Yazarlar Brogan Konuk, Michelle Weaver, ve Kristen Lippert kabul etmek istiyorum. Bu çalışma için Fon NIH Grant R03EB009237 yanı sıra, National Biomedical Görüntüleme ve Biyomühendislik Enstitüsü T32HL076124-05 NIH Eğitimi Teşvik T32EB001026-06 ile sağlanmıştır.

1. Doku Hazırlanması ve Deney Kurulumu <ol><li> Hemen bir mezbaha veya araştırma tesisi ötanazi sonra ve aşırı kan durulayın Hasat domuz organdır. Atrium ve aorta sağlam tutmak, aşırı yağ ve doku kalp kesin. Uzaklıkta aortadan pulmoner arter ayırmak için yağ kesin. Dokusunda herhangi bir kesik varsa, uygun atın. </li><li> Dondurucu kağıt ayrı ayrı her kalp sarın ve tam donma sağlamak için en az 24 saat için -80 ° C derin dondurucuda tüm doku saklayın. </li><li> Zaman kullanımı (genellikle 3 aydan daha az), gecede 4 ° C&#39;de 4 L beher batık Tip 1 su çözülme biri sağlam dondurulmuş domuz kalbi hazır </li><li> Kalbi tamamen çözülmüş sonra, kalp kurulayın kalp tartmak ve ağırlığı kaydetmek. Bir pazar kilo domuz kalbi yaklaşık 375-450 g tartmak gerekir. </li><li> Bir dikenli redüktör ¼ &quot;sonuna kadar boyutu 18 Masterflex hortumu bağlayın. Dikenli redüktör yerleştirin veaort içine hortum. Sadece brakiyosefalik gövde altındaki Sıra 2 hortum kelepçeleri veya aort etrafında güvenli zip bağları,. Redüktör ve hortum aort kapak üzerinde kalmalıdır, koroner arterler (Şekil 1) perfüze böylece. </li><li> Tip I su boruları doldurmak için 30 ya da 60 ml şırınga kullanın. Onun yaklaşık orta noktasında bir Masterflex silindir pompa kartuş içinde tüp yerleştirin. Akış 2.5 L su ile doldurulmuş bir 4 L ölçeğinin alt boru ucu ve boru güvenli daldırmak. </li><li> Su ile doldurulmuş behere kalp ve hava kabarcıklarını çıkarmak için pompayı yerleştirin. Kabarcıklar tüp eklenen aort gelen gözlenirse, aort ilave bağları ile yerleştirilmesi veya temin edilmesi gerekebilir. Hava geçirmez bir mühür decellularization işlemi (Şekil 2) sırasında yeterli basınç sağlamak için önemlidir. </li><li> % 0.2 Trypsin/0.05% EDTA/0.05% NaN 3 L içeren 4 L kabı yerleştirinPlaka karıştırın ve 37 ısıtın üzerine ° C decellularization sürecinin hazırlanmasında 3 çözüm. </li></ol> 2. Doku durulamak <ol><li> Doğru boru boyutu seçilir sağlanması, 400 ml / dak &#39;lık bir akış oranı ile pompa ayarlayın. 15-25 dk Ben su yazın ile kalp yıkayın. Pompa çalışmaya başladığında, kalp şişer ve karıncıklar kan dökmek gerekir. Taze çözelti her 5-10 dakikada ikame edilmiş, ya da kalp kaldırılır kan miktarına dayalı olarak gerek yoktur. Kan kalp effused ise, gerekli olduğu gibi tüp ve kelepçeler ayarlayın. </li><li> Pompa durdurun ve 2X Fosfat dolu ayrı bir behere kalp aktarmak Salin (PBS) Tamponlanmış. Boru çözelti içine daldırılmış sonra, pompa ve 700 ml / dk &#39;ya kadar akış oranını artırmaktadır. Kalp çözümü her 5 dk değişen, 15 dakika için çözüm kalmalıdır. Her çözüm değişim doku ve boru hareket ederken geçici olarak kesilmelidir pompa gerektirirYeni behere. </li><li> 10 dakika boyunca bir su tipi ve 750 ml / dakika akış hızı ile artmaya kalp aktarın. </li></ol> 3. Decellularization ve Çözüm Perfüzyon <ol><li> 37 NaN 3 0.2% Trypsin/0.05% EDTA/0.05% içeren behere kalp aktarın ° C. 1.200 ml / dk pompa hızını artırın ve pompa başlar. Beher içinde çözelti sirküle beher altına yerleştirilmiş bir karıştırma çubuğu kullanın. Kalp üç saat olmak üzere toplam 37 ° C&#39;de% 0.2 Trypsin/0.05% EDTA/0.05% NaN 3 çözüm kalmalıdır. 1 saat sonra, 1.500 ml / dk pompa hızını artırın. Ek bir saat sonra, 1.800 ml / dk pompa hızını artırın. Doku yavaş koşulu doku artmış perfüzyon hızları tabi ve damarların yırtılması önlemek edilir. Kalp protokolün bu adımı sırasında boyutu şişer ve neredeyse çift olacaktır. Doku inci boyunca atriyumlar apekse ilerliyor, doğal rengini kaybedere protokolü (Şekil 3). </li><li> Her çözüm perfüzyon sonra durulama iki adım hücresel enkaz, kimyasal kalıntı ve yardım hücre lizis kaldırmak için yapılır. Her durulama bir 10 dakika oluşan bir 10 dakika, ardından oda sıcaklığında su 2X PBS çözeltisi ile durulama Tip durulama. Her bir yıkama durulama çözüm, ekleme ve batık kalp içeren deney şişesi içinde dolaşan perfüzat, ilk kaptan çıkarılmasını çözelti oluşur. Ardından% 0.2 Trypsin/0.05% EDTA/0.05% NaN3 çözeltisi ardından, perfuse 1,900 ml / dakika, su ve 1950 ml / dk &#39;da PBS 2X serpmek. </li><li> % 3 oda sıcaklığında, Triton X-100/0.05% EDTA/0.05% NaN3 ihtiva eden bir çözeltiye kalp aktarın. 2.000 ml / bir saat dakika ve perfuse çözüm pompa hızını artırın. Kaptan çözüm çıkarın ve taze solüsyon ile değiştirin, 2100 ml / dk pompa hızını artırmak, ve ek bir buçuk saat için taze solüsyon serpmek, toplam zamanı getirerek3% Triton X-100/0.05% EDTA/0.05% NaN 3 2.5 hr. </li><li> 2180 ml / 10 dk için her bir dakikada 2150 ml / dakika ve 2x PBS de Tip I su içinde doku durulayın. </li><li> Oda sıcaklığında, bir% 4 Sodyum deoksikolatın çözeltisine kalp aktarın. 2.200 ml / dk ve 3 saat süreyle perfuse çözüm pompa hızını artırın. </li><li> De Tip I su içinde doku durulayın ve 2x PBS 2.200 her bir çözelti için 5-10 dakika sonra çözüm değişen 15 dakika her biri için, ml / dakika. Açıklanan perfüzyon adımları iki kez durulayın adımı gerçekleştirdikten ve 4 ° C gecede ekli boru ile kalp depolayarak birden fazla gün içinde bölünmüş ve Tip I suya sokulmasına olabilir. </li><li> Ertesi gün, 5 dakika ml / dk 1,500 1X PBS ile durulayın, 5 dakika, ardından 750 ml / dk hızla Tip I su ile durulama gerçekleştirmek. Bu protokol daha sonra uygun bir solüsyon içinde tarif akış hızında devam edilebilir. </li></ol> 4. Dezenfeksiyon ve Nihai İşleme <ol><li> % 0.1 &#39;lik perac için kalp aktarınetic asit / 2,200 ml / dk &#39;da% 4 etanol ve çözelti 1.5 saat için çözelti perfuse. </li><li> Doku için son çalkalamalar hepsi 2,200 ml / dk &#39;da gerçekleştirilir. Tip I su içinde iki yıkamadan 5 dakika, ardından 15 dk için 1X PBS ile doku serpmek. Durulama Bu bir dizi çözüm perfüzyon işlemi tamamlamak için bir kez daha tekrar edilir. </li><li> Pompayı kapatın ve kalp boşaltmak için çözüm kalp çıkarın. Aortadan bağları kes, tüm tüp kaldırın ve 1 saat boyunca boşaltmak için boş bir behere kalp yerleştirin. Aşırı sıvı periyodik tahliye edilmesi gerekir. Tam kalp (Şekil 4) boşaltmak için bir emici ped üzerine kalp yatırın. </li><li> Su en kaldırıldıktan sonra, kardiyak hücre dışı matris (C-ECM) ağırlığını kaydedin. Kalp decellularization işlemi sırasında iç ağırlığının yaklaşık olarak% 20-25 kaybetmeye beklenebilir. </li><li> Sağ ve sol ventrikül, hem de DNA quantificati için ventriküler septum teşrihve histolojik işlem sırasında doku tam decellularization (Şekil 5) teyit etmek için. </li><li> Liyofilizasyon önce en az 2 saat süreyle -80 ° C&#39;de C-ECM dondurun. </li></ol>

<ol>
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Gilbert çalışma yapılmakta idi ise ACell, Inc Bilimsel Danışma Kurulu, ve son zamanlarda Araştırma ve Geliştirme Başkan Yardımcısı oldu. ACell, Inc mesane matrisi satıyor ve bu çalışmada hiçbir ticari çıkarı vardır.

Çalışmada domuz kalp tutarlı ve verimli decellularization için metodoloji açıklanmalıdır. Protokol, daha önce yayımlanan bir raporunda, 1 bir değişiklik oldu ve daha fazla tekrarlanabilir sonuçlar elde akışına uzun pozlama ve artan basınç, dahil. Çıkan decellularized doku doku 2 başarılı decellularization için yayınlanmış tüm ölçütleri bir araya geldi. Sık çözüm değişikliklerin doku hücresel materyalin yeniden yerleştirilmesi sınırlamak için yapılmıştı...

<table border="1"><tbody><tr><td> Reaktif / Malzeme Adı </td><td> Şirket </td><td> Katalog Numarası </td><td> Yorumlar </td></tr><tr><td> Tripsin </td><td> Gibco </td><td> 15090 </td><td></td></tr><tr><td> EDTA </td><td> Balıkçı </td><td> BP120-500 </td><td></td></tr><tr><td> NaN 3 </td><td> Sigma </td><td> S2002-500G </td><td></td></tr><tr><td> Triton X-100 </td><td> Sigma </td><td> X100-1L </td><td></td></tr><tr><td> 10X PBS </td><td> Balıkçı </td><td> BP399-20 </td><td></td></tr><tr><td> Sodyum deoksikolatın </td><td> Sigma </td><td> D6750-500G </td><td></td></tr><tr><td> Perasetik Asit </td><td> Pfaltz ve Bauer </td><td> P05020 </td><td> 35% CAS # 79-21-0 </td></tr><tr><td> Etanol </td><td> Pharmco </td><td> 111000200 </td><td></td&gt; </tr><tr><td> Masterflex Pompa Tahrik </td><td> Cole Parmer </td><td> SI-07524-50 </td><td></td></tr><tr><td> Masterflex Boru </td><td> Cole Parmer </td><td> 96400-18 </td><td> Boyutu 18 </td></tr><tr><td> Dikenli Redüktör </td><td> Cole Parmer </td><td> EW-30612-20 </td><td></td></tr><tr><td> 4L Beaker </td><td> Fisher Scientific </td><td> 02-540T </td><td></td></tr></tbody></table>

procedure for decellularization of porcine heart by retrograde coronary perfusion

Büyük ölçüde iskele yerli mimarisi koruyarak Perfüzyon tabanlı tüm organ decellularization son zamanlarda, siteye özgü ekstraselüler matriks iskelelerinin oluşturmak için bir araç olarak doku mühendisliği alanında ilgi kazanmıştır. Bugüne kadar, bu yaklaşım, kalp, akciğer, karaciğer ve 1-5 de dahil olmak üzere, organ sistemlerinin çeşitli kullanılmıştır. Kolay erişilebilir bir damar ağı olmadan dokuların Önceki decellularization yöntemleri deterjanlar, asitler veya çevredeki ekstraselüler ortamda 6-8 hücresel ve nükleer bileşenleri kaldırmak için bir araç olarak enzimatik tedavi çözümleri doku uzun süre maruz başvurmuşlardır. Bununla birlikte, çözeltilerin yeteneği bu yöntemlerin menteşeli etkinliğini difüzyon yoluyla doku nüfuz. Buna karşılık, doğal damar sistemi aracılığıyla organ perfüzyon etkili bir difüzyon mesafe ve decellularizati kolaylaştırılmıştır taşıma azaltılmışdoku dışarı doku ve hücresel bileşenlerine ajanlardır. Bu yazıda, tam koroner retrograd perfüzyon yoluyla sağlam bir domuz kalbi decellularize için bir yöntem açıklanmaktadır. Protokol bozulmamış kalp üç-boyutlu bir yapıya sahip bir tam decellularized kardiyak hücre dışı matris (ECM c) iskele elde edilmiştir. Önerilen yöntem, enzimler, deterjan ve hücre lisisi ve çıkarmaya yardım etmek için hipertonik ve hipotonik durulama ile birlikte asit dizisi kullanılır. Protokol hücresel malzeme çıkarmaya yardım etmek için Triton X-100 ve sodyum deoksikolatın çözümler ardından matris hücreleri ayırmak için bir tripsin çözelti kullanılmıştır. Açıklanan protokol, aynı zamanda daha büyük 2 L / uzun süre için min perfüzyon hızlarını kullanır. Hücresel enkaz kirlenme olmadan dokuya ajanlar taşıma izin ve çözüm değişiklikler ile birleştiğinde yüksek debi, doku durulama etkili sağlanmalıdır. Burada tarif edilen yöntem nati tüm nükleer malzeme ayrılmıştırçeşitli uygulamalar için kullanılan bir site-spesifik kardiyak ECM iskele oluşturarak, domuz kardiyak doku ve.

Bütün domuz kalpleri üzerinde decellularization etkisi doğal boyutunda farklılıklar, baskılar ve damar düzenleme nedeniyle değişir. Bu nedenle, elde edilen hücre dışı matris iskelelerinin tam kompozisyon kalp kalp ile aynı olmaz. Açıklanan protokol tamamlanması hücresel malzeme kaybı gösteren beyaz ya da yarı saydam görünür bir kalp ortaya çıkarır. Ancak, yaygın bir doku bir kaç daha fazla kantitatif parametreleri 8 kombinasyonuna dayalı &quot;decellularized&quot; kabul edilebil...

Watch this Scientific Journal Video about Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion at JoVE.com

Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

Hızla ve tamamen retrograd perfüzyon yoluyla sağlam bir domuz kalbinden hücresel bileşenleri kaldırmak için bir yöntem tarif edilmiştir. Bu yöntem, çoklu klinik uygulamalarda kullanım için potansiyele sahip bir site spesifik kardiyak ekstraselüler matriks iskele verir.

Retrograd Koroner Perfüzyon tarafından Domuz Kalp Decellularization Prosedürü

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

procedure-for-decellularization-porcine-heart-retrograde-coronary

Research

JoVE Journal

Bioengineering

23.7K Görüntüleme.  McGowan Institute for Regenerative Medicine. Hızla ve tamamen retrograd perfüzyon yoluyla sağlam bir domuz kalbinden hücresel bileşenleri kaldırmak için bir yöntem tarif edilmiştir. Bu yöntem, çoklu klinik uygulamalarda kullanım için potansiyele sahip bir site spesifik kardiyak ekstraselüler matriks iskele verir.

Video: Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

Decellularization and Recellularization of Whole Livers

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

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

Tüm karaciğerleri Decellularization ve Recellularization

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 ...

Biyomühendislik

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.

Bir Salınım basınçlı perfüzyon Aygıt Sıçan karaciğerinin decellularization Prosedürü

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

Decellularization of Whole Human Heart Inside a Pressurized Pouch in an Inverted Orientation

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ultimately rejection can occur. Creating a functional, autologous bio-artificial heart could solve these challenges. Biofabrication of a heart comprised of scaffold and cells is one option. A natural scaffold with tissue-specific composition as well as micro- and macro-architecture can be obtained by decellularizing hearts from humans or large animals such as pigs. Decellularization involves washing out cellular debris while preserving 3D extracellular matrix and vasculature and allowing &#34;cellularization&#34; at a later timepoint. Capitalizing on our novel finding that perfusion decellularization of complex organs is possible, we developed a more &#34;physiological&#34; method to decellularize non-transplantable human hearts by placing them inside a pressurized pouch, in an inverted orientation, under controlled pressure. The purpose of using a pressurized pouch is to create pressure gradients across the aortic valve to keep it closed and improve myocardial perfusion. Simultaneous assessment of flow dynamics and cellular debris removal during decellularization allowed us to monitor both fluid inflow and debris outflow, thereby generating a scaffold that can be used either for simple cardiac repair (e.g. as a patch or valve scaffold) or as a whole-organ scaffold.

This method enables decellularization of a complex solid organ using a simple protocol based on osmotic shock and perfusion of ionic detergent with minimal organ matrix disruption. It comprises a novel decellularization technique for human hearts inside a pressurized pouch with real-time monitoring of flow dynamics and cellular debris outflow.

Decellularization ters yönde bir basınçlı bir kese içinde bütün insan kalp

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ...

Isolation and Decellularization of a Whole Porcine Pancreas

Tissue engineering of the whole pancreas can improve current treatments for diabetes mellitus. The ultimate goal is to tissue engineer pancreas from an allogeneic or xenogeneic source with human cells. A demonstration of methods for the efficient dissection, decellularization, and recellularization of porcine pancreas might benefit the field. Akin to human pancreases, porcine pancreases have a special anatomical arrangement with three lobes (splenic, duodenal, and connection) rounded by the duodenum and small intestine. The duodenal lobe of the pancreas connects to the duodenum by several small blood vessels. Tissue engineering of the pancreas is complicated because of its exocrine and endocrine nature. In this paper, we show a detailed protocol to dissect the whole porcine pancreas and decellularize it with detergents while saving its structure and some extracellular matrix components. To achieve complete perfusion, the aorta is chosen as inlet and the portal vein as outlet. The other blood vessels (hepatic artery, splenic vein, splenic artery,&#160;mesenteric artery and vein tree) and bile duct are ligated. To prevent the formation of thrombus, the pig is heparinized and, immediately after dissection, the organ is flushed with cold heparin. To inhibit the action of exocrine enzymes, the pancreas decellularization is set at 4 &#176;C. The decellularization is performed by perfusion of Triton X-100, sodium deoxycholate, and deoxyribonuclease, with an intermittent and final extensive washing. With a successful decellularization, the pancreas appears white, and a histological evaluation with hematoxylin and eosin shows an absence of nuclei with a preserved extracellular matrix structure. Thus, the proposed method can be used to successfully dissect and decellularize whole porcine pancreas.

Tissue engineering of the whole pancreas is a challenge because of its exocrine and endocrine functions. We show a method for the dissection of an intact porcine pancreas and the process of successful decellularization by perfusion of detergents Triton X-100, sodium deoxycholate, and deoxyribonuclease.

Yalıtım ve bütün domuz pankreas Decellularization

Tissue engineering of the whole pancreas can improve current treatments for diabetes mellitus. The ultimate goal is to tissue engineer pancreas from an ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

Nano-formülasyonları endotoksin tespiti Limulus Amoebocyte Lysate (LAL) deneyleri kullanarak

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Time-Resolved In Vivo Measurement of Neuropeptide Dynamics by Capacitive Immunoprobe in Porcine Heart

The ability to measure biomarkers in vivo relevant to the assessment of disease progression is of great interest to the scientific and medical communities. The resolution of results obtained from current methods of measuring certain biomarkers can take several days or weeks to obtain, as they can be limited in resolution both spatially and temporally (e.g., fluid compartment microdialysis of interstitial fluid analyzed by enzyme-linked immunosorbent assay [ELISA], high-performance liquid chromatography [HPLC], or mass spectrometry); thus, their guidance of timely diagnosis and treatment is disrupted. In the present study, a unique technique for detecting and measuring peptide transmitters in vivo through the use of a capacitive immunoprobe biosensor (CI probe) is reported. The fabrication protocol and in vitro characterization of these probes are described. Measurements of sympathetic stimulation-evoked neuropeptide Y (NPY) release in vivo are provided. NPY release is correlated to the sympathetic release of norepinephrine for reference. The data demonstrate an approach for the fast and localized measurement of neuropeptides in vivo. Future applications include intraoperative real-time assessment of disease progression and minimally invasive catheter-based deployment of these probes.

Time-Resolved In Vivo Measurement of Neuropeptide Dynamics by Capacitive Immunoprobe in Porcine Heart

Established immunochemical methods to measure peptide transmitters in vivo rely on microdialysis or bulk fluid draw to obtain the sample for offline analysis. However, these suffer from spatiotemporal limitations. The present protocol describes the fabrication and application of a capacitive immunoprobe biosensor that overcomes the limitations of the existing techniques.

Domuz Kalbinde Kapasitif İmmünoprob ile Nöropeptit Dinamiğinin İn Vivo Ölçümü

The ability to measure biomarkers in vivo relevant to the assessment of disease progression is of great interest to the scientific and medical ...

Procurement and Perfusion-Decellularization of Porcine Vascularized Flaps in a Customized Perfusion Bioreactor

Large volume soft tissue defects lead to functional deficits and can greatly impact the patient&#39;s quality of life. Although surgical reconstruction can be performed using autologous free flap transfer or vascularized composite allotransplantation (VCA), such methods also have disadvantages. Issues such as donor site morbidity and tissue availability limit autologous free flap transfer, while immunosuppression is a significant limitation of VCA. Engineered tissues in reconstructive surgery using decellularization/recellularization methods represent a possible solution. Decellularized tissues are generated using methods that remove native cellular material while preserving the underlying extracellular matrix (ECM) microarchitecture. These acellular scaffolds can then be subsequently recellularized with recipient-specific cells.
This protocol details the procurement and decellularization methods used to achieve acellular scaffolds in a pig model. In addition, it also provides a description of the perfusion bioreactor design and setup. The flaps include the porcine omentum, tensor fascia lata, and the radial forearm. Decellularization is performed via ex vivo perfusion of low concentration sodium dodecyl sulfate (SDS) detergent followed by DNase enzyme treatment and peracetic acid sterilization in a customized perfusion bioreactor.
Successful tissue decellularization is characterized by a white-opaque appearance of flaps macroscopically. Acellular flaps show the absence of nuclei on histological staining and a significant reduction in DNA content. This protocol can be used efficiently to generate decellularized soft tissue scaffolds with preserved ECM and vascular microarchitecture. Such scaffolds can be used in subsequent recellularization studies and have the potential for clinical translation in reconstructive surgery.

The protocol describes the surgical procurement and subsequent decellularization of vascularized porcine flaps by the perfusion of sodium dodecyl sulfate detergent through the flap vasculature in a customized perfusion bioreactor.

Özelleştirilmiş Perfüzyon Biyoreaktöründe Domuz Vaskülarize Fleplerinin Tedariği ve Perfüzyon-Desellülarizasyonu

Large volume soft tissue defects lead to functional deficits and can greatly impact the patient&#39;s quality of life. Although surgical reconstruction ...

Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation

Patients with severe traumatic injuries and tissue loss require complex surgical reconstruction. Vascularized composite allotransplantation (VCA) is an evolving reconstructive avenue for transferring multiple tissues as a composite subunit. Despite the promising nature of VCA, the long-term immunosuppressive requirements are a significant limitation due to the increased risk of malignancies, end-organ toxicity, and opportunistic infections. Tissue engineering of acellular composite scaffolds is a potential alternative in reducing the need for immunosuppression. Herein, the procurement of a rat hindlimb and its subsequent decellularization using sodium dodecyl sulfate (SDS) is described. The procurement strategy presented is based upon the common femoral artery. A machine perfusion-based bioreactor system was constructed and used for ex vivo decellularization of the hindlimb. Successful perfusion decellularization was performed, resulting in a white translucent-like appearance of the hindlimb. An intact, perfusable, vascular network throughout the hindlimb was observed. Histological analyses showed the removal of nuclear contents and the preservation of tissue architecture across all tissue compartments.

Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation

We describe the surgical technique and decellularization process for composite rat hindlimbs. Decellularization is conducted using low-concentration sodium dodecyl sulfate through an ex vivo machine perfusion system.

Vaskülarize Kompozit Allotransplantasyon için Ex Vivo Perfüzyon Bazlı Biyoreaktör Kullanılarak Sıçan Arka Bacaklarının Tedariği ve Desellülarizasyonu

Patients with severe traumatic injuries and tissue loss require complex surgical reconstruction. Vascularized composite allotransplantation (VCA) is an ...

Monte Edilmiş Domuz Aort Kapaklarında Hidrodinamik Özelliklerin Değerlendirilmesi

An Ex Vivo Porcine Model for Hydrodynamic Testing of Experimental Aortic Valve Procedures and Novel Medical Devices

The options for testing new cardiac procedures and investigative medical devices prior to use in an animal model are limited. In this study, we present a method for mounting a porcine aortic valve in a pulse duplicator to evaluate its hydrodynamic properties. These properties can then be evaluated before and after the procedure under investigation is performed and/or the investigative medical device is applied. Securing the inflow segment presents some difficulty owing to the lack of circumferential myocardium in the left ventricular outflow tract. This method addresses that issue by securing the inflow segment using the anterior leaflet of the mitral valve and then suturing the left ventricular free wall around the inflow fixture. The outflow segment is secured simply by inserting the fixture into an incision in the superior aspect of the aortic arch. We found that specimens had significantly different hydrodynamic properties before and after tissue fixation. This finding induced us to use fresh specimens in our testing and should be considered when using this method. In our work, we used this method to test novel intracardiac patch materials for use in the valvular position by performing an aortic valve neocuspidization procedure (Ozaki procedure) on the mounted porcine aortic valves. These valves were tested before and after the procedure to assess the change in hydrodynamic properties in comparison to the native valve. Herein, we report a platform for hydrodynamic testing of experimental aortic valve procedures that enables comparison with the native valve and between different devices and techniques used for the procedure under investigation.

Yazar Spot Işığı: Büyük Hayvan Çalışmalarına Başlamadan Önce Kardiyak Prosedür Testini İlerletmek 

We present a method for mounting a porcine aortic valve on a pulse duplicator to test its hydrodynamic properties. This method can be used to determine the change in hydrodynamics after the application of an experimental procedure or novel medical device prior to use in a large animal model.

Deneysel Aort Kapak Prosedürlerinin ve Yeni Tıbbi Cihazların Hidrodinamik Testi için Ex Vivo Domuz Modeli

The options for testing new cardiac procedures and investigative medical devices prior to use in an animal model are limited. In this study, we present a ...