Gli autori desiderano ringraziare Jack Milwid per la progettazione della camera di perfusione in vitro. Questo lavoro è stato supportato anche da finanziamenti del NIH americano, R01DK59766 e R01DK084053 a MY, R00DK080942 di KU, US NSF cbet-KU e 0.853.569 per il Shriners Hospitals for Children per BEU (non concedere. 8.503). Riconosciamo inoltre il supporto e il Shriners ospedali per bambini.

1. Fegato Decellularization <ol><li> Raccolto un fegato di ratto con incannulazione della vena porta e con 18-gauge catetere. Lasciare l&#39;inferiore e vena cava superiore aperto. Mantenere l&#39;organo idratata in tampone fosfato (PBS) in una di 10 cm capsula di Petri. </li><li> Istituire un sistema di perfusione costituito da serbatoio da 8 litri, pompa peristaltica e un gorgogliatore. </li><li> Riempire il sistema di perfusione con tampone fosfato e mantenerlo in funzione per 10 minuti. Riempire PBS in una di 10 cm capsula di Petri e di ridurre la portata di tampone fosfato a 1 ml / min. </li><li> Trasferire accuratamente l&#39;fegato raccolte al tampone fosfato pieno di 10 cm capsula di Petri. </li><li> Continuare con perfusione PBS durante la notte. </li><li> Inizio perfusione con 0,01% (w / v) dodecil solfato di sodio (SDS) in acqua distillata per 5 minuti. </li><li> Profumato con PBS per 1 ora. </li><li> Ripetere i passi 1,6 e 1,7 per altre tre volte aumentando il tempo di perfusione SDS a minuti 10, 15 e 20 in ciascun momento. </li><li> Continua perfusione con 0,01% (w / v) di SDS per 24 ore. </li><li> Continua perfusione con 0,1% (w / v) di SDS per 24 ore. </li><li> Profumato con il 0,2% (w / v) di SDS per 3 ore. </li><li> Profumato con il 0,5% (w / v) di SDS per 3 ore. </li><li> Profumato con acqua distillata per 15 minuti. </li><li> Profumato con l&#39;1% (w / v) Triton X-100 in acqua distillata per 30 minuti per eliminare gli acidi nucleici legati. </li><li> Per lavare il fegato decellularized matrice (DLM), profumato con PBS per 2 ore. </li><li> Opzionale: resecare tutti i lobi, tranne il lobo mediano. </li><li> Conservare il DLM in un piatto pulito e sigillato Petri immersi in PBS a 4 ° C fino al momento dell&#39;uso (Figura 1). </li><li> Per sterilizzare l&#39;DLM: 1) Lavare il DLM con PBS sterile contenente 0,1% (v / v) e acido peracetico al 4% (v / v) di etanolo e incubare per 3 ore a 4 ° C. 2) Lavare con PBS sterile 2 volte. 3) Lavare con PBS sterile contenente 2% di penicillina-streptomicina, 10ug / mL di gentamicina, e 2,5 ug / ml di amfotericina B. Conservare il fegato decellularized nella stessa soluzione a 4 ° C fino al momento dell&#39;uso per esperimenti recellularization. </li></ol> 2. Recellularization di Decellularized Fegato Matrix <ol><li> Istituire un sistema di perfusione costituito da una camera di perfusione, pompa peristaltica e un gorgogliatore in condizioni sterili. Riempire il sistema di perfusione con 200 ml di terreno di coltura, per esempio, elevata glicemia DMEM (Sigma), 10% siero fetale bovino (Hyclone,), 100 U ml -1 penicillina, e 100 mg ml -1 streptomicina (Invitrogen). </li><li> Posizionare la matrice decellularized fegato nella camera di perfusione e collegare il DLM al sistema di perfusione attraverso la cannula vena porta, mentre la pompa è in funzione a 5 mL / min per evitare la formazione di eventuali bolle d&#39;aria. </li><li> Lasciare perfusione del mezzo attraverso il DLM per 30 minuti. </li><li> Isolare epatociti primari di un ratto adulto con almeno il 90% vitalità. </li><li> Fermare il flusso nel sistema di perfusione e iniettare lentamente 50 milioni epatociti (in mL 1-3 su terreno di coltura) nel sistema di perfusione attraverso il gorgogliatore. </li><li> Avviare il flusso a 10 ml / min e ricircolo del mezzo per 10 minuti. </li><li> Ripetere i passi 2.5 e 2.6 fino a un totale di 200 milioni di cellule vengono introdotti nel DLM (Figura 2) 6. </li><li> Una volta che tutte le cellule sono perfusi in DLM, raccogliete il perfusato in quattro da 50 ml, provette da centrifuga e centrifugare a 600 giri per 10 minuti. Eliminare il surnatante e raccogliere il pellet in un unico tubo. Determinare il numero di cellule e vitalità attraverso l&#39;esclusione trypan blu per determinare l&#39;efficienza semina. </li></ol> 3. Coltura in vitro del trapianto di fegato Recellularized <ol><li> Istituire un sistema di perfusione costituito da una camera di perfusione, pompa peristaltica, ossigenatore ed un gorgogliatore in condizioni sterili (Figura 3). Riempire il sistema di perfusione con 50 ml di terreno di coltura, per esempio, Williams &#39;E (Sigma), il 5% siero fetale bovino (Hyclone,), 0,5 U di insulina mL -1 (Eli Lilly), 20 ng mL -1 FEG (Invitrogen) , 14 ng mL -1 glucagone (Bedford Laboratories), 7,5 ml mg di idrocortisone -1 (Pharmacia), 100 U ml -1 penicillina, e 100 mg ml -1 streptomicina (Invitrogen). </li><li> Posizionare il trapianto di fegato recellularized nella camera di perfusione e collegare il DLM al sistema di perfusione attraverso la cannula vena porta, mentre la pompa è in funzione a 5 mL / min per evitare la formazione di eventuali bolle d&#39;aria. </li><li> Asetticamente chiudere la camera di perfusione e sigilla ermeticamente per evitare perdite durante cultura. </li><li> Trasferire il sistema di perfusione di un incubatore che è 37 ° C e ha il 10% di CO 2 e aumentare il tasso di flusso di perfusione a 15 ml / min. </li><li> Collegare l&#39;ossigenatore ad un 95% di O 2 e il 5% di CO 2 Serbatoio miscela di gas e di impostare la velocità di flusso del gas di 0,5 litri / min. Questo dovrebbe raggiungere una pressione parziale di ossigeno di approssimazionetely 400 mmHg. </li><li> La cultura può continuare fino a 10 giorni, con variazioni giornaliere di terreno di coltura. Il terreno di coltura può essere campionato al giorno per il monitoraggio delle funzioni del fegato del trapianto, come l&#39;albumina, urea e totale secrezione degli acidi biliari. Al termine del periodo di cultura, il trapianto di fegato recellularized possono essere prelevati per analisi molecolari e istologiche. </li></ol> 4. Rappresentante dei risultati: Il decellularization completa di un fegato di ratto è di circa 72 ore utilizzando il protocollo descritto. La matrice risultante mantiene il 100% del collageno fibrillare, il 50% dei glicosaminoglicani e solo il 5% del DNA del fegato nativo (Tabella 1) 6. La struttura vascolare della matrice è conservato come evidenziato dalla corrosione casting e analisi microscopia elettronica a scansione (Figura 4) 6. La presenza della microarchitettura vascolare all&#39;interno del DLM facilita il suo ripopolamento con celle con un&#39;efficienza del 96% e la sua perfusione successive per coltura in vitro. Il trapianto di fegato recellularized può essere colta fino a 10 giorni in vitro e visualizza le funzioni del fegato corretto come confermato via albumina, urea e totale secrezione degli acidi biliari (Figura 5) 6. <img src="/files/ftp_upload/2394/2394fig1.jpg" alt="Figura 1" /> Figura 1. Decellularized matrice fegato al termine del processo di decellularization. (A) il fegato intero (b) il lobo mediano dopo la resezione. <img src="/files/ftp_upload/2394/2394fig2.jpg" alt="Figura 2" /> Figura 2. Rappresentazione schematica della recellularization del DLM. <img src="/files/ftp_upload/2394/2394fig3.jpg" alt="Figura 3" /> Figura 3. Configurazione del sistema di perfusione per coltura in vitro del trapianto di fegato recellularized. <img src="/files/ftp_upload/2394/2394fig4.jpg" alt="Figura 4" /> Figura 4. La struttura microvascolare viene mantenuta nella matrice fegato decellularized. Fusione di immagini corrosione a) un fegato normale b) una decellularized fegato, portale (rosso) e venoso (blu) vascolare. Microscopia elettronica a scansione di immagini del DLM c) una nave, d) una sezione con i piccoli vasi del dotto biliare-like (frecce), bar Scala (a, b) 5 mm (c, d) 20 micron. <img src="/files/ftp_upload/2394/2394fig5.jpg" alt="Figura 5" /> Figura 5. Fegato funzioni specifiche del trapianto di fegato recellularized durante la perfusione nella cultura in vitro. a) la secrezione di albumina (p = 0,5249), b) produzione di urea (p = 0,5271) e c) totale di acidi secrezione biliare (p = 0,0114). L&#39;analisi statistica della differenza tra l&#39;esperimento e il controllo è stato fatto nel periodo 10 cultura d dal test di Friedman per a = 0,01. Barre di errore rappresentano sem (n = 3). <table border="1"><tbody><tr><td></td><td> Fresco Livera </td><td> Decellularized fegato matrice A </td><td rowspan="2"> valori di p </td><td rowspan="2"> % Di fegato fresco </td></tr><tr><td></td><td> n = 4 </td><td> n = 8 </td></tr><tr><td> Collagene </td><td rowspan="2"> 0,07 ± 0,01 </td><td rowspan="2"> 0,08 ± 0,03 </td><td rowspan="2"> 0,56 </td><td rowspan="2"> 114% </td></tr><tr><td> (Mg per g di fegato) </td></tr><tr><td> Glicosaminoglicani </td><td rowspan="2"> 73,1 ± 6,7 </td><td rowspan="2"> 34,2 ± 2,9 </td><td rowspan="2"> 0,004 </td><td rowspan="2"> 47% </td></tr><tr><td> (Mg per g di fegato) </td></tr><tr><td> DNA </td><td rowspan="2"> 14,9 ± 5,6 </td><td rowspan="2"> 0,44 ± 0,08 </td><td rowspan="2"> 3.3 10 -5 </td><td rowspan="2"> 2,9% </td></tr><tr><td> (Mg per g di fegato) </td></tr></tbody></table> Tabella 1. La composizione biochimica della matrice fegato decellularized rispetto al fegato nativo. a I valori sono rappresentati come media ± sem

<ol>
	<li>Kulig, K. M., Vacanti, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+tissue+engineering.">Hepatic tissue engineering.</a> Transpl Immunol. 12, 303-310 (2004).</li><li>Lutolf, M. P., Hubbell, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+biomaterials+as+instructive+extracellular+microenvironments+for+morphogenesis+in+tissue+engineering.">Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering.</a> Nature biotechnology. 23, 47-55 (2005).</li><li>Dahl, S. L., Koh, J., Prabhakar, V., Niklason, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularized+native+and+engineered+arterial+scaffolds+for+transplantation.">Decellularized native and engineered arterial scaffolds for transplantation.</a> Cell Transplant. 12, 659-666 (2003).</li><li>Schechner, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engraftment+of+a+vascularized+human+skin+equivalent.">Engraftment of a vascularized human skin equivalent.</a> FASEB J. 17, 2250-2256 (2003).</li><li>Gilbert, T. W., Sellaro, T. L., Badylak, S. F., F, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularization+of+tissues+and+organs.">Decellularization of tissues and organs.</a> Biomaterials. 27, 3675-3683 (2006).</li><li>Uygun, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+reengineering+through+development+of+a+transplantable+recellularized+liver+graft+using+decellularized+liver+matrix.">Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix.</a> Nat Med. , (2010).</li></ol>

Nessun conflitto di interessi dichiarati.

Il metodo di perfusione decellularization qui descritto produce una intera impalcatura del fegato che ha la stessa struttura lordo e la microarchitettura vascolare del fegato nativo. Il ponteggio ha una composizione simile a quello della matrice extracellulare del fegato nativo. Il metodo recellularization raggiunge ripopolamento del patibolo con celle ad alta efficienza e le cellule rimangono vitali e funzionali durante il periodo di coltura in vitro testati. Con l&#39;aggiunta di cellule nonparenchymal nel tr...

A correction was made to <a href="http://jove.unicartagenaproxy.elogim.com/Details.php?ID=2394">Decellularization and Recellularization of Whole Livers</a>. There was an error with an author's name. The author's last name had a typo and was corrected to:
Nima Saeidi
instead of:
Nima Saedi.

Erratum: Decellularization and Recellularization of Whole Livers

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Sodium dodecyl sulfate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>L4390</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>T8787</td>
<td>&nbsp;</td>
<tr>
<td>Masterflex L/S Digital Drive</td>
<td>&nbsp;</td>
<td>Cole-Parmer</td>
<td>EW-07523-80</td>
<td>&nbsp;</td>
<tr>
<td>Masterflex L/S Standard pump head</td>
<td>&nbsp;</td>
<td>Cole-Parmer</td>
<td>EW- 07013-81</td>
<td>&nbsp;</td>
<tr>
<td>Bubble trap</td>
<td>&nbsp;</td>
<td>Radnoti</td>
<td>130149</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

decellularization and recellularization of whole livers

Il fegato è un organo complesso che richiede perfusione costante per la consegna di sostanze nutritive e di ossigeno e la rimozione dei rifiuti per sopravvivere 1. Gli sforzi per ricreare o imitare la microstruttura fegato con motivi up utilizzando l&#39;ingegneria dei tessuti e le tecniche di microfabbricazione non hanno avuto successo finora a causa di questa sfida di design. Inoltre, biomateriali sintetici utilizzati per creare impalcature per le applicazioni di tessuto epatico di ingegneria sono stati limitati nell&#39;indurre la rigenerazione dei tessuti e la riparazione in gran parte a causa della mancanza di motivi specifici delle cellule vincolante che avrebbe indotto le funzioni cellulari corretta 2. Tessuti Decellularized nativo vasi sanguigni della pelle come 3 e 4 invece hanno trovato molte applicazioni in ingegneria dei tessuti, e hanno fornito una soluzione pratica per alcune delle sfide. Il vantaggio di matrice decellularized nativa è che si conserva, in una certa misura, la composizione originale e la microstruttura, migliorando quindi l&#39;attaccamento delle cellule e riorganizzazione 5. In questo lavoro si descrivono i metodi per eseguire perfusione-decellularization del fegato, come ad esempio che un fegato intatto bioscaffold che mantiene la struttura dei vasi sanguigni principali si ottiene. Inoltre, si descrivono i metodi per recellularize questi bioscaffolds con adulti epatociti primari, la creazione di un trapianto di fegato che è funzionale in vitro, ed ha l&#39;accesso nave nella misura necessaria per il trapianto in vivo.

Watch this Scientific Journal Video about Decellularization and Recellularization of Whole Livers at JoVE.com

Decellularization and Recellularization of Whole Livers

Decellularization perfusione è una tecnica innovativa per la produzione di ponteggi fegato intero che conserva la composizione della matrice extracellulare l&#39;organo e la microarchitettura. Qui, il metodo di preparazione di ponteggi intero organo utilizzando decellularization perfusione e conseguente ripopolamento con epatociti è descritto. Trapianti di fegato funzionale e trapiantabili possono essere generati utilizzando questa tecnica.

Decellularization e Recellularization di fegati interi

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

decellularization-and-recellularization-of-whole-livers

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

21.9K Views.  Massachusetts General Hospital, Harvard Medical School, Shriners Hospitals for Children. Decellularization perfusione è una tecnica innovativa per la produzione di ponteggi fegato intero che conserva la composizione della matrice extracellulare l'organo e la microarchitettura. Qui, il metodo di preparazione di ponteggi intero organo utilizzando decellularization perfusione e conseguente ripopolamento con epatociti è descritto. Trapianti di fegato funzionale e trapiantabili possono essere generati utilizzando questa tecnica.

Video: Decellularization and Recellularization of Whole Livers

Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

Perfusion-based whole organ decellularization has recently gained interest in the field of tissue engineering as a means to create site-specific extracellular matrix scaffolds, while largely preserving the native architecture of the scaffold. To date, this approach has been utilized in a variety of organ systems, including the heart, lung, and liver 1-5. Previous decellularization methods for tissues without an easily accessible vascular network have relied upon prolonged exposure of tissue to solutions of detergents, acids, or enzymatic treatments as a means to remove the cellular and nuclear components from the surrounding extracellular environment6-8. However, the effectiveness of these methods hinged upon the ability of the solutions to permeate the tissue via diffusion. In contrast, perfusion of organs through the natural vascular system effectively reduced the diffusion distance and facilitated transport of decellularization agents into the tissue and cellular components out of the tissue. Herein, we describe a method to fully decellularize an intact porcine heart through coronary retrograde perfusion. The protocol yielded a fully decellularized cardiac extracellular matrix (c-ECM) scaffold with the three-dimensional structure of the heart intact. Our method used a series of enzymes, detergents, and acids coupled with hypertonic and hypotonic rinses to aid in the lysis and removal of cells. The protocol used a Trypsin solution to detach cells from the matrix followed by Triton X-100 and sodium deoxycholate solutions to aid in removal of cellular material. The described protocol also uses perfusion speeds of greater than 2 L/min for extended periods of time. The high flow rate, coupled with solution changes allowed transport of agents to the tissue without contamination of cellular debris and ensured effective rinsing of the tissue. The described method removed all nuclear material from native porcine cardiac tissue, creating a site-specific cardiac ECM scaffold that can be used for a variety of applications.

A method to rapidly and completely remove cellular components from an intact porcine heart through retrograde perfusion is described. This method yields a site specific cardiac extracellular matrix scaffold which has the potential for use in multiple clinical applications.

Procedura per decellularizzazione del Cuore di suino da retrograda perfusione coronarica

Perfusion-based  whole organ decellularization has recently gained interest in the field of  tissue engineering as a means to create site-specific ...

Ricerca

Bioingegneria

A Decellularization Methodology for the Production of a Natural Acellular Intestinal Matrix

Successful tissue engineering involves the combination of scaffolds with appropriate cells in vitro or in vivo. Scaffolds may be synthetic, naturally-derived or derived from tissues/organs. The latter are obtained using a technique called decellularization. Decellularization may involve a combination of physical, chemical, and enzymatic methods. The goal of this technique is to remove all cellular traces whilst maintaining the macro- and micro-architecture of the original tissue.
Intestinal tissue engineering has thus far used relatively simple scaffolds that do not replicate the complex architecture of the native organ. The focus of this paper is to describe an efficient decellularization technique for rat small intestine. The isolation of the small intestine so as to ensure the maintenance of a vascular connection is described. The combination of chemical and enzymatic solutions to remove the cells whilst preserving the villus-crypt axis in the luminal aspect of the scaffold is also set out. Finally, assessment of produced scaffolds for appropriate characteristics is discussed.

The article describes a methodology for the production of an acellular matrix from rat intestine. The derivation of intestinal scaffolds is important for future applications in tissue engineering, stem cell biology and drug testing.

Un Decellularization Metodologia per la produzione di un naturale acellulare intestinale Matrix

Successful tissue engineering involves the  combination of scaffolds with appropriate cells in vitro or in vivo.  Scaffolds may be synthetic, ...

Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an attractive alternative to classic organ transplantation. This technology utilizes an organ&#39;s natural biological extracellular matrix (ECM) as a scaffold onto which autologous or stem/progenitor cells may be seeded and cultured in such a way that facilitates regeneration of the original tissue. The natural ECM is isolated by a process called decellularization. Decellularization is accomplished by treating tissues with a series of detergents, salts, and enzymes to achieve effective removal of cellular material while leaving the ECM intact. Studies conducted utilizing decellularization and subsequent recellularization of rodent lungs demonstrated marginal success in generating pulmonary-like tissue which is capable of gas exchange in vivo. While offering essential proof-of-concept, rodent models are not directly translatable to human use. Nonhuman primates (NHP) offer a more suitable model in which to investigate the use of bioartificial organ production for eventual clinical use.
The protocols for achieving complete decellularization of lungs acquired from the NHP rhesus macaque are presented. The resulting acellular lungs can be seeded with a variety of cells including mesenchymal stem cells and endothelial cells. The manuscript also describes the development of a bioreactor system in which cell-seeded macaque lungs can be cultured under conditions of mechanical stretch and strain provided by negative pressure ventilation as well as pulsatile perfusion through the vasculature; these forces are known to direct differentiation along pulmonary and endothelial lineages, respectively. Representative results of decellularization and cell seeding are provided.

Whole-organ decellularization produces natural biological scaffolds that may be used for regenerative medicine. The description of a nonhuman primate model of lung regeneration in which whole lungs are decellularized and then seeded with adult stem cells and endothelial cells in a bioreactor that facilitates vascular circulation and liquid media ventilation is presented.

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an ...

Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher neutrophil counts in the blood are usually an indicator of ongoing infections, while low neutrophil counts are a warning sign for higher risks for infections. To accomplish their functions, neutrophils also have to be able to move effectively from the blood where they spend most of their life, into tissues, where infections occur. Consequently, any defects in the ability of neutrophils to migrate can increase the risks for infections, even when neutrophils are present in appropriate numbers in the blood. However, measuring neutrophil migration ability in the clinic is a challenging task, which is time consuming, requires large volume of blood, and expert knowledge. To address these limitations, we designed a robust microfluidic assays for neutrophil migration, which requires a single droplet of unprocessed blood, circumvents the need for neutrophil separation, and is easy to quantify on a simple microscope. In this assay, neutrophils migrate directly from the blood droplet, through small channels, towards the source of chemoattractant. To prevent the granular flow of red blood cells through the same channels, we implemented mechanical filters with right angle turns that selectively block the advance of red blood cells. We validated the assay by comparing neutrophil migration from blood droplets collected from finger prick and venous blood. We also compared these whole blood (WB) sources with neutrophil migration from samples of purified neutrophils and found consistent speed and directionality between the three sources. This microfluidic platform will enable the study of human neutrophil migration in the clinic and the research setting to help advance our understanding of neutrophil functions in health and disease.

This protocol details an assay designed to measure human neutrophil chemotaxis from one droplet of whole blood with robust reproducibility. This approach circumvents the need for neutrophil separation and requires only a few minutes of assay preparation time. The microfluidic chip enables the repeated measure of neutrophil chemotaxis over time in infants or small mammals, where sample volume is limited.

Piattaforma microfluidica per la misurazione dei neutrofili Chemiotassi dal Lordo Sangue intero

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher ...

Impact Assessment of Repeated Exposure of Organotypic 3D Bronchial and Nasal Tissue Culture Models to Whole Cigarette Smoke

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease or lung cancer. To understand the underlying mechanisms of disease development, it would be important to examine the impact of CS exposure directly on lung tissues. However, this approach is difficult to implement in epidemiological studies because lung tissue sampling is complex and invasive. Alternatively, tissue culture models can facilitate the assessment of exposure impacts on the lung tissue. Submerged 2D cell cultures, such as normal human bronchial epithelial (NHBE) cell cultures, have traditionally been used for this purpose. However, they cannot be exposed directly to smoke in a similar manner to the in vivo exposure situation. Recently developed 3D tissue culture models better reflect the in vivo situation because they can be cultured at the air-liquid interface (ALI). Their basal sides are immersed in the culture medium; whereas, their apical sides are exposed to air. Moreover, organotypic tissue cultures that contain different type of cells, better represent the physiology of the tissue in vivo. In this work, the utilization of an in vitro exposure system to expose human organotypic bronchial and nasal tissue models to mainstream CS is demonstrated. Ciliary beating frequency and the activity of cytochrome P450s (CYP) 1A1/1B1 were measured to assess functional impacts of CS on the tissues. Furthermore, to examine CS-induced alterations at the molecular level, gene expression profiles were generated from the tissues following exposure. A slight increase in CYP1A1/1B1 activity was observed in CS-exposed tissues compared with air-exposed tissues. A network-and transcriptomics-based systems biology approach was sufficiently robust to demonstrate CS-induced alterations of xenobiotic metabolism that were similar to those observed in the bronchial and nasal epithelial cells obtained from smokers.

The aim of this protocol is to expose human organotypic 3D bronchial and nasal tissue models to mainstream cigarette smoke (CS) at the air-liquid interface. The impact of CS on the tissues is then investigated using a cytochrome P450 activity assay, a cilia beating measurement, and a systems biology approach.

Valutazione dell&#39;impatto di esposizione ripetuta di organotipica 3D bronchiali e nasali coltura tissutale modelli di Whole fumo di sigaretta

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease ...

Procedure for Decellularization of Rat Livers in an Oscillating-pressure Perfusion Device

Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent liver decellularization have already been published. We aimed to improve the decellularization process by construction of a proprietary perfusion device enabling selective perfusion via the portal vein and/or the hepatic artery. Furthermore, we sought to perform perfusion under oscillating surrounding pressure conditions to improve the homogeneity of decellularization. The homogeneity of perfusion decellularization has been an underestimated factor to date. During decellularization, areas within the organ that are poorly perfused may still contain cells, whereas the extracellular matrix (ECM) in well-perfused areas may already be affected by alkaline detergents. Oscillating pressure changes can mimic the intraabdominal pressure changes that occur during respiration to optimize microperfusion inside the liver. In the study presented here, decellularized rat liver matrices were analyzed by histological staining, DNA content analysis and corrosion casting. Perfusion via the hepatic artery showed more homogenous results than portal venous perfusion did. The application of oscillating pressure conditions improved the effectiveness of perfusion decellularization. Livers perfused via the hepatic artery and under oscillating pressure conditions showed the best results. The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

Procedura per Decellularization di Rat Livers in un dispositivo di perfusione oscillante pressione

Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent ...

Real-time Monitoring of High Intensity Focused Ultrasound (HIFU) Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound (HMIFU)

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An oscillatory motion is generated at the focus of a 93-element and 4.5 MHz center frequency HIFU transducer by applying a 25 Hz amplitude-modulated signal using a function generator. A 64-element and 2.5 MHz imaging transducer with 68kPa peak pressure is confocally placed at the center of the HIFU transducer to acquire the radio-frequency (RF) channel data. In this protocol, real-time monitoring of thermal ablation using HIFU with an acoustic power of 7 W on canine livers in vitro is described. HIFU treatment is applied on the tissue during 2 min and the ablated region is imaged in real-time using diverging or plane wave imaging up to 1,000 frames/second. The matrix of RF channel data is multiplied by a sparse matrix for image reconstruction. The reconstructed field of view is of 90&#176; for diverging wave and 20 mm for plane wave imaging and the data are sampled at 80 MHz. The reconstruction is performed on a Graphical Processing Unit (GPU) in order to image in real-time at a 4.5 display frame rate. 1-D normalized cross-correlation of the reconstructed RF data is used to estimate axial displacements in the focal region. The magnitude of the peak-to-peak displacement at the focal depth decreases during the thermal ablation which denotes stiffening of the tissue due to the formation of a lesion. The displacement signal-to-noise ratio (SNRd) at the focal area for plane wave was 1.4 times higher than for diverging wave showing that plane wave imaging appears to produce better displacement maps quality for HMIFU than diverging wave imaging.

Real-time Monitoring of High Intensity Focused Ultrasound HIFU Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound HMIFU

This article describes real-time monitoring of HIFU ablation in canine liver with high frame rate ultrasound imaging using diverging and plane wave imaging. Harmonic Motion Imaging for Focused Ultrasound is used to image the decrease of acoustic radiation force induced displacement in the ablated region.

Monitoraggio in tempo reale di alta intensità ultrasuoni focalizzati (HIFU) Ablazione<em&gt; In Vitro</em&gt; Canine Livers Uso Harmonic Imaging Movimento per Focused Ultrasound (HMIFU)

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An ...

Comparable Decellularization of Fetal and Adult Cardiac Tissue Explants as 3D-like Platforms for In Vitro Studies

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components are presented as a surface coating. These culture systems constitute a simplification of the complex nature of the tissue ECM that encompasses biochemical composition, structure, and mechanical properties. To better emulate the ECM-cell communication shaping the cardiac microenvironment, we developed a protocol that allows for the decellularization of the whole fetal heart and adult left ventricle tissue explants simultaneously for comparative studies. The protocol combines the use of a hypotonic buffer, a detergent of anionic surfactant properties, and DNase treatment without any requirement for specialized skills or equipment. The application of the same decellularization strategy across tissue samples from subjects of various age is an alternative approach to perform comparative studies. The present protocol allows the identification of unique structural differences across fetal and adult cardiac ECM mesh and biological cellular responses. Furthermore, the herein methodology demonstrates a broader application being successfully applied in other tissues and species with minor adjustments, such as in human intestine biopsies and mouse lung.

The cardiac extracellular matrix (ECM) is a complex network of molecules that orchestrate key processes in tissues and organs while enduring physiological remodeling throughout life. Standardized decellularization of fetal and adult hearts permits comparative experimental studies of both tissues in a 3D context by capturing native architecture and biomechanical properties.

Paragonabile decellularizzazione degli espianti di tessuto cardiaco fetali ed adulti come 3D-come le piattaforme per studi In Vitro

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowing the user to select the protocol which better suits their intended application.

Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

Due metodi per decellularizzazione di tessuti vegetali per applicazioni di ingegneria del tessuto

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...

Decellularization and Recellularization Methodology for Human Saphenous Veins

Vascular conduits used during most vascular surgeries are allogeneic or synthetic grafts that often lead to complications caused by immunosuppression and poor patency. Tissue engineering offers a novel solution to generate personalized grafts with a natural extracellular matrix containing the recipient&#39;s cells using the method of decellularization and recellularization. We show a detailed method for performing decellularization of the human saphenous vein and recellularization by perfusion of peripheral blood. The vein was decellularized by perfusing 1% Triton X-100, 1% tri-n-butyl-phosphate (TnBP) and 2,000 Kunitz units of deoxyribonuclease (DNase). Triton X-100 and TnBP were perfused at 35 mL/min for 4 h while DNase was perfused at 10 mL/min at 37 &#176;C for 4 h. The vein was washed in ultrapure water and PBS and then sterilized in 0.1% peracetic acid. It was washed again in PBS and preconditioned in endothelial medium. The vein was connected to a bioreactor and perfused with endothelial medium containing 50 IU/mL heparin for 1 h. Recellularization was performed by filling the bioreactor with fresh blood, diluted 1:1 in Steen solution, and adding endocrine gland-derived vascular endothelial growth factors (80 ng/mL), basic fibroblast growth factors (4 &#181;L/mL), and acetyl salicylic acid (5 &#181;g/mL). The bioreactor was then moved into an incubator and perfused for 48 h at 2 mL/min while maintaining glucose between 3 - 9 mmol/L. Later, the vein was washed with PBS, filled with endothelial medium and perfused for 96 h in the incubator. Treatment with Triton X-100, TnBP and DNase decellularized the saphenous vein in 5 cycles. The decellularized vein looked white in contrast to normal and recellularized veins (light red). The hematoxylin &#38; eosin (H&#38;E) staining showed the presence of nuclei only in normal but not in decellularized veins. In the recellularized vein, H&#38;E-staining showed the presence of cells on the luminal surface of the vein.

Here, we describe a protocol for the saphenous vein decellularization using detergents and recellularization by perfusion of the peripheral blood and the endothelial medium.

Decellularizzazione e metodologia di ricellularizzazione per le vene safena umane

Vascular conduits used during most vascular surgeries are allogeneic or synthetic grafts that often lead to complications caused by immunosuppression and ...

Decellularization e Recellularization di fegati interi

Riepilogo

Esplora Altri Video

Capitoli in questo video