricerca riportata in questa pubblicazione è stato sostenuto dal National Institute of Biomedical Imaging e Bioingegneria, con il numero premio R21 EB015612 (SJS), e il National Heart, Lung, and Blood Institute, con il numero premio T32 HL007915 (JBS e RJL), della Nazionale Istituto Superiore di Sanità.

La chimica fotoattivazione (riassunto nella Figura 1) consente la modifica di qualsiasi superficie polimerica che ha idrocarburi sufficienti per facilitare il fissaggio PDT-BzPh e la successiva irradiazione UV per foto-attivare il PDT-BzPh. Funzionalizzare la superficie polimerica con gruppi tiolici reattivi consente la successiva collocazione di una serie di molecole di interesse testabili. Nei nostri studi particolari abbiamo scelto ricombinante CD47 11-13. La particolare coniugazione chimi...

Molte procedure cliniche, come bypass cardiopolmonare e dialisi renale, richiedono l&#39;uso di condotti sangue polimerici e sono spesso associati a complicanze post-procedurali 1. Quando perfusi con sangue, questi polimeri illeciti la reazione da corpo estraneo (FBR), con conseguente assorbimento di proteine ​​del sangue e piastrine, monociti / macrofagi adesione, e il rilascio di citochine pro-infiammatorie, tutti che contribuiscono complicanze post-procedurali e / o guasto del dispositivo 2,3. Pertanto, le strategie per affrontare la questione rimane un settore importante e costante della ricerca biomateriali. Gli investigatori hanno cercato di affrontare questo problema modificando superfici sangue contatto con bioattivi o bioinerte molecole 4-6. La ricerca nel nostro laboratorio si è concentrata sulla aggiungendo CD47 ricombinante (recCD47) per biomateriali polimerici come strategia per mitigare la FBR e aumentare l&#39;efficacia di questi materiali. CD47 è una transmembr ubiquitariamente espressaane proteina con un ruolo conosciuto in evasione immune, riconoscere il carattere &quot;self&quot; su cellule che esprimono 7-10 e mostra la promessa di conferire biocompatibilità quando aggiunto alle superfici polimeriche 11-13. Signal-normativo proteina alfa (SIRPα), il recettore affine per CD47, e un membro del motivo inibitorio a base di tirosina immunoreceptor (ITIM) -contenenti famiglia di proteine ​​transmembrana, è espresso sulle cellule di origine mieloide 14. Abbiamo precedentemente dimostrato che CD47, tramite segnalazione cellulare SIRPα-mediata, down-regola le risposte immunitarie a poliuretano (PU) e polivinilcloruro (PVC) in in vitro, ex vivo e in modelli in vivo 11-13. Centrale per le nostre indagini è una chimica fotoattivazione relativamente nuovo, qui descritto, in cui i gruppi tiolici chimicamente reattivi sono covalentemente aggiunti a tubo polimerico facendo reagire il tubo con un polimero multifunzionale (PDT-BzPh), composto da 2-pyridyldithio (PDT), il benzofenone fotoreattivo (BzPh) e un polyallylamine carbossi-modificata 11-13. Ridurre i gruppi PDT covalentemente allegati con tris (2-carbossietil) fosfina cloridrato (TCEP) 11 produce una superficie tiolati che può essere successivamente fatto reagire con porzioni terapeutiche. Particolareggiata in questa sede e in precedenza 12,13, recCD47, ulteriormente modificato con l&#39;aggiunta di una poli-lisina coda C-terminale 12,13, viene fatto reagire con Sulfosuccinimidyl-4-[N -maleimidomethyl] cicloesano-1-carbossilato (solfo-SMCC) per 1 ora a generare gruppi tiolici-reattiva, consentendo una formazione di legami monosulfide tra il tubo e il recCD47 11. La capacità anti-infiammatoria delle superfici funzionalizzate CD47 è stato testato, ex viv o, utilizzando l&#39;Apparato Chandler Loop con tutto il sangue umano, che è stato originariamente descritto nel 1958 come un modello in vitro della coagulazione trombotica 15. L&#39;apparato si basa su unSistema di tubo chiuso parzialmente riempito di aria e un motore rotativo per far circolare il sangue attraverso il tubo 15. Questo modello sperimentale fornisce l&#39;opportunità di esaminare l&#39;effetto dell&#39;esposizione sangue su superfici modificate e non modificate così come l&#39;effetto di tali modificazioni superficiali sulla fisiologia delle cellule del sangue. recCD47 può essere aggiunto a una varietà di superfici polimeriche utilizzando questa chimica fotoattivazione, e la sua capacità anti-infiammatoria può essere valutata utilizzando un modello clinicamente rilevante ex vivo imitando la perfusione di sangue sulle superfici polimeriche 11,12. Grade clinici condotti sanguigni modificati con recCD47 mostrano molto meno piastrinica e l&#39;adesione delle cellule infiammatorie rispetto a polimeri modificati se esposti a sangue umano nell&#39;apparato. Una descrizione step-by-step di questo processo di modifica è descritto di seguito.

<table border="0" cellpadding="0" cellspacing="0" width="802">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Name</td>
			<td style="width:175px;">Company</td>
			<td style="width:107px;">Catalog Number</td>
			<td style="width:251px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">16% Paraformaldehyde (PFA)</td>
			<td style="width:175px;">Thermo Scientific</td>
			<td style="width:107px;">58906</td>
			<td>Caution! Use in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">25% Glutaraldehyde</td>
			<td style="width:175px;">VWR</td>
			<td style="width:107px;">AAA17876-AP&#160;</td>
			<td>Caution! Use in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">2-pyridyldithio,benzophenone (PDT-BzPH)</td>
			<td style="width:175px;">Synthesized in lab</td>
			<td style="width:107px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:107px;">A3059-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Citrate</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:107px;">S5770-50ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Digital Camera</td>
			<td style="width:175px;">Leica</td>
			<td style="width:107px;">DC500</td>
			<td>Out of production</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Dimethylformamide (DMF)</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:107px;">270547-100ML</td>
			<td>Caution! Use in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Dulbecco&#8217;s Phosphate Buffered Saline (DPBS)</td>
			<td style="width:175px;">Gibco/Life Technologies</td>
			<td style="width:107px;">14190-136</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Fluorescent Microscope</td>
			<td style="width:175px;">Nikon</td>
			<td style="width:107px;">TE300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Glacial Acetic Acid</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:107px;">A38-212</td>
			<td>Caution! Use in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Human CD47 (B6H12) &#8211; FITC Antibody</td>
			<td style="width:175px;">Santa Cruz Biotechnology</td>
			<td style="width:107px;">SC-12730</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Osmium Tetroxide</td>
			<td style="width:175px;">Acros Organics</td>
			<td style="width:107px;">197450050</td>
			<td>Caution! Use in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Potassium Bicarbonate (KHCO3)</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:107px;">237205-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Potassium Phosphate Monobasic (KH2PO4)</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:107px;">P5655-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">PVC Tubing (Cardiovascular Procedure Kit)</td>
			<td style="width:175px;">Terumo Cardiovascular Systems</td>
			<td style="width:107px;">60050</td>
			<td>Most clinical-grade tubing will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Scanning Electron Microscope</td>
			<td>JEOL</td>
			<td>JSM-T330A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:269px;">Sodium Chloride (NaCl)</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td>BP358-212</td>
			<td></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:269px;">Microplate Reader</td>
			<td style="width:175px;">Molecular Devices</td>
			<td style="width:107px;">Spectramax Gemini EM&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Sulfo-SMCC</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:107px;">M6035-10MG</td>
			<td>Moisture Sensitive!</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">tris (2-carboxyethyl) phosphine (TCEP-HCl)</td>
			<td style="width:175px;">Thermo Scientific</td>
			<td style="width:107px;">20491</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:269px;">Tris Base</td>
			<td style="width:175px;">Sigma</td>
			<td>T1503-100G</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:269px;">Tween-20</td>
			<td>Bio-Rad</td>
			<td>170-6531</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Vectashield with DAPI</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:107px;">H-1200</td>
			<td>Light sensitive!</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Zeba Spin Desalt Columns &#8211; 7K MWCO</td>
			<td style="width:175px;">Thermo Scientific</td>
			<td style="width:107px;">89891</td>
			<td></td>
		</tr>
	</tbody>
</table>

the use of the ex vivo chandler loop apparatus to assess the biocompatibility of modified polymeric blood conduits

La reazione corpo estraneo si verifica quando una superficie sintetica viene introdotto al corpo. Essa è caratterizzata da adsorbimento di proteine ​​del sangue e la conseguente attaccamento e l&#39;attivazione delle piastrine, monociti / macrofagi adesione, e gli eventi di segnalazione cellulare infiammatori, portando a complicazioni post-procedurali. L&#39;Apparato Chandler Loop è un sistema sperimentale che permette ai ricercatori di studiare le interazioni molecolari e cellulari che si verificano quando grandi volumi di sangue sono perfusi su condotti polimerici. A tal fine, questo apparato è stato usato come modello ex vivo che consentano di valutare le proprietà anti-infiammatorie di varie modificazioni superficiali polimeriche. Il nostro laboratorio ha dimostrato che i condotti del sangue, covalentemente modificati via chimica fotoattivazione con CD47 ricombinante, possono conferire biocompatibilità alle superfici polimeriche. Aggiunta di CD47 alle superfici polimeriche potrebbe essere un mezzo efficace per promuovere l&#39;efficacia dei condotti di sangue polimerici. Leiein è la metodologia in dettaglio la chimica fotoattivazione utilizzato per aggiungere CD47 ricombinante per clinicamente rilevanti condotti sanguigni polimerici e l&#39;uso del Chandler Loop come modello sperimentale ex vivo per esaminare le interazioni di sangue con le CD47 modificato e controllo condotti.

Generazione di superfici polimeriche tiolo reattivo attraverso l&#39;uso di PDT-BzPh e TCEP insieme tiolo reattivo recCD47 poli-lisina utilizzando SMCC permette il fissaggio di recCD47 a superfici polimeriche. Il processo di modifica è riassunta schematicamente in figura 1. La comodità di questo processo di modifica è che può essere applicato a molte proteine ​​differenti e molteplici superfici polimeriche, assumendo la proteina può essere modificato con sufficienti gruppi chimicamente reattivi...

Watch this Scientific Journal Video about The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits at JoVE.com

The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits

Blood exposure to polymeric blood conduits initiates the foreign body reaction that has been implicated in clinical complications. Here, the Chandler Loop Apparatus, an experimental tool mimicking blood perfusion through these conduits, is described. Appendage of recombinant CD47 results in decreased evidence of the foreign body reaction on these conduits.

L&#39;uso della<em&gt; Ex Vivo</em&gt; Chandler Loop Apparecchio per valutare la biocompatibilità di Modificati Condotti sangue Polimerici

The foreign body reaction occurs when a synthetic surface is introduced to the body. It is characterized by adsorption of blood proteins and the ...

the-use-ex-vivo-chandler-loop-apparatus-to-assess-biocompatibility

Research

JoVE Journal

Bioengineering

The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits

11.5K Views.  The Children's Hospital of Philadelphia. Blood exposure to polymeric blood conduits initiates the foreign body reaction that has been implicated in clinical complications. Here, the Chandler Loop Apparatus, an experimental tool mimicking blood perfusion through these conduits, is described. Appendage of recombinant CD47 results in decreased evidence of the foreign body reaction on these conduits. 

Video: The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

Organotipiche Collagene I test: una piattaforma malleabile per valutare Comportamento cella in un 3-dimensionale Contesto

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

Ricerca

Bioingegneria

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

Progettazione di un bioreattore per la pressione ciclica Ex Vivo Studio delle valvole cardiache aortica

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

Determinazione del tasso di trasporto di xenobiotici e nanomateriali attraverso la placenta con il<em&gt; Ex vivo</em&gt; Umano Modello perfusione placentare

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

Migliorata metodo per la preparazione di un cell-based, Contact modello umano della barriera sanguigna del cervello

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 &#181;m diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

Gene Transfection verso Celle sferoide su lastre micropatterned coltura per trapianto di cellule geneticamente modificati

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected within in vivo oral biofilms. Furthermore, a system that uses natural human saliva as the nutrient source, instead of artificial media, is particularly desirable in order to support the expression of cellular and biofilm-specific properties that mimic the in vivo communities. We describe a method for the development of multi-species oral biofilms that are comparable, with respect to species composition, to supragingival dental plaque, under conditions similar to the human oral cavity. Specifically, this methods article will describe how a commercially available microfluidic system can be adapted to facilitate the development of multi-species oral biofilms derived from and grown within pooled saliva. Furthermore, a description of how the system can be used in conjunction with a confocal laser scanning microscope to generate 3-D biofilm reconstructions for architectural and viability analyses will be presented. Given the broad diversity of microorganisms that grow within biofilms in the microfluidic system (including Streptococcus, Neisseria, Veillonella, Gemella, and Porphyromonas), a protocol will also be presented describing how to harvest the biofilm cells for further subculture&#160;or DNA extraction and analysis. The limits of both the microfluidic biofilm system and the current state-of-the-art data analyses will be addressed. Ultimately, it is envisioned that this article will provide a baseline technique that will improve the study of oral biofilms and aid in the development of additional technologies that can be integrated with the microfluidic platform.

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque.&#160;Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted.

L&#39;uso di un high-throughput<em&gt; In Vitro</em&gt; Microfluidic sistema per sviluppare orale biofilm Multi-specie

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential to establish an ex vivo model that can evaluate SIM performance accurately, for developing high-performance SIMs. In our previous study, we developed a new ex vivo model that can be used to evaluate the performance of various SIMs in detail by applying constant tension to the specimen&#39;s ends. We also confirmed that the proposed new ex vivo model allows accurate submucosal elevation height (SEH) measurement under uniform conditions and detailed comparisons of the performances of various types of SIMs. Here, we describe the new ex vivo model and explain the detailed setup methodology of this model. Since all parts of the new model were easy to obtain, the setup of the new model could be completed quickly. SEH of various SIMs could be measured more accurately by using the new model. The critical factor that determines SIM performance can be identified using the new model. SIM development speed will drastically increase after the factor has been identified.

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

We developed a new ex vivo model that applies constant tension to the porcine gastric specimen. This development made it possible to evaluate the performance (the height and duration of the submucosal elevation) of various SIMs accurately.&#160; The detailed setup methodology of this new model is explained.

Un nuovo modello Ex Vivo per la valutazione delle prestazioni del materiale di iniezione sottomucosa endoscopica

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential ...

Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome absorbance respectively. In addition, numerous aspects of the mitochondrial metabolic status such as membrane potential and substrate entry can also be estimated. To perform cardiac wall transmission optical spectroscopy, a commercially available side-firing optical fiber catheter is placed in the left ventricle of the isolated perfused heart as a light source. Light passing through the heart wall is collected with an external optical fiber to perform optical spectroscopy of the heart in near real- time. The transmission approach avoids numerous surface scattering interference occurring in widely used reflection approaches. Changes in transmural absorbance spectra were deconvolved using a library of chromophore reference spectra, providing quantitative measures of all the known cardiac chromophores simultaneously. This spectral deconvolution approach eliminated intrinsic errors that may result from using common dual wavelength methods applied to overlapping absorbance spectra, as well as provided a quantitative evaluation of the goodness of fit. A custom program was designed for data acquisition and analysis, which permitted the investigator to monitor the metabolic state of the preparation during the experiment. These relatively simple additions to the standard heart perfusion system provide a unique insight into the metabolic state of the heart wall in addition to conventional measures of contraction, perfusion, and substrate/oxygen extraction.

Here we introduce a method for using an intra-ventricle optical catheter in perfused hearts to perform absorbance spectroscopy across the heart wall. The data obtained provides robust information on tissue oxygen tension as well as substrate utilization and membrane potential simultaneously with cardiac performance measures in this ubiquitous preparation.

Catetere leggero intra-cardiaco per il monitoraggio del metabolismo cellulare utilizzando la spettroscopia transmurale di riambente di cuori mammiferi perfusi

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome ...

Mitigation of Blood Borne Cell Attachment to Metal Implants through CD47-Derived Peptide Immobilization

The key complications associated with bare metal stents and drug eluting stents are in-stent restenosis and late stent thrombosis, respectively. Thus, improving the biocompatibility of metal stents remains a significant challenge. The goal of this protocol is to describe a robust technique of metal surface modification by biologically active peptides to increase biocompatibility of blood contacting medical implants, including endovascular stents. CD47 is an immunological species-specific marker of self and has anti-inflammatory properties. Studies have shown that a 22 amino acid peptide corresponding to the Ig domain of CD47 in the extracellular region (pepCD47), has anti-inflammatory properties like the full-length protein. In vivo studies in rats, and ex vivo studies in rabbit and human blood experimental systems from our lab have demonstrated that pepCD47 immobilization on metals improves their biocompatibility by preventing inflammatory cell attachment and activation. This paper describes the step-by step protocol for the functionalization of metal surfaces and peptide attachment. The metal surfaces are modified using polyallylamine bisphosphate with latent thiol groups (PABT) followed by deprotection of thiols and amplification of thiol-reactive sites via reaction with polyethyleneimine installed with pyridyldithio groups (PEI-PDT). Finally, pepCD47, incorporating terminal cysteine residues connected to the core peptide sequence through a dual 8-amino-3,6-dioxa-octanoyl spacer, are attached to the metal surface via disulfide bonds. This methodology of peptide attachment to metal surface is efficient and relatively inexpensive and thus can be applied to improve biocompatibility of several metallic biomaterials.

Presented here is a protocol for appending peptide CD47 (pepCD47) to metal stents using polybisphosphonate chemistry. Functionalization of metal stents using pepCD47 prevents the attachment and activation of inflammatory cells thus improving their biocompatibility.

Mitigazione dell'attaccamento delle cellule trasmesse dal sangue agli impianti metallici attraverso l'immobilizzazione peptidica derivata da CD47

The key complications associated with bare metal stents and drug eluting stents are in-stent restenosis and late stent thrombosis, respectively. Thus, ...

Blood Flow Imaging with Ultrafast Doppler

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler modes, it has several limits. Firstly, a finely tuned signal filtering operation is needed to distinguish blood flows from surrounding moving tissues. Secondly, the operator must choose between localizing the blood flows or quantifying them. In the last two decades, ultrasound imaging has undergone a paradigm shift with the emergence of ultrafast ultrasound using unfocused waves. In addition to a hundredfold increase in framerate (up to 10000 Hz), this new technique also breaks the conventional quantification/localization trade-off, offering a complete blood flow mapping of the field of view and a simultaneous access to fine velocities measurements at the single-pixel level (down to 50 &#181;m). This data continuity in both spatial and temporal dimensions strongly improves the tissue/blood filtering process, which results in an increase sensitivity to small blood flow velocities (down to 1 mm/s). In this method paper, we aim to introduce the concept of ultrafast Doppler as well as its main parameters. Firstly, we summarize the physical principles of unfocused wave imaging. Then, we present the Doppler signal processing main steps. Particularly, we explain the practical implementation of the critical tissue/blood flow separation algorithms and on the extraction of velocities from these filtered data. This theoretical description is supplemented by in vitro experiences. A tissue phantom embedding a canal with flowing blood-mimicking fluid is imaged with a research programmable ultrasound system. A blood flow image is obtained and the flow characteristics are displayed for several pixels in the canal. Finally, a review of in vivo applications is proposed, showing examples in several organs such as carotids, kidney, thyroid, brain and heart.

This protocol shows how to apply ultrafast ultrasound Doppler imaging to quantify blood flows. After a 1 s long acquisition, the experimenter has access to a movie of the full field of view with axial velocity values for each pixel every &#8776;0.3 ms (depending on the ultrasound time of flight).

Imaging del flusso sanguigno con Doppler ultraveloce

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler ...

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