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

23.8K 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 Lung Engineering

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths annually; chronic obstructive pulmonary disease is currently the fourth leading cause of death in the United States1. Contributing to this mortality is the fact that lungs do not generally repair or regenerate beyond the microscopic, cellular level. Therefore, lung tissue that is damaged by degeneration or infection, or lung tissue that is surgically resected is not functionally replaced in vivo. To explore whether lung tissue can be generated in vitro, we treated lungs from adult rats using a procedure that removes cellular components to produce an acellular lung extracellular matrix scaffold. This scaffold retains the hierarchical branching structures of airways and vasculature, as well as a largely intact basement membrane, which comprises collagen IV, laminin, and fibronectin. The scaffold is mounted in a bioreactor designed to mimic critical aspects of lung physiology, such as negative pressure ventilation and pulsatile vascular perfusion. By culturing pulmonary epithelium and vascular endothelium within the bioreactor-mounted scaffold, we are able to generate lung tissue that is phenotypically comparable to native lung tissue and that is able to participate in gas exchange for short time intervals (45-120 minutes). These results are encouraging, and suggest that repopulation of lung matrix is a viable strategy for lung regeneration. This possibility presents an opportunity not only to work toward increasing the supply of lung tissue for transplantation, but also to study respiratory cell and molecular biology in vitro for longer time periods and in a more accurate microenvironment than has previously been possible.

We have developed a decellularized lung extracellular matrix and novel biomimetic bioreactor that can be used to generate functional lung tissue. By seeding cells into the matrix and culturing in the bioreactor, we generate tissue that demonstrates effective gas exchange when transplanted in vivo for short periods of time.

Akciğer Mühendisliği Prosedürü

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths ...

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.

Doğal Aselüler Bağırsak Matrix&#39;in üretimi için bir Decellularization Metodoloji

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 ...

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 ...

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.

Doku mühendisliği uygulamaları için bitki dokuların Decellularization için iki yöntem

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 ve Recellularization metodoloji insan safen damarlar için

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

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.