Os autores gostariam de agradecer a Leica Microsystems para os seus conselhos valiosos sobre seções de imagens de titânio e Gemini Bio Produtos para fornecer o soro de suínos utilizados neste estudo. Temos também de agradecer aos NIH para o seu apoio através de Grant &quot;forro autólogo EPC para melhorar a biocompatibilidade de dispositivos de assistência circulatória&quot;, RC1HL099863-01. Além disso, estamos gratos pelo apoio da National Science Foundation Graduate Research Fellowship de Alexandra Jantzen. Também gostaria de agradecer George rápida, Mike Lowe e Ianthia Parker para ajudar com muitos aspectos do procedimento cirúrgico e manipulação dos animais de nossa pesquisa. Steven Owen tem sido inestimável para usinagem de peças muitas das nossas dispositivo semeadura e corte de tubos de titânio.

<ol>
	<li>Achneck, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+of+bleeding+and+clotting+in+the+cardiac+surgery+patient:+from+vascular+endothelium+to+circulatory+assist+device+surface.">Pathophysiology of bleeding and clotting in the cardiac surgery patient: from vascular endothelium to circulatory assist device surface.</a> Circulation. 122, 2068-2077 (2010).</li><li>Achneck, H. E., Sileshi, B., Lawson, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+the+biology+of+bleeding+and+clotting+in+the+surgical+patient.">Review of the biology of bleeding and clotting in the surgical patient.</a> Vascular. 16, 6-13 (2008).</li><li>Arvidsson, S., Askendal, A., Tengvall, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+plasma+contact+activation+on+silicon,+titanium+and+aluminium.">Blood plasma contact activation on silicon, titanium and aluminium.</a> Biomaterials. 28, 1346-1354 (2007).</li><li>Achneck, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biocompatibility+of+titanium+cardiovascular+devices+seeded+with+autologous+blood-derived+endothelial+progenitor+cells:+EPC-seeded+antithrombotic+Ti+Implants.">The biocompatibility of titanium cardiovascular devices seeded with autologous blood-derived endothelial progenitor cells: EPC-seeded antithrombotic Ti Implants.</a> Biomaterials. 32, 10-18 (2011).</li><li>Kang, C., Bonneau, M., Brouland, J. P., Bal dit Sollier, C., Drouet, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+pig+models+of+venous+thrombosis+mimicking+human+disease.">In vivo pig models of venous thrombosis mimicking human disease.</a> Thromb Haemost. 89, 256-263 (2003).</li><li>Ueberrueck, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+ovine+and+porcine+animal+models+for+biocompatibility+testing+of+vascular+prostheses.">Comparison of the ovine and porcine animal models for biocompatibility testing of vascular prostheses.</a> J Surg Res. 124, 305-311 (2005).</li><li>Velik-Salchner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+values+for+thrombelastography+(ROTEM)+and+selected+coagulation+parameters+in+porcine+blood.">Normal values for thrombelastography (ROTEM) and selected coagulation parameters in porcine blood.</a> Thromb Res. 117, 597-602 (2006).</li><li>Yoder, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redefining+endothelial+progenitor+cells+via+clonal+analysis+and+hematopoietic+stem/progenitor+cell+principals.">Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem/progenitor cell principals.</a> Blood. 109, 1801-1809 (2007).</li><li>Achneck, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> American Heart Association Scientific Sessions, Abstract Oral Sessions, Medical Aspects End Stage Heart Failure: Transplantation and Device Therapies. , (2010).</li><li>Achneck, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerating+titanium+ventricular+assist+device+surfaces+after+gold/palladium+coating+for+scanning+electron+microscopy.">Regenerating titanium ventricular assist device surfaces after gold/palladium coating for scanning electron microscopy.</a> Microsc Res Tech. 73, 71-76 (2010).</li><li>Ford, J. W., Welling, T. H., Stanley, J. C., Messina, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PKH26+and+125I-PKH95:+characterization+and+efficacy+as+labels+for+in+vitro+and+in+vivo+endothelial+cell+localization+and+tracking.">PKH26 and 125I-PKH95: characterization and efficacy as labels for in vitro and in vivo endothelial cell localization and tracking.</a> J Surg Res. 62, 23-28 (1996).</li></ol>

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O método de tubos das células de semeadura de Ti aqui apresentada permite que médicos e cientistas de forma rápida e uniformemente endothelialize sangue entrar em contato com as superfícies dos dispositivos implantáveis. Desde que isolar e expandir EPCs a partir de amostras de sangue periférico, nenhum procedimento invasivo principais é necessária para a colheita destas células. Além disso, o CPE são autólogos e, portanto, o risco de qualquer reação imunológica ao implante de células-semeada é eliminado. Os princípios demonstrados em seu protocolo pode ser utilizado não só para os tubos de Ti, mas para muitos outros biomateriais, que são utilizados na medicina cardiovascular. Passos críticos neste protocolo são a limpeza meticulosa dos tubos de Ti, como descobrimos que qualquer filme aderente de adesão de contaminantes compromissos celular. Além disso, uma velocidade de rotação lenta (inversamente proporcional ao diâmetro do tubo) é essencial durante o processo de semeadura, de modo que EPCs pode lentamente se estabelecer e aderir à superfície como o tubo de Ti está se movendo. Nosso método de semeadura imediatamente antes da implantação evita impraticável ex tempos de cultivo in vivo, as células aderem individualmente e, posteriormente, formar uma folha confluentes in vivo, evitando a possibilidade de embolização como uma folha imediatamente após o restabelecimento do fluxo. Nossos estudos anteriores mostram que uma vez que o CPE tem crescido a uma camada confluente, eles fazem uma matriz extra-celular para o qual eles aderem firmemente, adicionalmente, minimizando qualquer sloughing possível de uma folha endoteliais. Embora a possibilidade de descolamento embólicos não pode ser inteiramente descartada, parece ser de risco multifold inferior trombose da superfície dispositivo inteiro, o problema que esta terapia é projetado para impedir. Nossa abordagem de implantação na corte baixo, o ambiente pró-trombótico da veia cava inferior utiliza um dos mais confiáveis ​​modelos animais de grande porte para a pesquisa de compatibilidade de sangue e trombose de dispositivos de 5,6. Note-se que todos os cuidados com animais de experimentação e foi conduzido de acordo com o Instituto Nacional de Saúde Diretrizes para o Cuidado e Uso de Animais de Laboratório e somente após a aprovação do Animal Care Duke University Institucional e Comitê de uso. A fim de conseguir utilizar este método para testar o implante de biomateriais e dispositivos, é importante pacientemente esqueletizar o segmento da VCI e ligar todos os vasos venosos filial em preparação para a inserção do dispositivo, tal que nenhum sangramento em torno do dispositivo tem origem em uma &quot;falsa luz&quot;. Outro passo fundamental é a adição de DPBS no lúmen dispositivo de tal forma que as células na superfície do tubo dentro de permanecer úmida durante o fechamento do veinotomy e antes da reperfusão é iniciada. Se o dispositivo não pode ser encontrado no local onde foi implantado, ele pode ter migrado &#39;upstream&#39; na VCI. Isto pode ser evitado colocando uma sutura (Prolene 4-O), através da parede da veia e através de um 2-3 mm de seção da tubulação de PVC de modo que o tubo está firmemente ancorada em sua posição atual. O pesquisador deve ter dificuldade em encontrar as células fluorescente pré-rotulados mostrado na Figura 8, após explante em um tubo de outra forma patente, é provável que as células têm descascada como uma folha coerente. Isto pode ser evitado por meio de dissecção muito gentil da veia circundante e dis-montagem das três secções tubulares Ti, depois da fixação da veia juntamente com o tubo. Nossa tecnologia fornece a prova de conceito para prevenção de trombose dispositivo cardiovascular através de EPC-semeadura. Esta tecnologia pode ser usada no desenvolvimento de &quot;biogênicos&quot; implantes alinhados com os próprios pacientes, as células progenitoras endoteliais. O estudo de viabilidade em nosso modelo animal porcino prevê os primeiros passos em direção a tradução deste &quot;medicina personalizada&quot; na prática clínica.

<table border="1"><tbody><tr><td> Reagente </td><td> Nome da Empresa </td><td> Número Catálogo </td><td> Comentários </td></tr><tr><td> Acepromazina </td><td> Boehringer-Ingelheim </td><td> BIC670025 </td><td> NAC # 10280002 </td></tr><tr><td> Alconox Cleaner Precision Produzido </td><td> Alconox </td><td> 1104 </td><td></td></tr><tr><td> Balfour Retractor Cirúrgica </td><td> Adler </td><td> N / A </td><td></td></tr><tr><td> Baytril </td><td> Bayer </td><td> APVMA 46028/0705 </td><td></td></tr><tr><td> Borboleta Needle (19G) </td><td> Terumo </td><td> SV19CLK </td><td></td></tr><tr><td> Clorexidina </td><td> 3M </td><td> 9200 </td><td></td></tr><tr><td> Grampos (45 graus) </td><td> Aesculap </td><td> FC339T </td><td></td></tr><tr><td> DPBS (-/-) </td><td> Gibco </td><td> 14190-144 </td><td></td></tr><tr><td> DPBS (+/+) </td><td> Gibco </td><td> 14040-133 </td><td></td></tr><tr><td> DuraPrep </td><td> 3M </td><td> 8635 </td><td></td></tr><tr><td> EBM-2 Medium </td><td> Lonza </td><td> CC-3156 </td><td> Base para as médias de crescimento sérico livre e pleno </td></tr><tr><td> EGM-2 SingleQuots </td><td> Lonza </td><td> CC-3162 </td><td> Usado com EBM-2 para ambos meio de crescimento sérico livre e pleno </td></tr><tr><td> Ferramenta de eletrocautério </td><td> Valleylab </td><td> SurgII-20 </td><td></td></tr><tr><td> Lixa de esmeril </td><td> 3M </td><td> 60-0700-0425-8 </td><td> 240 grit </td></tr><tr><td> Euthasol Solução Eutanásia </td><td> Virback Saúde Animal </td><td></td><td> Anada #: 200-071 </td></tr><tr><td> Fentanilo </td><td> Actavis </td><td></td><td> NDC # 67767-120-18 </td></tr><tr><td> Flunixin </td><td> Schering-Plough </td><td></td><td> NAC #: 10470183 </td></tr><tr><td> Foley Cateter (16F) </td><td> Bardo </td><td> 730116 </td><td></td></tr><tr><td> HBSS </td><td> Sigma </td><td> H8264-500ML </td><td></td></tr><tr><td> Heat Gun </td><td> Milwaukee </td><td> 8988-20 </td><td></td></tr><tr><td> Heparina </td><td></td><td></td><td> NDC #: 25021 </td></tr><tr><td> Histopaque-1077 </td><td> Sigma </td><td> H8889-500ML </td><td></td></tr><tr><td> Tubo de intubação </td><td> Mallinckrodt </td><td> 86113 </td><td></td></tr><tr><td> Isoflurano </td><td> MWI, Meridian </td><td></td><td> NDC # 13985-030 </td></tr><tr><td> IV cateter (18G) </td><td> Becton-Dickinson </td><td> 381547 </td><td></td></tr><tr><td> Ketamina </td><td> Fort Dodge </td><td></td><td> NDC # 0856-2013 </td></tr><tr><td> Nível </td><td> Swanson </td><td> LLA001 </td><td></td></tr><tr><td> Luer-Lock ponta cap </td><td> CML Abastecimento </td><td> 909-001 </td><td></td></tr><tr><td> Marcaine </td><td> Hospira </td><td></td><td> NDC #: 0409-1560-10 </td></tr><tr><td> Tesoura Metzenbaum </td><td> Adler </td><td> N / A </td><td></td></tr><tr><td> Micro-introdutor (5F) </td><td> Galt </td><td> KIT 002-01 </td><td></td></tr><tr><td> Pinça Mosquito </td><td> Adler </td><td> N / A </td><td></td></tr><tr><td> Motor </td><td> Herbach e Rademan </td><td> H1-08 </td><td></td></tr><tr><td> Oximorfona </td><td> Endo Labs </td><td></td><td> NDC: 63481-624-10 </td></tr><tr><td> PKH26 Dye Kit </td><td> Sigma </td><td> PKH26GL-1KT </td><td></td></tr><tr><td> Serum suína </td><td> Gémeos Bio-Products </td><td> 100-115 </td><td> Concentração de 2% em meio de crescimento total </td></tr><tr><td> Tesoura Potts </td><td> Adler </td><td> N / A </td><td></td></tr><tr><td> Pele precisa Vista grampeador </td><td> 3M </td><td> 3998 </td><td></td></tr><tr><td> Tubos de PVC </td><td> McMaster-Carr/Insultab </td><td> 7132K117 </td><td> expandiu 15,88 mm de diâmetro, ID recuperado 7,95 milímetros </td></tr><tr><td> Tamanho Médio direito Angle </td><td> Adler </td><td> N / A </td><td></td></tr><tr><td> Bisturi Blade (# 10-15) </td><td> Bardo </td><td> 373910 </td><td></td></tr><tr><td> Tubulação de silicone </td><td> McMaster-Carr </td><td> 51735K26 </td><td> 16,64 milímetros OD, 9,52 mm de diâmetro </td></tr><tr><td> Seringa (5 ml) </td><td> Becton-Dickinson (BD) </td><td> 309603 </td><td></td></tr><tr><td> Tegaderm </td><td> 3M </td><td> 90001 </td><td></td></tr><tr><td> Torneira de três vias </td><td> Kendall </td><td> 170060 </td><td></td></tr><tr><td> Tubo de Ti </td><td> Tico Titanium </td><td> N / A </td><td> Especificado como ½ &quot;OD, 0,065&quot; de parede, 0,370 ID &quot;, £ 0,1737 / ft </td></tr><tr><td> Tripsina </td><td> Lonza </td><td> CC-5012 </td><td></td></tr><tr><td> Tripsina solução neutralizante (TNS) </td><td> Lonza </td><td> CC-5002 </td><td> 0,03% </td></tr><tr><td> Vetropolycin </td><td> Pharmaderm Saúde Animal </td><td></td><td> NAC #: 12920110 </td></tr><tr><td> Vicryl sutura (3-0) </td><td> Ethicon </td><td> J808T </td><td></td></tr><tr><td> Aparelho de ultrasons de água </td><td> Branson </td><td> B200 </td><td></td></tr></tbody></table>

autologous endothelial progenitor cell-seeding technology and biocompatibility testing for cardiovascular devices in large animal model

Implantáveis ​​cardiovasculares são fabricados a partir de materiais artificiais (por exemplo, titânio (Ti), politetrafluoroetileno expandido), que representam o risco de formação tromboêmbolos 1,2,3. Nós desenvolvemos um método para a linha de superfície interna de tubos de Ti com autólogo derivados do sangue humano ou de suíno células progenitoras endoteliais (EPCs) 4. Através da implantação de tubos de Ti contendo uma camada confluente de EPCs suína na veia cava inferior (VCI) de porcos, testamos a biocompatibilidade melhorada da superfície da célula-semeado no ambiente pró-trombótico de um modelo animal de grande porte e comparou-a inalterada superfícies de metal nu 5,6,7 (Figura 1). Este método pode ser usado para endothelialize dispositivos dentro de poucos minutos de implantação e teste sua função antitrombótica in vivo. Sangue periférico foi obtido a partir de 50 kg Yorkshire suína e sua fração de células mononucleares de cultura para isolar EPCs 4,8. Ti tubos (9,4 mm de diâmetro) foram pré-cortados em três seções 4,5 centímetro longitudinal e remontado com calor-encolhem a tubulação. Um dispositivo de semeadura foi construído, que permite a rotação lenta dos tubos de Ti. Foi realizada uma laparotomia na porcos e exteriorizada do intestino e bexiga urinária. Dissecção cortante e contundente foi usado para esqueletizar da VCI de sua bifurcação distal à artéria renal direita proximal. Os tubos de Ti foram então enchidos com fluorescente marcado autólogo suspensão EPC e rodado em 10 RPH x 30 min para alcançar uniforme de células de revestimento-9. Após a administração de heparina 100 USP / kg, ambas as extremidades do IVC e uma veia lombar foram presos. A veinotomy 4 cm foi realizada eo dispositivo inserido e preenchido com tampão fosfato. Como o veinotomy foi fechada com uma sutura Prolene 4-0, um grampo foi removido para o IVC de-ar. No final do procedimento, a fáscia foi aproximado com 0-PDS (polidioxanona sutura), o espaço subcutâneo fechado com Vicryl 2-0 ea pele fechados com grampos. Após 3-21 dias, os porcos foram sacrificados, o dispositivo explantado en-bloco e fixa. Os tubos foram desmontados Ti e as superfícies internas fotografada com um microscópio de fluorescência. Descobrimos que os tubos de metal simples Ti totalmente ocluído enquanto os tubos EPC-semeado permaneceu patente. Além disso, fomos capazes de demonstrar uma camada confluente de EPCs na superfície de sangue entrar em contato com seu interior. Concluindo, a nossa tecnologia pode ser usada para endothelialize tubos de Ti em poucos minutos de implantação com EPCs autólogo para prevenir a trombose do dispositivo. Nosso método cirúrgico permite testar a biocompatibilidade melhorada de tais dispositivos modificados com a perda de sangue mínima e EPC-semeado interrupção superfície.

Watch this Scientific Journal Video about Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model at JoVE.com

Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model

Um método para semeadura de titânio de sangue entrar em contato com biomateriais com células autólogas e biocompatibilidade teste é descrito. Este método utiliza células progenitoras endoteliais e tubos de titânio, semeadas em poucos minutos de implante cirúrgico em suínos as cavas. Esta técnica é adaptável a muitas outras implantáveis ​​dispositivos biomédicos.

Autólogas progenitoras endoteliais Cell Technology Semear e Teste de biocompatibilidade para dispositivos Cardiovascular em Modelo Animal Grande

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk ...

autologous-endothelial-progenitor-cell-seeding-technology

Research

JoVE Journal

Bioengineering

20.3K visualizações.  Duke University. Um método para semeadura de titânio de sangue entrar em contato com biomateriais com células autólogas e biocompatibilidade teste é descrito. Este método utiliza células progenitoras endoteliais e tubos de titânio, semeadas em poucos minutos de implante cirúrgico em suínos as cavas. Esta técnica é adaptável a muitas outras implantáveis ​​dispositivos biomédicos.

Vídeo: Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model

Electric Cell-substrate Impedance Sensing for the Quantification of Endothelial Proliferation, Barrier Function, and Motility

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. To this end, cells are grown in special culture chambers on top of opposing, circular gold electrodes. A constant small alternating current is applied between the electrodes and the potential across is measured. The insulating properties of the cell membrane create a resistance towards the electrical current flow resulting in an increased electrical potential between the electrodes. Measuring cellular impedance in this manner allows the automated study of cell attachment, growth, morphology, function, and motility. Although the ECIS measurement itself is straightforward and easy to learn, the underlying theory is complex and selection of the right settings and correct analysis and interpretation of the data is not self-evident. Yet, a clear protocol describing the individual steps from the experimental design to preparation, realization, and analysis of the experiment is not available. In this article the basic measurement principle as well as possible applications, experimental considerations, advantages and limitations of the ECIS system are discussed. A guide is provided for the study of cell attachment, spreading and proliferation; quantification of cell behavior in a confluent layer, with regard to barrier function, cell motility, quality of cell-cell and cell-substrate adhesions; and quantification of wound healing and cellular responses to vasoactive stimuli. Representative results are discussed based on human microvascular (MVEC) and human umbilical vein endothelial cells (HUVEC), but are applicable to all adherent growing cells.

This protocol reviews Electric Cell-substrate Impedance Sensing, a method to record and analyze the impedance spectrum of adherent cells for the quantification of cell attachment, proliferation, motility, and cellular responses to pharmacological and toxic stimuli. Detection of endothelial permeability and assessment of cell-cell and cell-substrate contacts are emphasized.

Elétrica Cell-substrato impedância Sensing para a quantificação do endotelial Proliferação, Barreira Function, e Motilidade

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. ...

Bioengenharia

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to model well differentiated human airway epithelium and heart muscle cells. This could be an invaluable tool to study the deleterious effects of toxic inhaled chemicals, such as chlorine, that can normally interact with the cell surfaces, and form various byproducts upon reacting with water, and limiting their effects in submerged cultures. Our model using well differentiated human airway epithelial cell cultures at air-liqiuid interface circumvents this limitation as well as provides an opportunity to evaluate critical mechanisms of toxicity of potential poisonous inhaled chemicals. We describe enhanced loss of membrane integrity, caspase release and death upon toxic inhaled chemical such as chlorine exposure. In this article, we propose methods to model chlorine exposure in mammalian heart and airway epithelial cells in culture and simple tests to evaluate its effect on these cell types.

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

This protocol is designed to demonstrate exposure method of cell cultures to inhaled toxic chemicals. Exposure of differentiated air-liquid&#160;interface (ALI) cultures of airway epithelial cells provides a unique model of airway exposure to toxic gases such as chlorine. In this manuscript we describe effect of chlorine exposure on air-liquid&#160;interface cultures of epithelial cells and submerged culture of cardiomyocytes. In vitro exposure systems allow important mechanistic studies to evaluate pathways that could then be utilized to develop novel therapeutic agents.

A cultura in vitro modelo celular para Toxic Chemical Testing Inhaled

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

Camada por camada de colágeno Deposição em microfluídicos para Microtissue Estabilização

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

Ferromagnetic Bare Metal Stent for Endothelial Cell Capture and Retention

Rapid endothelialization of cardiovascular stents is needed to reduce stent thrombosis and to avoid anti-platelet therapy which can reduce bleeding risk. The feasibility of using magnetic forces to capture and retain endothelial outgrowth cells (EOC) labeled with super paramagnetic iron oxide nanoparticles (SPION) has been shown previously. But this technique requires the development of a mechanically functional stent from a magnetic and biocompatible material followed by in-vitro and in-vivo testing to prove rapid endothelialization. We developed a weakly ferromagnetic stent from 2205 duplex stainless steel using computer aided design (CAD) and its design was further refined using finite element analysis (FEA). The final design of the stent exhibited a principal strain below the fracture limit of the material during mechanical crimping and expansion. One hundred stents were manufactured and a subset of them was used for mechanical testing, retained magnetic field measurements, in-vitro cell capture studies, and in-vivo implantation studies. Ten stents were tested for deployment to verify if they sustained crimping and expansion cycle without failure. Another 10 stents were magnetized using a strong neodymium magnet and their retained magnetic field was measured. The stents showed that the retained magnetism was sufficient to capture SPION-labeled EOC in our in-vitro studies. SPION-labeled EOC capture and retention was verified in large animal models by implanting 1 magnetized stent and 1 non-magnetized control stent in each of 4 pigs. The stented arteries were explanted after 7 days and analyzed histologically. The weakly magnetic stents developed in this study were capable of attracting and retaining SPION-labeled endothelial cells which can promote rapid healing.

Our goals were to design, manufacture and test ferromagnetic stents for endothelial cell capture. Ten stents were tested for fracture and 10 more stents were tested for retained magnetism. Finally, 10 stents were tested in-vitro and 8 more stents were implanted in 4 pigs to show cell capture and retention.

Ferromagnético Bare Metal Stent para captura de células endoteliais e Retenção

Rapid endothelialization of cardiovascular stents is needed to reduce stent thrombosis and to avoid anti-platelet therapy which can reduce bleeding risk. ...

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold. Previously, we developed a system for fabricating self-assembled tissue rings by seeding cells into non-adhesive agarose wells. A polydimethylsiloxane (PDMS) negative was first cast in a machined polycarbonate mold, and then agarose was gelled in the PDMS negative to create ring-shaped cell seeding wells. However, the versatility of this approach was limited by the resolution of the tools available for machining the polycarbonate mold. Here, we demonstrate that 3D-printed plastic can be used as an alternative to machined polycarbonate for fabricating PDMS negatives. The 3D-printed mold and revised mold design is simpler to use, inexpensive to produce, and requires significantly less agarose and PDMS per cell seeding well. We have demonstrated that the resulting agarose wells can be used to create self-assembled tissue rings with customized diameters from a variety of different cell types. Rings can then be used for mechanical, functional, and histological analysis, or for fabricating larger and more complex tubular tissues.

This protocol describes a platform for fabricating self-assembled tissue rings in variable sizes using a customized 3D-printed plastic mold. PDMS negatives are cured in the 3D-printed mold; then agarose is cast in the cured PDMS negatives. Cells are seeded into the resulting agarose wells where they aggregate into tissue rings.

Fabricação de poços de Agarose personalizado para a propagação de células e tecido anel Self-assembly usando moldes 3D-impresso

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, differentiation, morphology, and molecular synthesis. However, variables such stimuli strength and stimulation times need to be considered when stimulating either cells, tissues or scaffolds. Given that EFs and MFs vary according to cellular response, it remains unclear how to build devices that generate adequate biophysical stimuli to stimulate biological samples. In fact, there is a lack of evidence regarding the calculation and distribution when biophysical stimuli are applied. This protocol is focused on the design and manufacture of devices to generate EFs and MFs and implementation of a computational methodology to predict biophysical stimuli distribution inside and outside of biological samples. The EF device was composed of two parallel stainless-steel electrodes located at the top and bottom of biological cultures. Electrodes were connected to an oscillator to generate voltages (50, 100, 150 and 200 Vp-p) at 60 kHz. The MF device was composed of a coil, which was energized with a transformer to generate a current (1 A) and voltage (6 V) at 60 Hz. A polymethyl methacrylate support was built to locate the biological cultures in the middle of the coil. The computational simulation elucidated the homogeneous distribution of EFs and MFs inside and outside of biological tissues. This computational model is a promising tool that can modify parameters such as voltages, frequencies, tissue morphologies, well plate types, electrodes and coil size to estimate the EFs and MFs to achieve a cellular response.

This protocol describes the step-by-step process to build both electrical and magnetic stimulators used to stimulate biological tissues. The protocol includes a guideline to simulate computationally electric and magnetic fields and manufacture of stimulator devices.

Dispositivos de campo elétrico e magnético para estimulação de tecidos biológicos

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...

A Large Animal Model for Pulmonary Hypertension and Right Ventricular Failure: Left Pulmonary Artery Ligation and Progressive Main Pulmonary Artery Banding in Sheep

Decompensated right ventricular failure (RVF) in pulmonary hypertension (PH) is fatal, with limited medical treatment options. Developing and testing novel therapeutics for PH requires a clinically relevant large animal model of increased pulmonary vascular resistance and RVF. This manuscript discusses the latest development of the previously published ovine PH-RVF model that utilizes left pulmonary artery (PA) ligation and main PA occlusion. This model of PH-RVF is a versatile platform to control not only the disease severity but also the RV&#39;s phenotypic response.
Adult sheep (60-80 kg) underwent left PA (LPA) ligation, placement of main PA cuff, and insertion of RV pressure monitor. PA cuff and RV pressure monitor were connected to subcutaneous ports. Subjects underwent progressive PA banding twice per week for 9 weeks with sequential measures of RV pressure, PA cuff pressures, and mixed venous blood gas (SvO2). At the initiation and endpoint of this model, ventricular function and dimensions were assessed using echocardiography. In a representative group of 12 animal subjects, RV mean and systolic pressure increased from 28 &#177; 5 and 57 &#177; 7 mmHg at week 1, respectively, to 44 &#177; 7 and 93 &#177; 18 mmHg (mean &#177; standard deviation) by week 9. Echocardiography demonstrated characteristic findings of PH-RVF, notably RV dilation, increased wall thickness, and septal bowing. The longitudinal trend of SvO2 and PA cuff pressure demonstrates that the rate of PA banding can be titrated to elicit varying RV phenotypes. A faster PA banding strategy led to a precipitous decline in SvO2 &#60; 65%, indicating RV decompensation, whereas a slower, more paced strategy led to the maintenance of physiologic SvO2 at 70%-80%. One animal that experienced the accelerated strategy developed several liters of pleural effusion and ascites by week 9. This chronic PH-RVF model provides a valuable tool for studying molecular mechanisms, developing diagnostic biomarkers, and enabling therapeutic innovation to manage RV adaptation and maladaptation from PH.

This manuscript describes the surgical technique and experimental approach to develop severe right ventricular pressure overload to model their adaptive and maladaptive phenotypes.

Um grande modelo animal para hipertensão pulmonar e falha ventricular direita: Ligação da Artéria Pulmonar Esquerda e Banda de Artéria Pulmonar Principal Progressiva em Ovelhas

Decompensated right ventricular failure (RVF) in pulmonary hypertension (PH) is fatal, with limited medical treatment options. Developing and testing ...

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.

Produção automatizada em larga escala de esferoides de células-tronco derivados de adiposo para bioimpressão 3D

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue ...

In Silico Clinical Trials for Cardiovascular Disease

The SILICOFCM project mainly aims to develop a computational platform for in silico clinical trials of familial cardiomyopathies (FCMs). The unique characteristic of the platform is the integration of patient-specific biological, genetic, and clinical imaging data. The platform allows the testing and optimization of medical treatment to maximize positive therapeutic outcomes. Thus, adverse effects and drug interactions can be avoided, sudden cardiac death can be prevented, and the time between the commencement of drug treatment and the desired result can be shortened. This article presents a parametric model of the left ventricle automatically generated from patient-specific ultrasound images by applying an electromechanical model of the heart. Drug effects were prescribed through specific boundary conditions for inlet and outlet flow, ECG measurements, and calcium function for heart muscle properties. Genetic data from patients were incorporated through the material property of the ventricle wall. Apical view analysis involves segmenting the left ventricle using a previously trained U-net framework and calculating the bordering rectangle based on the length of the left ventricle in the diastolic and systolic cycle. M-mode view analysis includes bordering of the characteristic areas of the left ventricle in the M-mode view. After extracting the dimensions of the left ventricle, a finite elements mesh was generated based on mesh options, and a finite element analysis simulation was run with user-provided inlet and outlet velocities. Users can directly visualize on the platform various simulation results such as pressure-volume, pressure-strain, and myocardial work-time diagrams, as well as animations of different fields such as displacements, pressures, velocity, and shear stresses.

In Silico Clinical Trials for Cardiovascular Disease

This protocol demonstrates the workflow of the SILICOFCM platform for automatically generating a parametric model of the left ventricle from patient-specific ultrasound images by applying a multi-scale electromechanical model of the heart. This platform enables in silico clinical trials intended to reduce real clinical trials and maximize positive therapeutic outcomes.

em Silico Ensaios clínicos para doenças cardiovasculares

The SILICOFCM project mainly aims to develop a computational platform for in silico clinical trials of familial cardiomyopathies (FCMs). The unique ...

Avaliação das propriedades hidrodinâmicas em valvas aórticas suínas montadas

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.

Autor em destaque: Avançando nos testes de procedimentos cardíacos antes de embarcar em estudos com animais de grande porte 

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.

Um Modelo Ex Vivo Porcino para Testes Hidrodinâmicos de Procedimentos Experimentais de Valva Aórtica e Novos Dispositivos Médicos

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 ...