Os autores gostariam de agradecer a Jack Milwid para o desenho da câmara de perfusão in vitro. Este trabalho foi financiado por concessões do NIH dos EUA, R01DK59766 e R01DK084053 para MY, R00DK080942 para KU, EUA NSF CBET-0853569 para KU e os Hospitais Shriners para Crianças, a BEU (concessão não. 8503). Reconhecemos também o apoio e os Hospitais Shriners para Crianças.

1. Decelularização fígado <ol><li> Colheita de um fígado de ratos com canulação da veia porta utilizando e 18 de calibre do cateter. Deixe o inferior e veia cava superior aberta. Manter o órgão hidratado em tampão fosfato salino (PBS) numa placa de Petri de 10 cm. </li><li> Criação de um sistema de perfusão, que consiste em reservatório de 8 litros, bomba peristáltica e um borbulhador. </li><li> Preencher o sistema de perfusão com tampão fosfato e mantê-lo funcionando por 10 minutos. Preencha PBS em uma placa de Petri de 10 cm e reduzir a taxa de fluxo de tampão fosfato salino a 1 mL / min. </li><li> Transferir cuidadosamente o fígado colhidas ao tampão fosfato salina preenchidos de 10 cm placa de Petri. </li><li> Continue com perfusão PBS durante a noite. </li><li> Iniciar a perfusão com 0,01% (w / v) dodecil sulfato de sódio (SDS) em água destilada por 5 minutos. </li><li> Perfundir com PBS por 1 hora. </li><li> Repita os passos 1.6 e 1.7 mais três vezes o aumento do tempo de perfusão SDS para minutos 10, 15 e 20 em cada tempo. </li><li> Continue a perfusão com 0,01% (w / v) SDS por 24 horas. </li><li> Continue a perfusão com 0,1% (w / v) SDS por 24 horas. </li><li> Perfundir com 0,2% (w / v) SDS por 3 horas. </li><li> Perfundir com 0,5% (w / v) SDS por 3 horas. </li><li> Perfundir com água destilada por 15 minutos. </li><li> Perfundir com 1% (w / v) de Triton X-100 em água destilada por 30 minutos para remover qualquer ácidos nucléicos ligado. </li><li> Para lavar o fígado decelularizado matriz (DLM), perfundir com PBS por 2 horas. </li><li> Opcional: ressecar todos os lobos, exceto o lobo mediano. </li><li> Armazenar o DLM numa placa de Petri limpa e selada embebido em PBS a 4 ° C até que esteja pronto para uso (Figura 1). </li><li> Para esterilizar o DLM: 1) Lave o DLM com PBS estéril contendo 0,1% (v / v) de ácido peracético e 4% (v / v) de etanol e incubar durante 3 horas a 4 o C. 2) Lavar com PBS esterilizado duas vezes. 3) Lavar com PBS estéril contendo 2% de penicilina-estreptomicina, 10ug / mL de gentamicina e 2,5 ug / ml anfotericina B. loja o fígado decelularizado na mesma solução a 4 ° C até ser utilizado para experimentos recellularization. </li></ol> 2. Recellularization de decelularizados Fígado Matrix <ol><li> Criação de um sistema de perfusão, que consiste de uma câmara de perfusão, bomba peristáltica e um borbulhador em condições estéreis. Preencher o sistema de perfusão com 200 mL de meio de cultura, por exemplo, alta de glicose DMEM (Sigma), 10% soro fetal bovino (Hyclone), 100 U mL -1 de penicilina e 100 mg mL -1 de estreptomicina (Invitrogen). </li><li> Coloque a matriz fígado decelularizado na câmara de perfusão e conectar o DLM ao sistema de perfusão através da cânula da veia porta, enquanto a bomba está funcionando em 5 mL / min para evitar a formação de bolhas de ar. </li><li> Permitir a perfusão do meio através da DLM por 30 minutos. </li><li> Isolar hepatócitos primários de um rato adulto com viabilidade, pelo menos, 90%. </li><li> Interromper o fluxo no sistema de perfusão e injecte lentamente 50000000 hepatócitos (em mL 1-3 de meio de cultura) em sistema de perfusão através do borbulhador. </li><li> Iniciar o fluxo de 10 mL / min e recircular o meio por 10 minutos. </li><li> Repita os passos 2.5 e 2.6, até um total de 200 milhões de células são introduzidas no DLM (Figura 2) 6. </li><li> Uma vez que todas as células são perfundidos para a DLM, coletar o perfusato em quatro tubos de 50 ml de centrífuga e centrifugar a 600 rpm por 10 minutos. Descartar o sobrenadante e recolher as bolinhas em um único tubo. Determinar o número de células e viabilidade através de exclusão do azul tripan para determinar a eficiência da semeadura. </li></ol> 3. Cultivo in vitro do Enxerto Fígado Recellularized <ol><li> Criação de um sistema de perfusão, que consiste de uma câmara de perfusão, bomba peristáltica, oxigenador e um borbulhador em condições estéreis (Figura 3). Preencher o sistema de perfusão com 50 mL de meio de cultura, por exemplo, E Williams (Sigma), 5% soro fetal bovino (Hyclone), 0,5 mL de insulina U -1 (Eli Lilly), 20 ng mL -1 EGF (Invitrogen) , 14 ng mL -1 glucagon (Bedford Laboratories), 7,5 mg mL -1 hidrocortisona (Pharmacia), 100 U mL -1 de penicilina e estreptomicina 100 mg mL -1 (Invitrogen). </li><li> Coloque o enxerto de fígado recellularized na câmara de perfusão e conectar o DLM ao sistema de perfusão através da cânula da veia porta, enquanto a bomba está funcionando em 5 mL / min para evitar a formação de bolhas de ar. </li><li> Assepticamente fechar a câmara de perfusão e lacre firmemente para evitar qualquer vazamento durante a cultura. </li><li> Transferir o sistema de perfusão de uma incubadora que é de 37 º C e tem 10% de CO 2 e aumentar a taxa de fluxo de perfusão a 15 mL / min. </li><li> Conecte o oxigenador a um 95% O 2 e 5% CO 2 tanque de mistura de gás e definir a taxa de fluxo de gás para 0,5 litros / min. Isto deve atingir uma pressão parcial de oxigênio de aproximaçãotely mmHg 400. </li><li> A cultura pode continuar até 10 dias, com variações diárias de meio de cultura. O meio de cultura podem ser amostrados diariamente para monitoramento das funções hepática do enxerto, tais como uréia, albumina e secreção de ácido total de bile. No final do período de cultura, o enxerto de fígado recellularized podem ser amostrados para análise molecular e histológica. </li></ol> 4. Resultados representativos: A decelularização completa de um fígado de rato leva cerca de 72 horas usando o protocolo descrito. A matriz resultante mantém 100% do colágeno fibrilar, 50% dos glicosaminoglicanos e apenas 5% do DNA do fígado nativo (Tabela 1) 6. A estrutura vascular da matriz é preservada como evidenciado pela corrosão de fundição e digitalização análise de microscopia eletrônica (Figura 4) 6. A presença da microarquitetura vascular dentro da DLM facilita o seu repovoamento com células com uma eficiência de 96% e sua subseqüente perfusão de cultura in vitro. O enxerto de fígado recellularized podem ser cultivadas até 10 dias in vitro e exibe as funções do fígado adequada como foi confirmado através de uréia, albumina e secreção de bile ácida total (Figura 5) 6. <img src="/files/ftp_upload/2394/2394fig1.jpg" alt="Figura 1" /> Figura 1. Decelularizados matriz de fígado no final do processo de decelularização. (A) todo o fígado (b) a mediana do lobo após a ressecção. <img src="/files/ftp_upload/2394/2394fig2.jpg" alt="Figura 2" /> Figura 2. Representação esquemática do recellularization da DLM. <img src="/files/ftp_upload/2394/2394fig3.jpg" alt="Figura 3" /> Figura 3. Perfusão configuração do sistema para a cultura in vitro do enxerto hepático recellularized. <img src="/files/ftp_upload/2394/2394fig4.jpg" alt="Figura 4" /> Figura 4. A estrutura microvascular é retida na matriz do fígado descelularizados. Corrosão imagens elenco de a) b) um fígado decelularizado um fígado normal, portal (vermelho) e venosa vasculatura (azul). Digitalização de imagens de microscopia eletrônica do c DLM) um navio, d) apresentando uma seção do ducto biliar, como pequenos vasos (setas), barras de escala (a, b) 5 mm (c, d) 20 mM. <img src="/files/ftp_upload/2394/2394fig5.jpg" alt="Figura 5" /> Figura 5. Liver funções específicas do enxerto hepático recellularized durante o cultivo in vitro de perfusão. uma secreção) de albumina (p = 0,5249), b) produção de uréia (p = 0,5271) e c) a secreção de bile ácida total (p = 0,0114). Análise estatística da diferença entre a experiência eo controle foi feito durante o período de 10 d cultura pelo teste de Friedman em a = 0,01. Barras de erro representam sem (n = 3). <table border="1"><tbody><tr><td></td><td> Livera fresco </td><td> Decelularizado fígado uma matriz </td><td rowspan="2"> p-valores </td><td rowspan="2"> % De fígado fresco </td></tr><tr><td></td><td> n = 4 </td><td> n = 8 </td></tr><tr><td> Colágeno </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 por fígado g) </td></tr><tr><td> Glicosaminoglicanos </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 por fígado g) </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 por fígado g) </td></tr></tbody></table> Tabela 1. A composição bioquímica da matriz fígado decelularizado em comparação com o fígado nativo. uma Os valores são representados como média ± epm

<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>

Não há conflitos de interesse declarados.

O método descrito aqui decelularização perfusão produz um todo fígado andaime que tem a mesma estrutura bruta ea microarquitetura vascular do fígado nativo. O andaime tem uma composição da matriz extracelular semelhante ao fígado nativo. O método recellularization atinge repovoamento do andaime com células de alta eficiência e as células permanecem viáveis ​​e funcionais durante o período de cultivo in vitro testados. Com a adição de células não parenquimatosos para o enxerto de fígado r...

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

Erratum: Decellularization and Recellularization of Whole Livers

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decellularization and recellularization of whole livers

O fígado é um órgão complexo que requer constante de perfusão para a entrega de nutrientes e oxigênio e remoção de resíduos, a fim de sobreviver 1. Esforços para recriar ou imitar a microestrutura fígado com motivos de abordagem utilizando a engenharia de tecidos e técnicas de microfabricação não tenham sido bem sucedida até agora devido a este desafio do projeto. Além disso, biomateriais sintéticos usados ​​para criar andaimes para aplicações de engenharia de tecidos do fígado têm sido limitados em induzir a regeneração dos tecidos e reparação, em grande parte devido à falta de motivos de células específicas de ligação que induziria as funções das células adequada 2. Tecidos decelularizados nativa vasos sanguíneos, tais 3 e 4 de pele, por outro lado têm encontrado muitas aplicações na engenharia de tecidos, e forneceram uma solução prática para alguns dos desafios. A vantagem de decelularizado matriz nativa é que ele mantém, até certo ponto, a composição original, a microestrutura e, conseqüentemente aumentando adesão celular ea reorganização 5. Neste trabalho, descrever os métodos para realizar a perfusão decelularização do fígado, de tal forma que um fígado intacto bioscaffold que mantém a estrutura dos principais vasos sanguíneos é obtido. Além disso, descrevemos métodos para recellularize esses bioscaffolds com adultos hepatócitos primários, a criação de um enxerto de fígado que é funcional in vitro, e tem o acesso dos navios necessários para o transplante em vivo.

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

Decellularization and Recellularization of Whole Livers

Decelularização perfusão é uma nova técnica para produzir scaffolds fígado inteiro que mantém a composição do órgão matriz extracelular e microarquitetura. Aqui, o método de preparação de andaimes órgão inteiro usando decelularização perfusão e repovoamento posterior com hepatócitos é descrito. Enxertos de fígado para transplante funcional e pode ser gerado usando esta técnica.

Decelularização e Recellularization de Fígados inteiros

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

Research

JoVE Journal

Bioengineering

21.9K visualizações.  Massachusetts General Hospital, Harvard Medical School, Shriners Hospitals for Children. Decelularização perfusão é uma nova técnica para produzir scaffolds fígado inteiro que mantém a composição do órgão matriz extracelular e microarquitetura. Aqui, o método de preparação de andaimes órgão inteiro usando decelularização perfusão e repovoamento posterior com hepatócitos é descrito. Enxertos de fígado para transplante funcional e pode ser gerado usando esta técnica.

Vídeo: 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.

Procedimento para decelularização do Coração Suínos por perfusão coronariana retrógrada

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

Bioengenharia

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.

A Metodologia Decelularização para a produção de um Intestinal Matrix Natural Acellular

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.

Procedimento para Decelularização de fígados de rato em uma Perfusão dispositivo oscilante pressão

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

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.

Decellularization comparável de explantes de tecido cardíaco Fetal e adulto como plataformas 3D-como para estudos de Vitro

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

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

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

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

Dois métodos para Decellularization de tecidos vegetais para aplicações de engenharia de tecidos

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 e Recellularization metodologia para as veias safena humanas

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 do coração humana dentro de uma bolsa pressurizada em uma orientação invertida

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.

Isolamento e Decellularization de um pâncreas todo porcino

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

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.

Aquisição e Perfusão-Descelularização de Flaps Vascularizados Suínos em Biorreator de Perfusão Customizado

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