Die Autoren bedanken sich bei Jack Milwid für die Gestaltung der in vitro Perfusionskammer danken. Diese Arbeit wurde durch Zuschüsse der US NIH, R01DK59766 und R01DK084053 zu MY, R00DK080942 zu KU, US NSF CBET-0853569 zu KU und die Shriners Hospitals for Children zu BEU (Grant No. 8503) unterstützt. Wir danken für die Unterstützung und die Shriners Hospitals for Children.

1. Liver Dezellularisierung <ol><li> Ernte einer Rattenleber mit Pfortader Kanülierung mit und 18-Gauge-Katheter. Lassen Sie die untere und obere Hohlvene zu öffnen. Halten Sie die Orgel hydratisiert in Phosphat-gepufferter Kochsalzlösung (PBS) in einer 10-cm-Petrischale. </li><li> Einrichten einer Perfusion System, das von 8-Liter-Behälter, Schlauchpumpe und einer Blasenfalle besteht. </li><li> Füllen Sie die Perfusion mit Phosphat-gepufferter Kochsalzlösung und halten Sie es läuft für 10 Minuten. Füllen PBS in einem 10-cm-Petrischale und reduzieren die Fließgeschwindigkeit des Phosphat-gepufferter Kochsalzlösung zu 1 mL / min. </li><li> Sorgfältig Übertragung der geernteten Leber zu den Phosphat-gepufferter Kochsalzlösung gefüllt 10-cm-Petrischale. </li><li> Fahren Sie mit PBS Perfusion über Nacht. </li><li> Starten Perfusion mit 0,01% (w / v) Natriumdodecylsulfat (SDS) in destilliertem Wasser für 5 Minuten. </li><li> Perfundieren mit PBS für 1 Stunde. </li><li> Wiederholen Sie die Schritte 1,6 und 1,7 noch dreimal die Erhöhung der SDS Perfusion auf 10, 15 und 20 Minuten zu jeder Zeit. </li><li> Weiter Perfusion mit 0,01% (w / v) SDS für 24 Stunden. </li><li> Weiter Perfusion mit 0,1% (w / v) SDS für 24 Stunden. </li><li> Perfundieren mit 0,2% (w / v) SDS für 3 Stunden. </li><li> Perfundieren mit 0,5% (w / v) SDS für 3 Stunden. </li><li> Perfundieren mit destilliertem Wasser für 15 Minuten. </li><li> Perfundieren mit 1% (w / v) Triton X-100 in destilliertem Wasser für 30 Minuten keine gebundenen Nukleinsäuren zu entfernen. </li><li> Zum Waschen der dezellularisierte Leber-Matrix (DLM), perfuse mit PBS für 2 Stunden. </li><li> Optional: reseziert allen Lappen mit Ausnahme der Mittellappen. </li><li> Bewahren Sie die DLM in einer sauberen und verschlossenen Petrischale in PBS bei 4 o C eingeweicht, bis sie verwendet (Abbildung 1). </li><li> Um sterilisieren DLM: 1) Spülen Sie die DLM mit sterilem PBS mit 0,1% (v / v) Peressigsäure und 4% (v / v) Ethanol und Inkubation für 3 Stunden bei 4 o C. 2) mit sterilem PBS 2 mal waschen. 3) mit sterilem PBS mit 2% Penicillin-Streptomycin, 10ug / mL Gentamicin und 2,5 ug / ml Amphotericin B. Bewahren Sie die dezellularisierte Leber in der gleichen Lösung bei 4 o C bis bereit, für recellularization Experimenten Wash. </li></ol> 2. Recellularization von dezellularisierte Leber Matrix <ol><li> Einrichten einer Perfusion System, das von einer Perfusionskammer, Schlauchpumpe und einer Blasenfalle unter sterilen Bedingungen besteht. Füllen Sie die Perfusion mit 200 ml Kulturmedium, z. B. hohe Glukose-DMEM (Sigma), 10% fötalem Rinderserum (Hyclone,), 100 U mL -1 Penicillin und 100 ug mL -1 Streptomycin (Invitrogen). </li><li> Legen Sie die dezellularisierte Leber-Matrix in der Perfusionskammer und verbinden Sie die DLM die Perfusion System durch die Pfortader Kanüle, während die Pumpe mit 5 ml / min läuft, um die Bildung von Luftblasen zu vermeiden. </li><li> Lassen Perfusion des Mediums durch die DLM für 30 Minuten. </li><li> Isolieren primären Hepatozyten aus einer erwachsenen Ratte mit mindestens 90% Lebensfähigkeit. </li><li> Stoppen Sie die Strömung in der Perfusion und injizieren Sie langsam 50 Millionen Hepatozyten (in 1-3 ml Kulturmedium) in Perfusionssystem durch die Blasenfalle. </li><li> Starten Sie den Durchfluss bei 10 mL / min und rezirkulieren das Medium für 10 Minuten. </li><li> Wiederholen Sie die Schritte 2.5 und 2.6 bis insgesamt 200 Millionen Zellen in die DLM (Abbildung 2) 6 eingeführt werden. </li><li> Wenn alle Zellen in die DLM perfundiert werden, sammeln das Perfusat in vier 50-ml-Zentrifugenröhrchen und Zentrifuge bei 600 rpm für 10 Minuten. Entsorgen Sie die Überstände und sammeln Sie die Pellets in einem einzigen Rohr. Bestimmen Sie die Anzahl der Zellen und die Lebensfähigkeit über Trypanblau-Ausschluss für die Aussaat Effizienz zu bestimmen. </li></ol> 3. In-vitro-Kultur der Recellularized Leber Graft <ol><li> Einrichten einer Perfusion System, das von einer Perfusionskammer, Schlauchpumpe, Oxygenator und eine Blasenfalle unter sterilen Bedingungen (Abbildung 3) besteht. Füllen Sie die Perfusion mit 50 ml Kulturmedium, z. B. Williams &#39;E (Sigma), 5% fötalem Rinderserum (Hyclone,), 0,5 U mL -1 Insulin (Eli Lilly), 20 ng mL -1 EGF (Invitrogen) , 14 ng mL -1 Glukagon (Bedford Laboratories), 7,5 pg mL -1 Hydrocortison (Pharmacia), 100 U mL -1 Penicillin und 100 ug mL -1 Streptomycin (Invitrogen). </li><li> Legen Sie die recellularized Lebertransplantation in der Perfusionskammer und verbinden Sie die DLM die Perfusion System durch die Pfortader Kanüle, während die Pumpe mit 5 ml / min läuft, um die Bildung von Luftblasen zu vermeiden. </li><li> Aseptischen Bedingungen in der Nähe der Perfusionskammer und dicht schließen, um ein Auslaufen während der Kultur zu vermeiden. </li><li> Übertragen Sie die Perfusion System in einen Inkubator, dass 37 o C ist und 10% CO 2 und erhöhen die Perfusionsflussrate bis 15 mL / min. </li><li> Verbinden Sie den Oxygenator zu einem 95% O 2 und 5% CO 2-Gasgemisch Tank und stellen Sie den Gas-Durchfluss bis 0,5 Liter / min. Dies sollte erreicht eine Sauerstoff-Partialdruck von NaherungTely 400 mmHg. </li><li> Die Kultur kann bis zu 10 Tagen mit täglich wechselnden Kulturmedium weiter. Das Kulturmedium kann täglich zur Überwachung der Leberfunktion des Transplantats, wie Albumin, Harnstoff und insgesamt Gallensäure Sekretion abgetastet werden. Am Ende der Kultur Frist kann der recellularized Lebertransplantation für molekulare und histologische Analyse beprobt werden. </li></ol> 4. Repräsentative Ergebnisse: Die komplette Dezellularisierung einer Rattenleber dauert etwa 72 Stunden mit dem beschriebenen Protokoll. Die resultierende Matrix behält 100% der fibrillären Kollagen, 50% der Glykosaminoglykane und nur 5% der DNA des nativen Leber (Tabelle 1) 6. Die vaskuläre Struktur der Matrix bleibt erhalten, wie durch Korrosion Gieß-und Rasterelektronenmikroskopie Analyse (Abbildung 4) 6 belegt. Die Anwesenheit des vaskulären Mikroarchitektur innerhalb der DLM erleichtert die Wiederbesiedlung mit Zellen mit einem Wirkungsgrad von 96% und die anschließende Perfusion in vitro-Kultur. Die recellularized Leber Transplantat kann bis zu 10 Tage in vitro kultiviert werden und zeigt geeignete Leber funktioniert als über Albumin, Harnstoff und insgesamt Gallensäure Sekretion (Abbildung 5) 6 bestätigt. <img src="/files/ftp_upload/2394/2394fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Dezellularisierte Leber-Matrix am Ende des Dezellularisierung Prozess. (A) die ganze Leber (b) der Mittellappen nach Resektion. <img src="/files/ftp_upload/2394/2394fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Schematische Darstellung der recellularization der DLM. <img src="/files/ftp_upload/2394/2394fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Perfusion System-Setup für In-vitro-Kultur der recellularized Lebertransplantation. <img src="/files/ftp_upload/2394/2394fig4.jpg" alt="Abbildung 4" /> Abbildung 4. Die mikrovaskuläre Struktur wird in der Leber dezellularisierte Matrix beibehalten. Ausgusspräparat Bilder aus a) einer normalen Leber b) eine dezellularisierte Leber-, Portal-(rot) und venösen (blau) Gefäßsystem. Rasterelektronenmikroskopie Bilder des DLM c) ein Gefäß, d) ein Abschnitt mit Gallengang-like kleinen Gefäße (Pfeile), Scale Bars (a, b) 5 mm (c, d) 20 um. <img src="/files/ftp_upload/2394/2394fig5.jpg" alt="Abbildung 5" /> Abbildung 5. Liver spezifischen Funktionen des recellularized Lebertransplantation während der in vitro Perfusion Kultur. a) Albumin-Sekretion (p = 0,5249), b) zur Herstellung von Harnstoff (p = 0,5271) und c) insgesamt Gallensäure-Sekretion (p = 0.0114). Die statistische Analyse der Differenz zwischen dem Experiment und der Kontrolle wurde über die 10 d Kultur Zeitraum von Friedman-Test bei a = 0,01 durchgeführt. Fehlerbalken stellen sem (n = 3). <table border="1"><tbody><tr><td></td><td> Frische Livera </td><td> Dezellularisierte Leber Matrix ein </td><td rowspan="2"> p-Werte </td><td rowspan="2"> % Der frischen Leber </td></tr><tr><td></td><td> n = 4 </td><td> n = 8 </td></tr><tr><td> Collagen </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 pro g Leber) </td></tr><tr><td> Glykosaminoglykane </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 pro g Leber) </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 pro g Leber) </td></tr></tbody></table> Tabelle 1. Die biochemische Zusammensetzung der dezellularisierte Leber-Matrix auf die native Leber verglichen. a Werte werden als Mittelwert ± SEM dargestellt

<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>

Die Perfusion Dezellularisierung hier beschriebene Methode produziert eine ganze Leber Gerüst, das die gleiche grobe Struktur und die vaskuläre Mikroarchitektur des nativen Leber hat. Das Gerüst hat eine extrazelluläre Matrix Zusammensetzung ähnlich der nativen Leber. Die recellularization Methode erreicht Wiederbesiedlung des Gerüsts mit Zellen mit hohem Wirkungsgrad und die Zellen lebensfähig bleiben und funktionelle während der in vitro-Kultur Zeitraum getestet. Mit dem Zusatz von nonparenchymal Zellen in die Leber recellularized Transplantat, planen wir, dass die recellularized Lebertransplantation als Plattform für die verschiedenen Funktionen der Leber, wie Arzneimittel-Metabolismus, Regeneration und Entwicklung Studie verwendet werden. Darüber hinaus kann der recellularized Transplantat zu finden klinische Anwendung, denn es kann möglicherweise durch den Anschluss an den Blutkreislauf des Empfängers Tier dank der Anwesenheit von vaskulären Strukturen in das Transplantat 6 transplantiert werden.

A correction was made to <a href="https://www-jove-com.remotexs.ntu.edu.sg/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

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decellularization and recellularization of whole livers

Die Leber ist ein komplexes Organ, das konstante Perfusion erfordert für die Lieferung von Sauerstoff und Nährstoffen und Beseitigung von Abfällen, um 1 überleben. Die Bemühungen zu erstellen oder zu imitieren die Leber Mikrostruktur mit Gründen up-Ansatz mit Tissue Engineering und Mikrofabrikationstechniken sind nicht erfolgreich gewesen, so weit durch, um diesen Entwurf Herausforderung. Darüber hinaus haben synthetische Biomaterialien verwendet werden, um Gerüste für Leber Tissue Engineering-Anwendungen zu erstellen bei der Induktion der Geweberegeneration und Reparatur zu einem großen Teil durch das Fehlen von spezifischen Zell-Bindung Motive, die die richtige Zelle Funktionen 2 induzieren wäre begrenzt. Dezellularisierte nativen Gewebe wie Blutgefäße 3 und Haut 4 auf der anderen Seite haben viele Anwendungen im Tissue Engineering gefunden, und sofern eine praktische Lösung für einige der Herausforderungen. Der Vorteil der dezellularisierte nativen Matrix ist, dass es, bewahrt in einem Umfang, der ursprünglichen Zusammensetzung und die Mikrostruktur, also die Verbesserung Zelladhäsion und Reorganisation 5. In dieser Arbeit beschreiben wir die Methoden zur Perfusion-Dezellularisierung der Leber, so dass eine intakte Leber biologisches Gerüst, dass die Struktur der großen Blutgefäße behält erhalten ist durchzuführen. Des Weiteren beschreiben wir Methoden, um diese bioscaffolds mit adulten primären Hepatozyten recellularize, die Schaffung eines Lebertransplantation, die funktionelle in vitro ist, und hat das Schiff den Zugang erforderlich für die Transplantation in vivo.

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

Decellularization and Recellularization of Whole Livers

Perfusion Dezellularisierung ist eine neuartige Technik, um ganze Leber Gerüste, dass die Orgel extrazellulären Matrix Zusammensetzung und Mikroarchitektur behält produzieren. Hierbei wird das Verfahren zur Herstellung von ganzen Organs Gerüste mit Perfusion Dezellularisierung und anschließende Wiederaufforstung mit Hepatozyten beschrieben. Funktionelle und transplantierbaren Leber Transplantate kann mit Hilfe dieser Technik werden.

Dezellularisierung und Recellularization von Whole Livers

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

decellularization-and-recellularization-of-whole-livers

Nanyang Technological University

Research

JoVE Journal

Bioengineering

21.9K Aufrufe.  Massachusetts General Hospital, Harvard Medical School, Shriners Hospitals for Children. Perfusion Dezellularisierung ist eine neuartige Technik, um ganze Leber Gerüste, dass die Orgel extrazellulären Matrix Zusammensetzung und Mikroarchitektur behält produzieren. Hierbei wird das Verfahren zur Herstellung von ganzen Organs Gerüste mit Perfusion Dezellularisierung und anschließende Wiederaufforstung mit Hepatozyten beschrieben. Funktionelle und transplantierbaren Leber Transplantate kann mit Hilfe dieser Technik werden.

Serie: 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.

Verfahren für Dezellularisierung der Porcine Heart von 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 ...

Forschung

Biotechnik

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.

Ein Dezellularisierung Methodik für die Herstellung von Naturdarm Azelluläre 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.

Mikrofluidik-Plattform zur Messung der Neutrophilen-Chemotaxis von Rohboden Vollblut

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.

Folgenabschätzung Wiederholte Exposition von organotypischen 3D bronchiale und nasale Zellkulturmodelle zu Whole Zigarettenrauch

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.

Verfahren für Dezellularisierung von Rattenlebern in einer oszillierenden Druck Perfusionsvorrichtung

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.

Echtzeit-Überwachung von hochintensiven fokussierten Ultraschall (HIFU) Ablation<em&gt; In-vitro-</em&gt; Canine Livers Verwendung harmonische Bewegung Imaging für fokussierten Ultraschall (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.

Vergleichbare Decellularization von fetalen und adulten Herzgewebe Explantaten als 3D-ähnliche Plattformen für In-vitro-Studien

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.

Zwei Methoden zur Decellularization von Pflanzengewebe für Tissue-Engineering-Anwendungen

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.

Decellularization und Recellularization Methoden für menschliche Saphenous Adern

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