The authors acknowledge support from a Translational Breast Cancer Research Grant (14-60-26-BROC to AB) from the Breast Cancer Research Foundation and the American Association for Cancer Research.

Procedimentos usando indivíduos animais foram aprovados pelo Comitê de Cuidado e Uso Institucional Animal da Universidade do Texas em Austin. 1. Pré-operatório Preparação <ol><li> Registe o peso do corpo de cada coelho. Tal como acontece com todos os estudos pré-clínicos, monitorar os pesos dos animais regularmente para avaliar a toxicidade potencial. </li><li> Antes de se anestesiar o coelho, encher um tubo de 50 mL cónico com gel de ultra-sons comercialmente disponível e de centrifugação a 500 xg durante 30 s; não deve haver bolhas visíveis no gel após a conclusão da centrifugação. </li><li> Administrar glicopirrolato por via subcutânea numa dose de 0,1 mg / kg e acepromazina por via intramuscular numa dose de 0,75 mg / kg. Aguarde 15-20 minutos para o sedativo para entrar em vigor durante o acompanhamento de sinais e comportamentos vitais. NOTA: O glicopirrolato é um agente anti-colinérgico que impede bradicardia e reduz respiratórias e secreções gastrointestinais. Acepromazina é um sedativo que serve como um premedicatipor anestesia. </li><li> Administrar 35 mg / kg de cetamina e 5 mg / kg de xilazina por via subcutânea como um anestésico. No entanto, como o operador torna-se mais experiente e pode realizar a entrega intraductal mais rapidamente, diminuir as doses de droga a 15 mg / kg de cetamina e 3 mg / kg de xilazina por via subcutânea; isto irá diminuir o tempo de anestesia e o tempo necessário para o animal a recuperar da anestesia. </li><li> Verificar e documentar a frequência cardíaca, SPO 2, temperatura, frequência respiratória, e cor membrana mucosa a cada 15 minutos. Aplique lubrificante olho em ambos os olhos. NOTA: Apenas pessoal que tenha recebido uma formação adequada e tenham sido aprovados pelo IACUC da sua instituição deve administrar ou controlar a anestesia. A utilização de um monitor de anestesia veterinária pode auxiliar na aquisição de sinais vitais, é recomendada. O monitor de anestesia veterinária, no entanto, não substitui a necessidade de verificar manualmente o animal a cada 15 minutos. Veja as instruções do fabricante fou o uso adequado de um monitor de veterinária. </li><li> Verifique se o início da anestesia por uma pitada toe suave; o coelho deve ser não-responsivo antes de continuar. </li><li> Cuidadosamente raspar o abdome caudal do coelho na área em torno do terceiro e quarto pares de tetas inguinais. </li><li> Com a maioria dos cabelos removidos, aplicar um creme de depilação over-the-counter à área rapada. Retire o creme de 10 min após a aplicação usar toalhas de papel húmidas molhadas com água morna. NOTA: pele de coelho é muito frágil / sensível. O creme de depilação deve ser usado para não mais do que 10 min. De facto, é mais seguro para tentar um &quot;ponto de teste&quot; em 5 min e apenas deixar no local já não se teste indica um tempo mais longo é necessário período. </li><li> Limpe a área com compressas de gaze embebido em álcool para limpar o local da injeção. </li><li> Coloque o coelho no seu dorso em uma calha em forma de v forrada com um cobertor água morna recirculação e uma almofada absorvente. </li></ol> 2. Preparation do agente de contraste <ol><li> Reconstituir o reagente não-alvo de contraste de acordo com as instruções do fabricante. Pipeta cima e para baixo suavemente para misturar. NOTA: O agente de contraste utilizado na presente protocolo é estável à temperatura ambiente durante 4-6 h depois da reconstituição. Agite suavemente o frasco entre cada recuperação. NOTA: O volume da solução requerida irá depender do número de condutas a ser injectada. O coelho tem aberturas 4 ductais por tetina, e 0,2 mL de solução é suficiente para preencher uma árvore ductal de um adulto coelho branco da Nova Zelândia (Oryctolaguscuniculus). Assim, um volume total de 0,8 mL pode ser entregue a uma glândula mamaria. </li></ol> 3. Entrega Intraductal <ol><li> Localize as tetas apropriadas a serem injetados; os 3º e 4º pares de tetas inguinais são recomendados, pois eles são facilmente visualizados quando o animal é posicionado sobre seu dorso. </li><li> Carga 0,2 ml de solução salina estéril 0,9% emuma seringa de tuberculina de 1 ml de Luer-lock com uma agulha 22 G. Descarte a agulha G 22 uma vez que a solução salina é na seringa e substituí-lo com uma agulha de calibre 25 estéril. Limpe cuidadosamente a área com 85% de álcool isopropílico em uma gaze. </li><li> Com o bisel da agulha para cima e a seringa paralela ao corpo do animal, inserir o bisel da agulha para o lado da tetina e injectar lentamente 0,1-0,2 mL de solução salina; isso vai permitir uma melhor visualização das aberturas ductal. </li><li> Carregar 0,2 ml de solução de injecção para uma seringa de tuberculina de 1 ml de bloqueio Luer. </li><li> Segure a tetina suavemente com o polegar eo dedo indicador e levante-o ligeiramente para posicioná-la para a injeção intraductal; uma lupa de ocular wearable pode ajudar a visualizar as aberturas ductal. </li><li> Enquanto se mantém a posição elevada do teto, canular cuidadosamente a conduta de interesse usando uma agulha de ponta romba 25 G. </li><li> Após punção, gire suavemente a seringa Luer-lock no cubo da franco-tagulha da infusão ip até travar no lugar. </li><li> Levantar a tetina e injectar a solução lentamente para minimizar o dano potencial causado pelo movimento rápido do fluido no interior da conduta; em nenhum momento deve haver resistência ao injetar a solução. </li></ol> 4. Ecografia <ol><li> Aplique uma quantidade generosa de gel de ultra-som centrifugado para a pele da área de interesse. Certifique-se de que não existem bolhas no gel, como estes irão comprometer a qualidade da imagem. </li><li> Defina a profundidade da imagem a 6 mm. Posicionar o transdutor de 21 MHz em contacto com o gel e digitalizar a área de interesse em modo-B. Observar o meio de contraste na região digitalizada, inclusive a abertura ductal e ao longo da conduta. NOTA: Estas definições foram desenvolvidos para uso com uma máquina de ultra-som fotoacústica específico. Consulte a tabela de materiais para mais detalhes. Pode ser necessário ajustar a potência de transmissão e a profundidade da imagem de outros sistemas de imagem para optimizar mamáriavisualização glândula. </li><li> Remover o gel de ultra-som da pele do animal com uma almofada de gaze. </li></ol> 5. Cuidados no pós-operatório <ol><li> Observar o local da injecção: não deve haver sinais de trauma para a região da tetina ou para o tecido circundante, e inchaço na área circundante da tetina provavelmente indica uma injecção de almofada de gordura mamaria em vez de uma injecção intraductal bem sucedida. </li><li> Coloque o coelho numa posição esternal. Se necessário, dar 0,2 mg / kg de ioimbina por via intravenosa na veia marginal da orelha; isto irá reverter o efeito da xilazina e permitir que o animal a recuperar mais rapidamente da anestesia. </li><li> Monitorar o coelho a cada 15 min durante todo o período de recuperação até que o animal está alerta, ágil e mantém uma posição esternal. NOTA: Este procedimento não deve resultar em danos nos tecidos ou inchaço. Se vermelhidão ou inchaço são observados, administrar uma dose de meloxicam de 0,1-0,2 mg / kg, PO, uma vez que o animal é alerta e capazde tomar a medicação por via oral. Entre em contato com o pessoal veterinário da instituição para obter mais orientações. </li></ol>

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Feasibility+Study+of+the+Intraductal+Administration+of+Chemotherapy.">A Feasibility Study of the Intraductal Administration of Chemotherapy.</a> Cancer Prevention Research. 6 (1), 51-58 (2013).</li><li>Stearns, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+and+Clinical+Evaluation+of+Intraductally+Administered+Agents+in+Early+Breast+Cancer.">Preclinical and Clinical Evaluation of Intraductally Administered Agents in Early Breast Cancer.</a> Sci Transl Med. 3 (106), 106ra108 (2011).</li><li>Zhang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Safety+Parameters+of+the+Study+on+Intraductal+Cytotoxic+Agent+Delivery+to+the+Breast+before+Mastectomy.">The Safety Parameters of the Study on Intraductal Cytotoxic Agent Delivery to the Breast before Mastectomy.</a> Chin J Cancer Res. 26 (5), 579-587 (2014).</li><li>Mahoney, M. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Intraductal+Approach+to+the+Breast:+Raison+d'Etre.">The Intraductal Approach to the Breast: Raison d'Etre.</a> Breast Cancer Res. 8 (2), 206 (2006).</li><li>Bu, W., Xin, L., Toneff, M., Li, L., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentivirus+vectors+for+stably+introducing+genes+into+mammary+epithelial+cells+in+vivo+.">Lentivirus vectors for stably introducing genes into mammary epithelial cells in vivo .</a> J Mammary Gland Biol Neoplasia. 14, 401-404 (2009).</li><li>Lyons, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Direct+Mammotrophic+Action+of+Lactogenic+Hormone.">The Direct Mammotrophic Action of Lactogenic Hormone.</a> Proc. Soc. Exp. Bio. Med. 51 (2), 308-311 (1942).</li><li>Fiddler, T. J., Birkinshaw, M., Falconer, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+Of+Intraductal+Prolactin+On+Some+Aspects+of+the+Ultrastructure+and+Biochemistry+of+Mammary+Tissue+in+the+Pseudopregnant+Rabbit.">Effects Of Intraductal Prolactin On Some Aspects of the Ultrastructure and Biochemistry of Mammary Tissue in the Pseudopregnant Rabbit.</a> J Endocrinol. 49 (3), 459-469 (1971).</li><li>Falconer, I. R., Fiddler, T. 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Long-term Follow-up After Treatment by Biopsy Alone.</a> JAMA. 239 (18), 1863-1867 (1978).</li><li>Eusebi, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+Follow-up+of+In+Situ+Carcinoma+of+the+Breast.">Long-term Follow-up of In Situ Carcinoma of the Breast.</a> Semin Diagn Pathol. 11 (3), 223-235 (1994).</li><li>Sanders, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Natural+History+Of+Low-Grade+Ductal+Carcinoma+In+Situ+Of+The+Breast+In+Women+Treated+By+Biopsy+Only+Revealed+Over+30+Years+Of+Long-Term+Follow-Up.">The Natural History Of Low-Grade Ductal Carcinoma In Situ Of The Breast In Women Treated By Biopsy Only Revealed Over 30 Years Of Long-Term Follow-Up.</a> Cancer. 103 (12), 2481-2484 (2005).</li><li>Esserman, L. 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The authors have nothing to disclose.

Este método de entrega intraductal para a glândula mamaria de coelho pode ser utilizada para reagentes de contraste para ultrassons e muitas outras soluções aquosas, incluindo corantes vitais e terapêutica. Estudos anteriores demonstraram a entrega intraductal de hormônios 17, 18, 19. Em modelos de roedores, a entrega intraductal de ácidos nucleicos 8, quimioterápicos 6,</s...

<html> A entrega intraductal de agentes terapêuticos tem sido estudada em modelos de roedores e em início de carreira testes em humanos 3, 4, 5, 6, 11, 12. A Fase I recente estudo demonstrou a segurança ea viabilidade de carboplatina intraductal ou intraductal doxorrubicina lipossomal peguilada em mulheres que aguardam mastectomia para o tratamento de câncer invasivo 2. Protocolos anteriores para entrega intraductal foram desenvolvidos para rato e mamárias de ratos glândulas 6, 7, 8, 9. Para fins de investigação, as injecções de células de tumor intraductal e a entrega de vector lentiviral de oncogenes também foram realizados em modelos de roedoresref &quot;&gt; 13, 14, 15, 16. No entanto, um ideal em modelo vivo do processo de entrega intraductal deve permitir o desenvolvimento de novas classes de compostos terapêuticos e facilitar a avaliação pré-clínica. diferenças anatômicas entre roedores e humanos têm complicado a tradução destes estudos. Ao contrário dos ratos, em que cada conduta termina a uma tetina separada, da mama humano consiste de 5 a 9 ductais sistemas independentes, cada um com uma abertura separada terminando na tetina. Coelho glândulas mamárias abrigar quatro sistemas ductais independentes, cada um separadamente, acessível através de um de quatro orifícios em uma única tetina. Um modelo de coelho corresponda melhor a anatomia humana e permite o estudo da entrega da droga intraductal em um contexto mais relevante. Aqui, usamos duas técnicas para avaliar a prestação intraductal. A co-administração de umcorante vital permite a visualização através da pele e fornece uma confirmação simples e rápida do método. Para algumas aplicações, o mapeamento de resolução mais elevado dos ductos podem ser preferidos. Nós apresentamos aqui um protocolo para ultra-sonografia das condutas através da entrega intraductal de um reagente não-alvo contraste.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>MicroMarker non-targeted contrast reagent</td>
			<td>VisualSonics</td>
			<td>VS-11694</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer Lock 1mL Syringes</td>
			<td>BD</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycopyrrolate 0.2mg/mL</td>
			<td>Wedgewood Compounding Pharmacy</td>
			<td>GLYCOP-INJ013VC</td>
			<td>6 month shelf life, supply may be limited.&#160;</td>
		</tr>
		<tr>
			<td>Atropine Sulfate 0.5 mg/mL</td>
			<td>Animal Health International</td>
			<td>15320764</td>
			<td>If glycopyrrolate is unavailable. Not to be combined with glycopyrrolate.</td>
		</tr>
		<tr>
			<td>Ketamine HCL 100mg/mL</td>
			<td>Animal Health International</td>
			<td>21250699</td>
			<td><a href="http://www.animalhealthinternational.com/">http://www.animalhealthinternational.com/</a></td>
		</tr>
		<tr>
			<td>Acepromazine 10mg/mL</td>
			<td>Animal Health International</td>
			<td>17640541</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine 20mg/mL</td>
			<td>Animal Health International</td>
			<td>20101547</td>
			<td></td>
		</tr>
		<tr>
			<td>Yohimbine 0.2mg/mL</td>
			<td>Animal Health International</td>
			<td>14588965</td>
			<td></td>
		</tr>
		<tr>
			<td>Hair Removing Cream</td>
			<td>Veet</td>
			<td></td>
			<td>Sensitive skin solution. Available through local retailers.</td>
		</tr>
		<tr>
			<td>Blunt tip infusion needles</td>
			<td>Sai Infusion Technology</td>
			<td>B14-50</td>
			<td><a href="http://www.sai-infusion.com/collections/blunt-needles">http://www.sai-infusion.com/collections/blunt-needles</a></td>
		</tr>
		<tr>
			<td>Veterinary Pulse Oximeter</td>
			<td>EdanUSA</td>
			<td>VE-H100B</td>
			<td><a href="http://www.edanusa.com/Product/VE-H100B-Veterinary-Pulse-Oximeter.html">http://www.edanusa.com/Product/VE-H100B-Veterinary-Pulse-Oximeter.html</a></td>
		</tr>
		<tr>
			<td>Warm Water Pump</td>
			<td>Gaymar</td>
			<td>TP700</td>
			<td></td>
		</tr>
		<tr>
			<td>Warm Water Blanket</td>
			<td>Animal Health International</td>
			<td>21232696</td>
			<td>Maxi-Therm Lite Warming Pads</td>
		</tr>
		<tr>
			<td>Ultrasound system</td>
			<td>VisualSonics</td>
			<td>Vevo 2100</td>
			<td></td>
		</tr>
	</tbody>
</table>

intraductal delivery to the rabbit mammary gland

Intraductais tratamentos localizados para o cancro da mama oferecem vantagens potenciais, incluindo a entrega eficiente para o tumor e reduziu a toxicidade sistémica e efeitos adversos 1, 2, 3, 4, 5, 6, 7. No entanto, vários desafios permanecem antes que esses tratamentos podem ser aplicados de forma mais ampla. O desenvolvimento e validação de terapêuticas intraductais em um modelo animal apropriado facilitar o desenvolvimento de estratégias terapêuticas intraductais para os pacientes. Enquanto a glândula mamaria do rato tem sido amplamente utilizada como um sistema modelo de desenvolvimento mamário e a tumorigénese, a anatomia é distinta da glândula humano. Um modelo animal maior, tal como o coelho, pode servir como modelo para uma melhor estrutura de glândula mamaria e no desenvolvimento terapêutico intraductal. em cOntrast para ratos, em que dez árvores ductal são espacialmente distribuídos ao longo do eixo do corpo, cada uma terminando em uma teta separada, a glândula mamária de coelho mais se assemelha a glândula humana, com vários sistemas sobrepostos ductal que a saída através de aberturas separadas em uma teta. Aqui, apresentamos métodos minimamente invasivos para a entrega de reagentes diretamente no ducto mamário coelho e para visualização do próprio parto com a ultra-sonografia de alta resolução.

Aqui, mostra-se que a entrega de reagentes intraductal contraste com as condutas mamarias de um coelho pode ser alcançado sem trauma para o tecido (Figura 2). Em coelhos, quatro sistemas ductais separadas convergem para uma tetina e, assim, podem ser acedidos individualmente e fotografada utilizando este método. aberturas ductal individuais são facilmente visualizado; Observe a marcação de uma segunda abertura ductal adjacente à conduta canulada na Figura 2...

Watch this Scientific Journal Video about Intraductal Delivery to the Rabbit Mammary Gland at JoVE.com

Intraductal Delivery to the Rabbit Mammary Gland

Here, we describe a technique for the localized delivery of reagents to the rabbit mammary gland via an intraductal injection. In addition, we describe a protocol for visualization and the confirmation of delivery by high-resolution ultrasound imaging of contrast agents.

Entrega Intraductal ao Coelho glândula mamária

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and ...

intraductal-delivery-to-the-rabbit-mammary-gland

Research

JoVE Journal

Bioengineering

9.2K visualizações.  The University of Texas at Austin. Here, we describe a technique for the localized delivery of reagents to the rabbit mammary gland via an intraductal injection. In addition, we describe a protocol for visualization and the confirmation of delivery by high-resolution ultrasound imaging of contrast agents. 

Vídeo: Intraductal Delivery to the Rabbit Mammary Gland

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches1. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible1. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization1. 
The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart2. It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling3. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.
Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia14-16.

This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.

Multiparamétrico Mapping Optical do Coração Coelho Langendorff-perfundidos

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

Bioengenharia

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

Projeto de um biorreator de pressão cíclica para o Ex Vivo Estudo de válvulas cardíacas Aórtica

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively for tissue regeneration, a number of variables must be taken into account. These variables include: the total volume and surface area of the implantation site, the mechanical properties of the tissue and the tissue microenvironment, which includes the amount of vascularization and the components of the extracellular matrix. Therefore, the materials being used to deliver these cells must be biocompatible with a defined chemical composition while maintaining a mechanical strength that mimics the host tissue. These materials must also be permeable to oxygen and nutrients to provide a favorable microenvironment for cells to attach and proliferate. Chitosan, a cationic polysaccharide with excellent biocompatibility, can be easily chemically modified and has a high affinity to bind with in vivo macromolecules4-5. Chitosan mimics the glycosaminoglycan portion of the extracellular matrix, enabling it to function as a substrate for cell adhesion, migration and proliferation. In this study we utilize chitosan in the form of microspheres to deliver adipose-derived stem cells (ASC) into a collagen based three-dimensional scaffold6. An ideal cell-to-microsphere ratio was determined with respect to incubation time and cell density to achieve maximum number of cells that could be loaded. Once ASC are seeded onto the chitosan microspheres (CSM), they are embedded in a collagen scaffold and can be maintained in culture for extended periods. In summary, this study provides a method to precisely deliver stem cells within a three dimensional biomaterial scaffold.

A major hurdle in current stem cell therapies is determining the most effective method to deliver these cells to host tissues. Here, we describe a chitosan-based delivery method that is efficient and simple in approach, while allowing adipose-derived stem cells to maintain their multipotency.

Construindo um hidrogel de colágeno para a entrega de células-tronco carregado microesferas

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively ...

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant exogenous molecular probes into cells. This work reports a microfluidic electroporation platform capable of performing multiple molecule delivery to mammalian cells with precise and molecular-dependent parameter control. The system&#8217;s ability to isolate cells with uniform size distribution allows for less variation in electroporation efficiency per given electric field strength; hence enhanced sample viability. Moreover, its process visualization feature allows for observation of the fluorescent molecular uptake process in real-time, which permits prompt molecular delivery parameter adjustments in situ for efficiency enhancement. To show the vast capabilities of the reported platform, macromolecules with different sizes and electrical charges (e.g., Dextran with MW of 3,000 and 70,000 Da) were delivered to metastatic breast cancer cells with high delivery efficiencies (&#62;70%) for all tested molecules. The developed platform has proven its potential for use in the expansion of research fields where on-chip electroporation techniques can be beneficial.

A microfluidic vortex assisted electroporation platform was developed for sequential delivery of multiple molecules into identical cell populations with precise and independent dosage control. The system&#8217;s size based target cell purification step preceding electroporation aided to enhance molecular delivery efficiency and processed cell viability.

Microescala Electroporator para entrega Sequential Molecular assistida por Vortex

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

Adaptação do Processo Electrospinning para fornecer três ambientes únicos para um Tri-camadas<em&gt; In Vitro</em&gt; Modelo da parede das vias aéreas

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is limited to cell proliferation and gene expression endpoints. However, in the organism, mammary morphogenesis occurs in a 3D environment. 3D culture systems help bridge the gap between monolayer cell culture (2D) and the complexity of the organism. Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action. It uses the commercially available hormone-responsive human breast epithelial cell line, T47D, and rat tail collagen type 1 as a matrix. This 3D culture model responds to the main mammotropic hormones: estradiol, progestins and prolactin. The influence of these hormones on epithelial morphogenesis can be observed after 1- or 2-week treatment according to the endpoint. The 3D cultures can be harvested for analysis of epithelial morphogenesis, cell proliferation and gene expression.

We describe a 3D culture model of the human breast epithelium that is suitable to study hormone action.

Um modelo de cultura 3D Hormone-responsive da Human glândula mamária Epitélio

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is ...

Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes. The emergence of prion diseases in wildlife populations and their increasing threat to human health has led to increased efforts to find a treatment for these diseases. Recent studies have found numerous anti-prion compounds that can either inhibit the infectious PrPRes isomer or down regulate the normal cellular prion protein. However, most of these compounds do not cross the blood brain barrier to effectively inhibit PrPRes formation in brain tissue, do not specifically target neuronal PrPC, and are often too toxic to use in animal or human subjects. 
We investigated whether siRNA delivered intravascularly and targeted towards neuronal PrPC is a safer and more effective anti-prion compound. This report outlines a protocol to produce two siRNA liposomal delivery vehicles, and to package and deliver PrP siRNA to neuronal cells. The two liposomal delivery vehicles are 1) complexed-siRNA liposome formulation using cationic liposomes (LSPCs), and 2) encapsulated-siRNA liposome formulation using cationic or anionic liposomes (PALETS). For the LSPCs, negatively charged siRNA is electrostatically bound to the cationic liposome. A positively charged peptide (RVG-9r [rabies virus glycoprotein]) is added to the complex, which specifically targets the liposome-siRNA-peptide complexes (LSPCs) across the blood brain barrier (BBB) to acetylcholine expressing neurons in the central nervous system (CNS). For the PALETS (peptide addressed liposome encapsulated therapeutic siRNA), the cationic and anionic lipids were rehydrated by the PrP siRNA. This procedure results in encapsulation of the siRNA within the cationic or anionic liposomes. Again, the RVG-9r neuropeptide was bound to the liposomes to target the siRNA/liposome complexes to the CNS. Using these formulations, we have successfully delivered PrP siRNA to AchR-expressing neurons, and decreased the PrPC expression of neurons in the CNS.

The goal of this protocol is to use cationic/anionic liposomes with a neuro-targeting peptide as a CNS delivery system to enable siRNA to cross the BBB. The optimization of a delivery system for treatments, like siRNA, would allow for more treatment options for prion and other neurodegenerative diseases.

Entrega de terapêutica siRNA para o SNC Usando catiônicos e aniônicos lipossomas

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes.  The ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

Fabricação de espumas de Matriz Extracelular derivados e Microcarregadores como cultura celular de tecidos específicos e plataformas de entrega

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Retroductal Nanoparticle Injection to the Murine Submandibular Gland

Two common goals of salivary gland therapeutics are prevention and cure of tissue dysfunction following either autoimmune or radiation injury. By locally delivering bioactive compounds to the salivary glands, greater tissue concentrations can be safely achieved versus systemic administration. Furthermore, off target tissue effects from extra-glandular accumulation of material can be dramatically reduced. In this regard, retroductal injection is a widely used method for investigating both salivary gland biology and pathophysiology. Retroductal administration of growth factors, primary cells, adenoviral vectors, and small molecule drugs has been shown to support gland function in the setting of injury. We have previously shown the efficacy of a retroductally injected nanoparticle-siRNA strategy to maintain gland function following irradiation. Here, a highly effective and reproducible method to administer nanomaterials to the murine submandibular gland through Wharton's duct is detailed (Figure 1). We describe accessing the oral cavity and outline the steps necessary to cannulate Wharton's duct, with further observations serving as quality checks throughout the procedure.

Local drug delivery to the submandibular glands is of interest in understanding salivary gland biology and for the development of novel therapeutics. We present an updated and detailed retroductal injection protocol, designed to improve delivery accuracy and experimental reproducibility. The application presented herein is the delivery of polymeric nanoparticles.

Injeção de Retroductal de nanopartículas para a glândula Submandibular murino

Two common goals of salivary gland therapeutics are prevention and cure of tissue dysfunction following either autoimmune or radiation injury. By locally ...

Growth and Characterization of Irradiated Organoids from Mammary Glands

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell monolayers. Although they cannot completely model in vivo complexity, they retain some functionality of the original organ. In cancer models, organoids are commonly used to study tumor cell invasion. This protocol aims to develop and characterize organoids from the normal and irradiated mouse mammary gland tissue to evaluate the radiation response in normal tissues. These organoids can be applied to future in vitro cancer studies to evaluate tumor cell interactions with irradiated organoids. Mammary glands were resected, irradiated to 20 Gy and digested in a collagenase VIII solution. Epithelial organoids were separated via centrifugal differentiation, and 3D organoids were developed in 96-well low-adhesion microplates. Organoids expressed the characteristic epithelial marker cytokeratin 14. Macrophage interaction with the organoids was observed in co-culture experiments. This model may be useful for studying tumor-stromal interactions, infiltration of immune cells, and macrophage polarization within an irradiated microenvironment.

Organoids developed from mouse mammary glands were irradiated and characterized to assess epithelial traits and interactions with immune cells. Irradiated organoids can be used to better evaluate cell-cell interactions that may lead to tumor cell recruitment in irradiated normal tissue.

Crescimento e caracterização de organóides irradiados de glândulas mamárias

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell ...