Questo lavoro è sostenuto finanziariamente dalla Fondazione nazionale svizzera, (PNR programma 64, concedere nessun 4064-131.232).

1. Preparazione del sistema di perfusione <ol><li> Configurare il sistema di perfusione costituito da un bagno d&#39;acqua, una camera di perfusione, due colonne per l&#39;ossigenazione, due pompe peristaltiche, due trappole bolla, due riscaldatori di flusso e un sensore di pressione (Figura 1). Collegare questi componenti con sezioni di tubi composti di materiali siliconici e cloruro di polivinile secondo lo schema della figura 2. Infine ci sono due circuiti che rappresentano rispettivamente il circuito fetale e materna. </li><li> Accendere il bagno d&#39;acqua, i riscaldatori di flusso ed il riscaldamento per la camera di perfusione. La temperatura dovrebbe essere di 37 ° C. </li><li> Riscaldare il fluido di perfusione (NCTC-135 coltura tissutale diluito 1:2 con tampone di Earle (6,8 g / l di cloruro di sodio, 0,4 g / l di cloruro di potassio, 0,14 g / L di fosfato monosodico, 0,2 g / L di magnesio solfato, 0,2 g / l di cloruro di calcio, 2 g / L di glucosio) supplementato con glucosio (1 g / L), destrano 40 (10 g / L), sieroalbumina bovina (10 g/ L), eparina sodica (2,500 IU / L), amoxicillina (250 mg / L) e bicarbonato di sodio (2,2 g / L); pH 7.4) nel bagnomaria. </li><li> Consecutivamente sciacquare i sistemi arteriosa del circuito fetale e materno with a) 200 ml di acqua distillata, b) 50 ml di idrossido di sodio 1%, c) 1% acido fosforico ed) ancora 200 ml di acqua distillata (portata: 15 - 20 ml / min). </li><li> Collegare la cannula fetale (Ø 1,2 millimetri; ago smussato deve essere collegata a un alato set di infusione ago modificato) per il tubo arterioso fetale. </li><li> Sciacquare i sistemi arteriosa del circuito fetale e materno con il mezzo di perfusione finché tutte le provette contengano medie (portata: 15-20 ml / min). Durante questa fase riempire le trappole bolla e rimuovere tutte le bolle a valle della trappola. Poi fermare le pompe. E &#39;davvero importante che i tubi arteriosi afferenti sono sempre liberi di bolle, altrimenti dopo incannulamento soprattutto le belle vasi fetali possono rompersi. </li><li> Accendere il flusso di gas. Il circuito materna è ossigenato with 5% di anidride carbonica e aria sintetica 95% e il circuito fetale con il 5% di biossido di carbonio e 95% di azoto. </li><li> Avviare la registrazione del sensore di pressione. </li></ol> 2. Cannulating la Placenta <ol><li> Ottenere placenta intatta da gravidanze non complicate termine dopo il taglio cesareo primario. Consenso scritto deve essere data (è stato ottenuto nel caso dei nostri studi) dalle madri prima della consegna e lo studio deve essere approvato dal comitato etico locale (è stato il caso nei nostri studi). Primo controllo visivo deve essere effettuato da ostetriche per assicurare una placenta sana e intatta. </li><li> Incannulamento della placenta è un passaggio fondamentale! Durante la perfusione ogni piccola perturbazione nel tessuto può portare ad una perdita tra la circolazione materna e fetale. La placenta deve essere ottenuto entro 30 minuti dopo la consegna. </li><li> Selezionare un cotiledone intatto nella zona marginale della placenta senza interruzioni visibili sul lato materno. Alla piastra corionica,legare entrambi i rami collegati al dell&#39;arteria ombelicale e vena a monte al lato incannulazione tardi (verso il cordone ombelicale) utilizzando materiale di sutura chirurgica. Fai sempre due nodi. </li><li> Incannulare l&#39;arteria fetale prima. Le arterie placentari fetali sono sempre più piccoli e più sottili delle vene. </li><li> Fai una sutura intorno all&#39;arteria del feto, ma non legarlo immediatamente. Tenere il recipiente con una pinza, tagliare accuratamente il vaso e mettere la piccola cannula (Ø 1,2 mm) in arteria. Poi legare la sutura (due nodi). </li><li> Procedere con la vena fetale nello stesso modo, ma usare una cannula più grande (Ø 1,5-1,8 mm; ago smussato deve essere collegata a un alato set di infusione ago modificato). </li><li> Accendere la pompa fetale (2 ml / min). Se non ci sono perdite visibili e sangue emana dalla cannula vena fetale, aumentare lentamente il flusso fino a 4 ml / min. Osservare la pressione nell&#39;arteria fetale, non dovrebbe superare i 70 mmHg. Se il liquido che fuoriesce al feto o di maternal cannula fissarli con un altro sutura. </li><li> Posizionare la placenta sul supporto del tessuto con il lato fetale e tirare la membrana placentare e tessuto sopra le punte. Alla fine il cotiledone perfuso dovrebbe essere al centro del foro nel supporto del tessuto. </li><li> Stabilizzare la parte dove solo la membrana contiene la placenta con una membrana di silicone (Ø 1 mm) o in alternativa ai due pezzi parafilm. </li><li> Montare il supporto del tessuto completo, stringere le viti e tagliare il tessuto sovrastante. Si prega di notare che il venoso e arterioso cannule non vengano schiacciati, ma invece giacevano nei piccoli canali del supporto del tessuto. </li><li> Ruotare il supporto del tessuto a testa in giù, metterlo nella camera di perfusione e di aggiungere la copertina. Ora, il lato materno dovrebbe essere al top. Controllare sempre se il circuito del feto è ancora intatta e il mezzo sta fluendo fuori del tubo vena fetale. </li><li> Accendere la pompa di materna (12 ml / min). Introdurre i tre smussato cannule (Ø 0.8 mm) e al secoloe estremità del tubo arteria materna nello spazio intervilloso penetrando la piastra deciduali. Per riportare il perfusato al circuito materna messo una provetta come drenaggio venoso anch&#39;esso collegato con la pompa materna nella posizione più bassa nella parte superiore della camera di perfusione. </li><li> Collegare la cannula venosa fetale al tubo vena fetale. </li></ol> 3. L&#39;esecuzione della fase di pre-e Sperimentale di perfusione <ol><li> Per consentire al tessuto di recuperare dal periodo ischemico dopo la consegna e per scovare il sangue nello spazio intervilloso, un open di pre-fase di 20 min è necessario. Ciò significa che la vena materna e fetale non stanno portando al serbatoio arteriosa contenente il medium di perfusione. Raccogliere il deflusso venoso fetale e materna in una bottiglia e gettarla via dopo la prima fase. </li><li> Per valutare l&#39;integrità della perfusione eseguire un&#39;altra pre-fase di 20 min, ma in un circuito chiuso. Utilizzare due serbatoi separati con il mezzo di perfusioneper il circuito fetale e materna e chiudere i circuiti guidando il deflusso venoso fetale torna nel serbatoio fetale e materno venoso torna nel serbatoio materna. </li><li> Per l&#39;esperimento perfusione principale preparare due beute da 120 ml di mezzo di perfusione (una per la materna e una per il serbatoio fetale). Aggiungere la radiomarcato 14 C-antipyrine (4 nCi / ml; serve come controllo positivo; ATTENZIONE: sostanza radioattiva) e la xenobiotici o nanoparticelle fluorescente che si vuole analizzare al serbatoio materna. Mescolare bene il perfusato materna. </li><li> Avviare l&#39;esperimento scambiando il medium di perfusione puro con i due palloni preparati (serbatoi fetale e materna). Chiudere i circuiti guidando il feto venoso torna nel serbatoio fetale e materno venoso torna nel serbatoio materna. </li><li> Continuare la perfusione per 6 ore e prelevare campioni regolarmente. Risospendere sempre il mezzo nel fetale e maternaserbatoio prima del ritiro. </li><li> Controllare la pressione in arteria fetale (non dovrebbe superare i 70 mmHg), pH in entrambi i circuiti (dovrebbe essere in un range fisiologico 7.2-7.4) e il volume di entrambi i serbatoi (perdita di volume fetale non deve superare 4 ml / hr) durante la perfusione . Se necessario regolare i valori di pH utilizzando sia l&#39;acido cloridrico o idrossido di sodio. </li><li> Se la perdita di volume nel serbatoio fetale supera 4 ml / hr c&#39;è una perdita nel tessuto e si deve interrompere la perfusione. Il tasso di successo di una perfusione per 6 ore senza perdita è di circa il 15-20%. </li><li> Fermare la perfusione dopo 6 ore. Spegni le pompe, bagno d&#39;acqua, riscaldatori di flusso e flusso di gas. </li><li> Rimuovere la placenta dal supporto del tessuto, tagliare il cotiledone perfuso (più luminoso del tessuto unperfused) e si pesa. </li><li> Prelevare campioni da unperfused (parte della placenta che è stato tagliato in principio; potrebbe essere già in fase di pre-fase) e perfusione tissutale (ciascuna di circa 1 g) e conservarli a -20 ° Cfino omogeneizzazione o in azoto liquido per la successiva analisi. Fissare un altro campione di tessuto in formalina 4% per la valutazione istopatologica. I campioni dovrebbero comprendere tutti gli strati della placenta. </li><li> Pulire le provette dopo perfusione ordine successivo risciacquo sistemi arteriosa del circuito fetale e materno con a) 200 ml di acqua distillata, b) 50 ml di idrossido di sodio 1%, c) 50 ml di 1% di acido fosforico e d) di nuovo 200 ml di acqua distillata (flusso: 15-20 ml / min). </li></ol> L&#39;intera procedura di lavoro dell&#39;esperimento perfusione placenta è mostrata in Figura 3. 4. Analizzando i campioni <ol><li> Centrifugare i campioni perfusato per 10 min a 800 xg prima dell&#39;analisi per rimuovere residui eritrociti. Prendete il surnatante per l&#39;ulteriore analisi. I campioni possono essere lasciati notte a 4 ° C. Per l&#39;analisi della produzione di leptina e hCG i campioni possono essere conservati a -20 ° C. </li><li> Per valutare la permeabilità delplacenta analizzare il C-14 antipyrine da scintillazione liquida. Mescolare 300 ml di campioni fetali e materni con 3 ml di cocktail di scintillazione e misura per 5 minuti in un contatore beta. </li><li> Per valutare il trasferimento delle nanoparticelle fluorescenti o xenobiotici di interesse leggere la fluorescenza a 485 nm di eccitazione e di emissione di 528 nm in un lettore di micropiastre (lunghezza d&#39;onda sono indicati per l&#39;analisi del marchio verde giallo che abbiamo usato per le nanoparticelle). </li><li> Per determinare la fattibilità del tessuto placentare durante la perfusione misura il consumo di glucosio e la produzione di lattato nel circuito fetale e materno con un sistema automatizzato di gas del sangue. Inoltre, valutare la produzione di ormoni placentare umano choriongonadotropin (hCG) e leptina nei campioni di tessuto omogeneizzati e le perfusati by enzyme-linked test immunoenzimatico (ELISA). </li></ol>

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Gli autori dichiarano di non avere interessi finanziari in competizione.

Sotto il duplice perfusione ricircolo mostrato qui, ci sono diverse altre configurazioni sperimentali possibili a seconda della questione che deve essere risolta. Perfusione placentare particolarmente aperta sono comunemente utilizzati per valutare la clearance del farmaco a concentrazioni steady-state 3. La perfusione ricircolo set-up può essere applicato anche per confermare trasporto attivo di sostanze endogene o esogene. Per questo approccio la stessa concentrazione del xenobiotici deve essere aggiunto a...

La placenta è un organo complesso che è responsabile per lo scambio di ossigeno, anidride carbonica, nutrienti e prodotti di scarto e allo stesso tempo in grado di mantenere i due circuiti di sangue della madre e la crescita del feto separati tra loro. Inoltre, impedisce il rifiuto del figlio da parte del sistema immunitario materno e secerne ormoni per mantenere la gravidanza. La barriera cellulare si forma dalle cellule cytotrophoblast che si fondono e formano un vero sincizio senza membrane cellulari laterali 4,5. Tutta la placenta è organizzato in diversi cotiledoni, che contengono un albero dei villi fetali e rappresentano una unità funzionale della placenta. Lo studio della funzione di barriera placentare è stata intensificata con la scoperta delle malformazioni indotte talidomide nel 1960. Per ovvie ragioni non possono essere eseguiti studi traslocazione con le donne in gravidanza. Di conseguenza, vari modelli alternativi sono stati sviluppati 6,7 </sup&gt;. Il modello in questione più promettente e probabilmente più clinico è il modello ex vivo umano placentare perfusione sviluppato da Panigel e collaboratori 2,3. Molte donne sono esposte a diversi xenobiotici quali farmaci o sostanze inquinanti ambientali durante la gravidanza 8. Per alcuni farmaci che sono stati già somministrati regolarmente durante la gravidanza, studi in vivo può essere eseguita confrontando la concentrazione ematica materna con quella nel sangue del cordone ombelicale. Tuttavia, in genere ci sono solo informazioni limitate sulla farmacocinetica e-dinamica nel feto e la teratogenicità di tali sostanze. Per esempio gli oppiacei come l&#39;eroina attraversa facilmente la barriera placentare e può portare alla restrizione della crescita intrauterina, parto prematuro o aborto spontaneo 9,10. Quindi, in caso di astinenza mancante durante la gravidanza si raccomanda una terapia sostitutiva con metadone. L&#39;exvivo modello perfusione placentare umano ha rivelato che il trasferimento di metadone nella circolazione fetale è trascurabile 11, che correla bene con la-a-sangue materno rapporto di concentrazione calcolata cordone sangue dopo la consegna 12. La nanotecnologia è un settore in crescita soprattutto in medicina. Così, sotto la naturale fine (&lt;2,5 micron di diametro) e le particelle ultrafini (&lt;0,1 micron di diametro) nei fumi degli incendi boschivi, eruzioni vulcaniche e in polvere del deserto, l&#39;esposizione a nanomateriali ingegnerizzati (almeno una dimensione &lt;0,1 micron 13 ) è in aumento. Questo ha sollevato interrogativi circa il potenziale tossicologico di nanomateriali ingegnerizzati. Anche se nessun pericolo umana potrebbe essere ancora dimostrata, ci sono i principali studi sperimentali indicano che le nanoparticelle ingegnerizzate possono causare reazioni avverse biologici che portano a risultati tossicologici 14. Recentemente, alcuni studi hanno indicato che l&#39;esposizione prenatale ainquinamento dell&#39;aria è collegato ad un bisogno respiratorio superiore e infiammazione delle vie aeree in neonati e bambini 15,16. Inoltre, le piccole nanoparticelle potrebbero essere utilizzati come trasportatori di farmaci per trattare specificamente, sia il feto o la madre. Pertanto, diventa evidente che sono necessari studi approfonditi di xenobiotici distinti o nanomateriali e la loro capacità di attraversare la barriera placentare. Una panoramica reale sugli studi in corso sulla permeabilità placentare di nanomateriali ingegnerizzati è riassunto nella Menezes et al. 2011 17 e Buerki-Thurnherr et al. 2012 7. La doppia ex vivo ricircolazione modello perfusione placentare umano fornisce un sistema controllato e affidabile per studiare il trasporto placentare di vari composti endogeni ed esogeni 3,11,12,18,19 e una vasta gamma di altre funzioni della placenta come meccanismi responsabili della sviluppo di stati patologici come preeclampsia &lt;sup&gt; 20-22. In questo protocollo ci concentriamo principalmente sul set up, la gestione e il metodo che permette lo studio di accumulo, effetti e tassi di traslocazione di un ampio set di xenobiotici o nanoparticelle.

<table border="1">
	<tbody>
 <tr>
 <td>Name of the Reagent</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>NCTC-135 medium</td>
 <td>ICN Biomedicals, Inc.</td>
 <td>10-911-22C</td>
 <td>could be replaced by Medium 199 from Sigma (M3769)</td>
 </tr>
 <tr>
 <td>Sodium chloride (NaCl)</td>
 <td>Sigma-Aldrich, Fluka</td>
 <td>71381</td>
 <td></td>
 </tr>
 <tr>
 <td>Potassium chloride (KCl)</td>
 <td>Hospital pharmacy</td>
 <td></td>
 <td>also possible: Sigma (P9541)</td>
 </tr>
 <tr>
 <td>Monosodium phosphate (NaH2PO4 &middot; H2O)</td>
 <td>Merck</td>
 <td>106346</td>
 <td></td>
 </tr>
 <tr>
 <td>Magnesium sulfate (MgSO4 &middot; H2O)</td>
 <td>Sigma-Aldrich, Fluka</td>
 <td>63139</td>
 <td></td>
 </tr>
 <tr>
 <td>Calcium chloride (CaCl, anhydrous)</td>
 <td>Merck</td>
 <td>102388</td>
 <td></td>
 </tr>
 <tr>
 <td>D(+) Glucose (anhydrous)</td>
 <td>Sigma-Aldrich, Fluka</td>
 <td>49138</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium bicarbonate (NaHCO3)</td>
 <td>Merck</td>
 <td>106329</td>
 <td></td>
 </tr>
 <tr>
 <td>Dextran from Leuconostoc spp.</td>
 <td>Sigma-Aldrich</td>
 <td>31389</td>
 <td></td>
 </tr>
 <tr>
 <td>Bovine serum albumin (BSA)</td>
 <td>Applichem</td>
 <td>A1391</td>
 <td></td>
 </tr>
 <tr>
 <td>Amoxicilline (Clamoxyl)</td>
 <td>GlaxoSmithKline AG</td>
 <td>2021101A</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium heparin</td>
 <td>B. Braun Medical AG</td>
 <td>3511014</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium hydoxide (NaOH) pellets</td>
 <td>Merck</td>
 <td>106498</td>
 <td>CAUTION: corrosive</td>
 </tr>
 <tr>
 <td>Ortho-phosphoric acid 85% (H3PO4)</td>
 <td>Merck</td>
 <td>100573</td>
 <td>CAUTION: corrosive</td>
 </tr>
 <tr>
 <td>Maternal gas mixture: 95% synthetic air, 5% CO2</td>
 <td>PanGas AG</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Fetal gas mixture: 95% N2, 5% CO2</td>
 <td>PanGas AG</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Antipyrine (N-methyl-14C)</td>
 <td>American Radiolabeled Chemicals, Inc.</td>
 <td>ARC 0108-50 &mu;Ci</td>
 <td>CAUTION: radioactive material (specific activity: 55mCi/mmol)</td>
 </tr>
 <tr>
 <td>Scintillation cocktail (IrgaSafe Plus)</td>
 <td>Zinsser Analytic GmbH</td>
 <td>1003100</td>
 <td></td>
 </tr>
 <tr>
 <td>Polystyrene particles 80 nm</td>
 <td>Polyscience, Inc.</td>
 <td>17150</td>
 <td></td>
 </tr>
 <tr>
 <td>Polystyrene particles 500 nm</td>
 <td>Polyscience, Inc.</td>
 <td>17152</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT</td>
 </tr>
 <tr>
 <td>Water bath</td>
 <td>VWR</td>
 <td>462-7001</td>
 <td></td>
 </tr>
 <tr>
 <td>Thermostat</td>
 <td>IKA-Werke GmbH &amp; Co. KG</td>
 <td>3164000</td>
 <td></td>
 </tr>
 <tr>
 <td>Peristaltic pumps</td>
 <td>Ismatec</td>
 <td>ISM 833</td>
 <td></td>
 </tr>
 <tr>
 <td>Bubble traps (glass)</td>
 <td>UNI-GLAS Laborbedarf</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Flow heater</td>
 <td>UNI-GLAS Laborbedarf</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Pressure sensor + Software for analyses</td>
 <td>MSR Electronics GmbH</td>
 <td>145B5</td>
 <td></td>
 </tr>
 <tr>
 <td>Notebook</td>
 <td>Hewlett Packard</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Miniature gas exchange oxygenator</td>
 <td>Living Systems Instrumentation</td>
 <td>LSI-OXR</td>
 <td></td>
 </tr>
 <tr>
 <td>Tygon Tube (ID: 1.6 mm; OD: 4.8 mm)</td>
 <td>Ismatec</td>
 <td>MF0028</td>
 <td></td>
 </tr>
 <tr>
 <td>Tubes for pumps (PharMed BPT; ID: 1.52 mm)</td>
 <td>Ismatec</td>
 <td>SC0744</td>
 <td></td>
 </tr>
 <tr>
 <td>Blunt cannulae (&Oslash; 0.8 mm)</td>
 <td>Polymed Medical Center</td>
 <td>03.592.81</td>
 <td></td>
 </tr>
 <tr>
 <td>Blunt cannulae (&Oslash; 1.2 mm)</td>
 <td>Polymed Medical Center</td>
 <td>03.592.90</td>
 <td></td>
 </tr>
 <tr>
 <td>Blunt cannulae (&Oslash; 1.5 mm)</td>
 <td>Polymed Medical Center</td>
 <td>03.592.94</td>
 <td></td>
 </tr>
 <tr>
 <td>Blunt cannulae (&Oslash; 1.8 mm)</td>
 <td>Polymed Medical Center</td>
 <td>03.952.82</td>
 <td></td>
 </tr>
 <tr>
 <td>Parafilm</td>
 <td>VWR</td>
 <td>291-1212</td>
 <td></td>
 </tr>
 <tr>
 <td>Perfusion chamber with tissue holder (plexiglass)</td>
 <td>Internal technical department</td>
 <td></td>
 <td>Similar equipment is available from Hemotek Limited, UK</td>
 </tr>
 <tr>
 <td>Surgical suture material (PremiCron)</td>
 <td>B. Braun Medical AG</td>
 <td>C0026005</td>
 <td></td>
 </tr>
 <tr>
 <td>Winged Needle Infusion Set (21G Butterfly)</td>
 <td>Hospira, Inc.</td>
 <td>ASN 2102</td>
 <td></td>
 </tr>
 <tr>
 <td>Multidirectional stopcock (Discofix C-3)</td>
 <td>B. Braun Medical AG</td>
 <td>16494C</td>
 <td></td>
 </tr>
 <tr>
 <td>Surgical scissors</td>
 <td>B. Braun Medical AG</td>
 <td>BC304R</td>
 <td></td>
 </tr>
 <tr>
 <td>Dissecting scissors</td>
 <td>B. Braun Medical AG</td>
 <td>BC162R</td>
 <td></td>
 </tr>
 <tr>
 <td>Needle holder</td>
 <td>B. Braun Medical AG</td>
 <td>BM200R</td>
 <td></td>
 </tr>
 <tr>
 <td>Dissecting forceps</td>
 <td>B. Braun Medical AG </td>
 <td>BD215R</td>
 <td></td>
 </tr>
 <tr>
 <td>Automated blood gas system </td>
 <td>Radiometer Medical ApS</td>
 <td>ABL800 FLEX</td>
 <td></td>
 </tr>
 <tr>
 <td>Multi-mode microplate reader</td>
 <td>BioTek</td>
 <td>Synergy HT</td>
 <td></td>
 </tr>
 <tr>
 <td>Liquid scintillation analyzer</td>
 <td>GMI, Inc.</td>
 <td>Packard Tri-Carb 2200</td>
 <td></td>
 </tr>
 <tr>
 <td>Scintillation tubes 5.5 ml</td>
 <td>Zinsser Analytic GmbH</td>
 <td>3020001</td>
 <td></td>
 </tr>
 <tr>
 <td>Tissue Homogenizer</td>
 <td>OMNI, Inc.</td>
 <td>TH-220</td>
 <td></td>
 </tr>
 <tr>
 <td>pH meter + electrode</td>
 <td>VWR</td>
 <td>662-2779 </td>
 <td></td>
 </tr>
	 </tbody>
 </table>

determination of the transport rate of xenobiotics and nanomaterials across the placenta using the ex vivo human placental perfusion model

Decenni fa, la placenta umana è stato pensato per essere una barriera impenetrabile tra madre e feto. Tuttavia, la scoperta di difetti alla nascita talidomide-indotta e molti studi successivi seguito si mostrò il contrario. Oggi diversi xenobiotici nocivi come la nicotina, eroina, metadone o droghe, nonché agenti inquinanti ambientali sono stati descritti per superare questa barriera. Con il crescente uso della nanotecnologia, la placenta rischia di entrare in contatto con nuove nanoparticelle sia accidentalmente attraverso l&#39;esposizione o intenzionalmente, nel caso di potenziali applicazioni nanomedical. I dati provenienti da esperimenti su animali non possono essere estrapolati per l&#39;uomo perché la placenta è la più specie-specifico organo dei mammiferi 1. Pertanto, l&#39;ex vivo doppio ricircolo di perfusione placentare umano, sviluppato da Panigel et al. Nel 1967 2 e continuamente modificato da Schneider et al. Nel 1972 3, può servire come un eccellente modello di tØ studiare il trasferimento di xenobiotici o particelle. Qui, ci concentriamo sul duplice ex vivo a ricircolo di protocollo di perfusione placentare umano e il suo ulteriore sviluppo per acquisire risultati riproducibili. La placenta sono stati ottenuti dopo il consenso informato delle madri di gravidanze non complicate termine sottoposti a parto cesareo. I vasi fetali e materni di un cotiledone intatto stati cannulati e perfusi almeno per cinque ore. Come un modello di particelle particelle di polistirene fluorescente con dimensioni di 80 e 500 nm di diametro sono stati aggiunti al circuito materna. Le 80 particelle nm sono stati in grado di attraversare la barriera placentare e fornire un esempio perfetto per una sostanza che viene trasferita attraverso la placenta al feto, mentre le particelle di 500 nm sono stati mantenuti nel tessuto placentare o circuito materna. Il modello di perfusione ex vivo umano placentare è uno dei pochi modelli che forniscono informazioni attendibili suil comportamento di trasporto di xenobiotici in una importante barriera tessuto che fornisce i dati rilevanti predittivi e clinici.

La Figura 4A mostra i profili di perfusione di piccole particelle di polistirene (80 nm) che sono stati trasportati attraverso la placenta più grandi rispetto a particelle di polistirene (500 nm), che non sono state trasferite al compartimento fetale. Ciascun punto di dati rappresenta la concentrazione di particella medio al dato punto di almeno tre esperimenti indipendenti tempo. Per polistirolo nanoparticelle il trasferimento placentare è formato-dipendente 19. Dopo 3 h di placenta perfus...

Watch this Scientific Journal Video about Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model at JoVE.com

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

Gli<em&gt; Ex vivo</em&gt; Dual ricircolo modello perfusione placentare umano può essere usato per studiare il trasferimento di xenobiotici e nanoparticelle attraverso la placenta umana. In questo protocollo video si descrive l&#39;attrezzatura e le tecniche necessarie per una corretta esecuzione di un perfusione placentare.

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 ...

determination-transport-rate-xenobiotics-nanomaterials-across

Research

JoVE Journal

Bioengineering

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

17.2K Views.  University Hospital Zurich. Gli Ex vivo Dual ricircolo modello perfusione placentare umano può essere usato per studiare il trasferimento di xenobiotici e nanoparticelle attraverso la placenta umana. In questo protocollo video si descrive l'attrezzatura e le tecniche necessarie per una corretta esecuzione di un perfusione placentare.

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

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 ...

Ricerca

Bioingegneria

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

Hematopoietic stem cells require a unique microenvironment in order to sustain blood cell formation1; the bone marrow (BM) is a complex three-dimensional (3D) tissue wherein hematopoiesis is regulated by spatially organized cellular microenvironments termed niches2-4. The organization of the BM niches is critical for the function or dysfunction of normal or malignant BM5. Therefore a better understanding of the in vivo microenvironment using an ex vivo mimicry would help us elucidate the molecular, cellular and microenvironmental determinants of leukemogenesis6.
Currently, hematopoietic cells are cultured in vitro in two-dimensional (2D) tissue culture flasks/well-plates7 requiring either co-culture with allogenic or xenogenic stromal cells or addition of exogenous cytokines8. These conditions are artificial and differ from the in vivo microenvironment in that they lack the 3D cellular niches and expose the cells to abnormally high cytokine concentrations which can result in differentiation and loss of pluripotency9,10.
Herein, we present a novel 3D bone marrow culture system that simulates the in vivo 3D growth environment and supports multilineage hematopoiesis in the absence of exogenous growth factors. The highly porous scaffold used in this system made of polyurethane (PU), facilitates high-density cell growth across a higher specific surface area than the conventional monolayer culture in 2D11. Our work has indicated that this model supported the growth of human cord blood (CB) mononuclear cells (MNC)12 and primary leukemic cells in the absence of exogenous cytokines. This novel 3D mimicry provides a viable platform for the development of a human experimental model to study hematopoiesis and to explore novel treatments for leukemia.

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

A 3D culture system for hematopoiesis is described using human cord blood and leukemic bone marrow cells. The method is based on the use of a porous synthetic polyurethane scaffold coated with extracellular matrix proteins. This scaffold is adaptable to accommodate a wide range of cells.

Ex vivo Mimicry di emopoiesi normale e anormale umana

Hematopoietic stem cells require a unique microenvironment in order to sustain blood cell formation1; the bone marrow (BM) is a complex three-dimensional ...

Anatomical Reconstructions of the Human Cardiac Venous System using Contrast-computed Tomography of Perfusion-fixed Specimens

A detailed understanding of the complexity and relative variability within the human cardiac venous system is crucial for the development of cardiac devices that require access to these vessels. For example, cardiac venous anatomy is known to be one of the key limitations for the proper delivery of cardiac resynchronization therapy (CRT)1 Therefore, the development of a database of anatomical parameters for human cardiac venous systems can aid in the design of CRT delivery devices to overcome such a limitation. In this research project, the anatomical parameters were obtained from 3D reconstructions of the venous system using contrast-computed tomography (CT) imaging and modeling software (Materialise, Leuven, Belgium). The following parameters were assessed for each vein: arc length, tortuousity, branching angle, distance to the coronary sinus ostium, and vessel diameter.
	CRT is a potential treatment for patients with electromechanical dyssynchrony. Approximately 10-20% of heart failure patients may benefit from CRT2. Electromechanical dyssynchrony implies that parts of the myocardium activate and contract earlier or later than the normal conduction pathway of the heart. In CRT, dyssynchronous areas of the myocardium are treated with electrical stimulation. CRT pacing typically involves pacing leads that stimulate the right atrium (RA), right ventricle (RV), and left ventricle (LV) to produce more resynchronized rhythms. The LV lead is typically implanted within a cardiac vein, with the aim to overlay it within the site of latest myocardial activation. 
	We believe that the models obtained and the analyses thereof will promote the anatomical education for patients, students, clinicians, and medical device designers. The methodologies employed here can also be utilized to study other anatomical features of our human heart specimens, such as the coronary arteries. To further encourage the educational value of this research, we have shared the venous models on our free access website: <a href="http://www.vhlab.umn.edu/atlas" target="_blank">www.vhlab.umn.edu/atlas</a>.

The objective of this research is to recreate and then access the anatomy of the human cardiac venous system using 3D reconstructions generated from contrast-computed tomography scans.

Ricostruzioni anatomiche del sistema umano venoso cardiaco utilizzando Contrasto-tomografia computerizzata di campioni di perfusione-fissi

A detailed  understanding of the complexity and relative variability within the human  cardiac venous system is crucial for the development of cardiac ...

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

The foreign body reaction occurs when a synthetic surface is introduced to the body. It is characterized by adsorption of blood proteins and the subsequent attachment and activation of platelets, monocyte/macrophage adhesion, and inflammatory cell signaling events, leading to post-procedural complications. The Chandler Loop Apparatus is an experimental system that allows researchers to study the molecular and cellular interactions that occur when large volumes of blood are perfused over polymeric conduits. To that end, this apparatus has been used as an ex vivo model allowing the assessment of the anti-inflammatory properties of various polymer surface modifications. Our laboratory has shown that blood conduits, covalently modified via photoactivation chemistry with recombinant CD47, can confer biocompatibility to polymeric surfaces. Appending CD47 to polymeric surfaces could be an effective means to promote the efficacy of polymeric blood conduits. Herein is the methodology detailing the photoactivation chemistry used to append recombinant CD47 to clinically relevant polymeric blood conduits and the use of the Chandler Loop as an ex vivo experimental model to examine blood interactions with the CD47 modified and control conduits.

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 ...

Determination of Zeta Potential via Nanoparticle Translocation Velocities through a Tunable Nanopore: Using DNA-modified Particles as an Example

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and nanoparticles. Tunable resistive pulse sensing (TRPS) is a relatively recent adaptation to RPS that incorporates a tunable pore that can be altered in real time. Here, we use TRPS to monitor the translocation times of DNA-modified nanoparticles as they traverse the tunable pore membrane as a function of DNA concentration and structure (i.e., single-stranded to double-stranded DNA).
TRPS is based on two Ag/AgCl electrodes, separated by an elastomeric pore membrane that establishes a stable ionic current upon an applied electric field. Unlike various optical-based particle characterization technologies, TRPS can characterize individual particles amongst a sample population, allowing for multimodal samples to be analyzed with ease. Here, we demonstrate zeta potential measurements via particle translocation velocities of known standards and apply these to sample analyte translocation times, thus resulting in measuring the zeta potential of those analytes.
As well as acquiring mean zeta potential values, the samples are all measured using a particle-by-particle perspective exhibiting more information on a given sample through sample population distributions, for example. Of such, this method demonstrates potential within sensing applications for both medical and environmental fields.

Here we use a polyurethane tunable nanopore integrated into a resistive pulse sensing technique to characterize nanoparticles surface chemistry via the measurement of particle translocation velocities, which can be used to determine the zeta potential of individual nanoparticles.

Determinazione della Zeta Potenziale tramite nanoparticelle Traslocazione Le velocità attraverso una Tunable Nanopore: usando le particelle di DNA modificato come esempio

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and ...

An Experimental and Finite Element Protocol to Investigate the Transport of Neutral and Charged Solutes across Articular Cartilage

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular cartilage impairs its load-bearing function substantially as it experiences tremendous chemical degradation, i.e. proteoglycan loss and collagen fibril disruption. One promising way to investigate chemical damage mechanisms during OA is to expose the cartilage specimens to an external solute and monitor the diffusion of the molecules. The degree of cartilage damage (i.e. concentration and configuration of essential macromolecules) is associated with collisional energy loss of external solutes while moving across articular cartilage creates different diffusion characteristics compared to healthy cartilage. In this study, we introduce a protocol, which consists of several steps and is based on previously developed experimental micro-Computed Tomography (micro-CT) and finite element modeling. The transport of charged and uncharged iodinated molecules is first recorded using micro-CT, which is followed by applying biphasic-solute and multiphasic finite element models to obtain diffusion coefficients and fixed charge densities across cartilage zones.

We propose a protocol to investigate the transport of charged and uncharged molecules across articular cartilage with the aid of recently developed experimental and numerical methods.

Uno Sperimentale e Finite Element protocollo per indagare il trasporto di soluti neutri e carichi attraverso cartilagine articolare

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular ...

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 ...

Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to the placenta while minimizing fetal and maternal side effects remains challenging. Targeted nanoparticle carriers provide new opportunities to treat placental disorders. We recently demonstrated that a synthetic placental chondroitin sulfate A binding peptide (plCSA-BP) could be used to guide nanoparticles to deliver drugs to the placenta. In this protocol, we describe in detail a system for assessing the efficiency of drug delivery to the placenta by plCSA-BP that employs three separate methods used in combination: in vivo imaging, high-frequency ultrasound (HFUS), and high-performance liquid chromatography (HPLC). Using in vivo imaging, plCSA-BP-guided nanoparticles were visualized in the placentas of live animals, while HFUS and HPLC demonstrated that plCSA-BP-conjugated nanoparticles efficiently and specifically delivered methotrexate to the placenta. Thus, a combination of these methods can be used as an effective tool for the targeted delivery of drugs to the placenta and development of new treatment strategies for several pregnancy complications.

We describe a system that utilizes three methods to evaluate the safety and effectiveness of placenta-targeted drug delivery: in vivo imaging to monitor nanoparticle accumulation, high-frequency ultrasound to monitor placental and fetal development, and HPLC to quantify drug delivery to tissue.

Valutazione globale dell'efficacia e della sicurezza di Placenta-Targeted Drug Delivery utilizzando tre metodi complementari

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to ...

Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically been studied in vivo in animal models showing characteristic patterns of cell migration. However, in vivo models have several limitations, including intercellular mediators that are difficult to access and analyze, as well as the inability to directly analyze human neutrophils. Because of these limitations, there is a need for an in vitro platform that studies swarming with human neutrophils and provides easy access to the molecular signals generated during swarming. Here, a multistep microstamping process is used to generate a bioparticle microarray that stimulates swarming by mimicking an in vivo infection. The bioparticle microarray induces neutrophils to swarm in a controlled and stable manner. On the microarray, neutrophils increase in speed and form stable swarms around bioparticle clusters. Additionally, supernatant generated by the neutrophils was analyzed and 16 proteins were discovered to have been differentially expressed over the course of swarming. This in vitro swarming platform facilitates direct analysis of neutrophil migration and protein release in a reproducible, spatially controlled manner.

This protocol generates bioparticle microarrays that provide spatially controlled neutrophil swarming. It provides easy access to the mediators that neutrophils release during migration and allows for quantitative imaging analysis.

Microarray di bioparticelle per l'analisi chemiotattica e molecolare dello sciame neutrofilo umano in vitro

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically ...

Biofunctionalization of Magnetic Nanomaterials

Magnetic nanomaterials have received great attention in different biomedical applications. Biofunctionalizing these nanomaterials with specific targeting agents is a crucial aspect to enhance their efficacy in diagnostics and treatments while minimizing the side effects. The benefit of magnetic nanomaterials compared to non-magnetic ones is their ability to respond to magnetic fields in a contact-free manner and over large distances. This allows to guide or accumulate them, while they can also be monitored. Recently, magnetic nanowires (NWs) with unique features were developed for biomedical applications. The large magnetic moment of these NWs enables a more efficient remote control of their movement by a magnetic field. This has been utilized with great success in cancer treatment, drug delivery, cell tracing, stem cell differentiation or magnetic resonance imaging. In addition, the NW fabrication by template-assisted electrochemical deposition provides a versatile method with tight control over the NW properties. Especially iron NWs and iron-iron oxide (core-shell) NWs are suitable for biomedical applications, due to their high magnetization and low toxicity.
In this work, we provide a method to biofunctionalize iron/iron oxide NWs with specific antibodies directed against a specific cell surface marker that is overexpressed in a large number of cancer cells. Since the method utilizes the properties of the iron oxide surface, it is also applicable to superparamagnetic iron oxide nanoparticles. The NWs are first coated with 3-aminopropyl-tri-ethoxy-silane (APTES) acting as a linker, which the antibodies are covalently attached to. The APTES coating and the antibody biofunctionalization are proven by electron energy loss spectroscopy (EELS) and zeta potential measurements. In addition, the antigenicity of the antibodies on the NWs is tested by using immunoprecipitation and western blot. The specific targeting of the biofunctionalized NWs and their biocompatibility are studied by confocal microscopy and a cell viability assay.

In this work, we provide a protocol to biofunctionalize magnetic nanomaterials with antibodies for specific cell targeting. As examples, we utilize iron nanowires to target cancer cells.

Biofunzionalizzazione di nanomateriali magnetici

Magnetic nanomaterials have received great attention in different biomedical applications. Biofunctionalizing these nanomaterials with specific targeting ...