Name: preparation of human myocardial tissue for long-term cultivation
Uploaded: 2022-06-02T14:10:00
Description: Watch this Scientific Journal Video about Preparation of Human Myocardial Tissue for Long-Term Cultivation at JoVE.com

A pesquisa foi financiada por bolsas DZHK 81Z0600207 (JH, PS e DM) e 81X2600253 (AD e TS).
Os autores gostariam de agradecer Claudia Fahney, Mei-Ping Wu e Matthias Semisch pelo apoio na preparação das configurações, bem como pela manutenção regular do cultivo de tecidos.

A coleta de tecidos para os experimentos aqui descritos foi aprovada pelos Conselhos de Revisão Institucional da Universidade de Munique e do Bochum ruhr-university. Os estudos foram realizados de acordo com as diretrizes da Declaração de Helsinque. Os pacientes deram seu consentimento por escrito informado antes da coleta de tecidos.
1. Aquisição de tecidos
<ol><li>Obtenha tecido humano de pacientes submetidos a transplante de coração ou cirurgia cardíaca.</li>
	<li...

<ol>
	<li>George, S. A., Brennan, J. A., Efimov, 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=Preclinical+cardiac+electrophysiology+assessment+by+dual+voltage+and+calcium+optical+mapping+of+human+organotypic+cardiac+slices.">Preclinical cardiac electrophysiology assessment by dual voltage and calcium optical mapping of human organotypic cardiac slices.</a> Journal of Visualized Expereiments: JoVE. (160), e60781 (2020).</li><li>Lu, K., 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=Progressive+stretch+enhances+growth+and+maturation+of+3D+stem-cell-derived+myocardium.">Progressive stretch enhances growth and maturation of 3D stem-cell-derived myocardium.</a> Theranostics. 11 (13), 6138-6153 (2021).</li><li>Pontes Soares, C., 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=2D+and+3D-organized+cardiac+cells+shows+differences+in+cellular+morphology,+adhesion+junctions,+presence+of+myofibrils+and+protein+expression.">2D and 3D-organized cardiac cells shows differences in cellular morphology, adhesion junctions, presence of myofibrils and protein expression.</a> PloS one. 7 (5), 38147 (2012).</li><li>Klumm, M. J., 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+cultivation+of+human+atrial+myocardium.">Long-term cultivation of human atrial myocardium.</a> Frontiers in Physiology. 13, 839139 (2022).</li><li>Krane, 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=Sequential+defects+in+cardiac+lineage+commitment+and+maturation+cause+hypoplastic+left+heart+syndrome.">Sequential defects in cardiac lineage commitment and maturation cause hypoplastic left heart syndrome.</a> Circulation. 144 (17), 1409-1428 (2021).</li><li>Miller, J. 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=Heart+slice+culture+system+reliably+demonstrates+clinical+drug-related+cardiotoxicity.">Heart slice culture system reliably demonstrates clinical drug-related cardiotoxicity.</a> Toxicology and Applied Pharmacology. , 406 (2020).</li><li>Fischer, C., 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+functional+and+structural+preservation+of+precision-cut+human+myocardium+under+continuous+electromechanical+stimulation+in+vitro.">Long-term functional and structural preservation of precision-cut human myocardium under continuous electromechanical stimulation in vitro.</a> Nature Communications. 10 (1), 1-12 (2019).</li><li>Kang, C., 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=Human+organotypic+cultured+cardiac+slices:+new+platform+for+high+throughput+preclinical+human+trials.">Human organotypic cultured cardiac slices: new platform for high throughput preclinical human trials.</a> Scientific Reports. 6 (1), 1-13 (2016).</li><li>Ou, Q., 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=Slicing+and+culturing+pig+hearts+under+physiological+conditions.">Slicing and culturing pig hearts under physiological conditions.</a> Journal of Visualized Experiments: JoVE. (157), e60913 (2020).</li><li>Ou, Q., 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=Physiological+biomimetic+culture+system+for+pig+and+human+heart+slices.">Physiological biomimetic culture system for pig and human heart slices.</a> Circulation research. 125 (6), 628-642 (2019).</li><li>Watson, S. A., 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=Biomimetic+electromechanical+stimulation+to+maintain+adult+myocardial+slices+in+vitro.">Biomimetic electromechanical stimulation to maintain adult myocardial slices in vitro.</a> Nature Communications. 10 (1), 1-15 (2019).</li><li>Abu-Khousa, 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+degree+of+t-system+remodeling+predicts+negative+force-frequency+relationship+and+prolonged+relaxation+time+in+failing+human+myocardium.">The degree of t-system remodeling predicts negative force-frequency relationship and prolonged relaxation time in failing human myocardium.</a> Frontiers in Physiology. 11, 182 (2020).</li><li>Bojkova, D., 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=SARS-CoV-2+infects+and+induces+cytotoxic+effects+in+human+cardiomyocytes.">SARS-CoV-2 infects and induces cytotoxic effects in human cardiomyocytes.</a> Cardiovascular Research. 116 (14), 2207-2215 (2020).</li><li>Esfandyari, D. A. -. O., 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=MicroRNA-365+regulates+human+cardiac+action+potential+duration.">MicroRNA-365 regulates human cardiac action potential duration.</a> Nature Communications. 13 (1), 1-15 (2022).</li><li>Moretti, A., 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=Somatic+gene+editing+ameliorates+skeletal+and+cardiac+muscle+failure+in+pig+and+human+models+of+Duchenne+muscular+dystrophy.">Somatic gene editing ameliorates skeletal and cardiac muscle failure in pig and human models of Duchenne muscular dystrophy.</a> Nature Medicine. 26 (2), 207-214 (2020).</li><li>Pitoulis, F. G., 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=Remodelling+of+adult+cardiac+tissue+subjected+to+physiological+and+pathological+mechanical+load+in+vitro.">Remodelling of adult cardiac tissue subjected to physiological and pathological mechanical load in vitro.</a> Cardiovascular Research. 118 (3), 814-827 (2021).</li><li>Curtis, T. M., Scholfield, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nifedipine+blocks+Ca2++store+refilling+through+a+pathway+not+involving+L-type+Ca2++channels+in+rabbit+arteriolar+smooth+muscle.">Nifedipine blocks Ca2+ store refilling through a pathway not involving L-type Ca2+ channels in rabbit arteriolar smooth muscle.</a> The Journal of Physiology. 532 (3), 609-623 (2001).</li><li>de Weille, J. R., Schweitz, H., Maes, P., Tartar, A., Lazdunski, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calciseptine,+a+peptide+isolated+from+black+mamba+venom,+is+a+specific+blocker+of+the+L-type+calcium+channel.">Calciseptine, a peptide isolated from black mamba venom, is a specific blocker of the L-type calcium channel.</a> Proceedings of the National Academy of Sciences. 88 (6), 2437-2440 (1991).</li><li>Schleifer, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+molecular+modelling+study+of+the+calcium+channel+blockers+nifedipine+and+black+mamba+toxin+FS2.">Comparative molecular modelling study of the calcium channel blockers nifedipine and black mamba toxin FS2.</a> Journal of Computer-Aided Molecular Design. 11 (5), 491-501 (1997).</li><li>Thomas, G., Chung, M., Cohen, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dihydropyridine+(Bay-K8644)+that+enhances+calcium+currents+in+guinea+pig+and+calf+myocardial+cells.+A+new+type+of+positive+inotropic+agent.">A dihydropyridine (Bay-K8644) that enhances calcium currents in guinea pig and calf myocardial cells. A new type of positive inotropic agent.</a> Circulation Research. 56 (1), 87-96 (1985).</li><li>Pitoulis, F. G., Watson, S. A., Perbellini, F., Terracciano, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+slices+come+to+age:+an+intermediate+complexity+in+vitro+cardiac+model+for+translational+research.">Myocardial slices come to age: an intermediate complexity in vitro cardiac model for translational research.</a> Cardiovascular Research. 116 (7), 1275-1287 (2020).</li><li>Watson, S. A., 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=Preparation+of+viable+adult+ventricular+myocardial+slices+from+large+and+small+mammals.">Preparation of viable adult ventricular myocardial slices from large and small mammals.</a> Nature Protocols. 12 (12), 2623-2639 (2017).</li></ol>

No passado, a pesquisa cardiovascular fez grandes avanços no cultivo de cardiomiócitos. No entanto, o cultivo 3D de cardiomiócitos com geometria intacta ainda não está bem estabelecido. Em comparação com os protocolos anteriores aplicados para o cultivo ex vivo do tecido miocárdio, o protocolo que descrevemos aqui se assemelha mais ao ambiente in vivo do tecido. Além disso, a aplicação de pré e pós-carga permite um ambiente mais biomimético. Somos capazes de analisar e entender completamen...

Na última década, o cultivo in vitro de células miocárdias fez grandes avanços, desde técnicas 2D e tridimensionais (3D) até o uso de organoides e células-tronco pluripotentes induzidas diferenciadas em miócitos cardíacos 1,2,3. Os cultivos de ex vivo e células primárias têm se mostrado de grande valor, especialmente para estudos genéticos e desenvolvimento de medicamentos 4,5,6. O uso de tecidos humanos melhora o valor translacional dos resultados. O cultivo 3D a longo prazo de tecidos miocárdios com geometria intacta, no entanto, não é bem estabelecido. A geometria intacta é uma característica fundamental para imitar condições in vivo, já que a função cardíaca adequada, a comunicação entre diferentes células, bem como as interações entre matriz celular são pré-requisitos. O cultivo de tecido miocárdio passou por várias fases de desenvolvimento. A taxa de sucesso e a estabilidade do cultivo de tecido miocárdio ex vivo foram inicialmente bastante baixas, mas abordagens recentes produziram resultados promissoresde 7,8,9,10,11.
Entre eles, Fischer et al. foram os primeiros a demonstrar que a viabilidade e o desempenho contítil do tecido miocárdio humano podem ser mantidos no cultivo de células ex vivo por muitas semanas7. Sua técnica foi baseada em fatias finas de tecido cortadas do miocárdio humano explantado, que foram montadas em câmaras de cultivo recém-desenvolvidas que forneceram condições biomecânicas definidas e estimulação elétrica contínua. Este método de cultivo se assemelha muito à função in vivo do tecido miocárdio, e foi reproduzido por vários grupos de pesquisa independentes 2,12,13,14,15. É importante ressaltar que as câmaras utilizadas por Fischer et al. também permitiram o registro contínuo de forças desenvolvidas por até 4 meses, e assim abriram oportunidades inéditas para pesquisas fisiológicas e farmacológicas sobre miocárdio humanointacto 7.
Técnicas semelhantes foram desenvolvidas independentemente por outros grupos e aplicadas ao miocárdio humano, rato, suíno e coelho 7,10,11. Pitoulis et al. desenvolveram posteriormente um método mais fisiológico, que reproduz a relação normal de comprimento de força durante um ciclo de contração, mas é menos adequado para análise de alto rendimento16. Como tal, a abordagem geral do cultivo biomimético pode ser considerada como mais um passo para a redução, refinamento e substituição (3R) de experimentos animais.
No entanto, a exploração desse potencial requer procedimentos padronizados, análises de alto conteúdo e um alto nível de rendimento. Apresentamos uma técnica que combina o corte automatizado do miocárdio humano vivo com a manutenção in vitro em um sistema de cultivo biomimético que se tornou comercialmente disponível (ver Tabela de Materiais). Com a abordagem proposta, o número de fatias individuais que podem ser geradas a partir de uma única amostra miocárdia transmural é limitado apenas pelo tempo de processamento. Uma amostra de tamanho e qualidade suficientes (3 cm x 3 cm) muitas vezes produz 20-40 fatias de tecido sendo convenientemente cortadas com um vibratome automatizado. Essas fatias podem ser colocadas em câmaras de cultivo pertencentes ao sistema. As câmaras permitem estimulação elétrica, cujas parâmetros podem ser moduladas (ou seja, duração do pulso, polaridade, taxa e corrente), bem como o ajuste da pré e pós-carga, utilizando fios de mola dentro das câmaras. A contração de cada fatia é registrada a partir do movimento de um pequeno ímã ligado a um fio de mola e exibido como um gráfico interpretável. Os dados podem ser registrados o tempo todo e analisados usando software livremente disponível. Além do ritmo constante da linha de base, protocolos programados podem ser realizados para avaliar funcionalmente seu período refratário, limiar de estimulação, potencialização pós-pausa e relação de frequência de força.
Este cultivo biomimético de longo prazo de múltiplas fatias de miocárdio de um coração individual abre caminho para futuras pesquisas ex vivo em tecido humano e animal, e facilita a triagem para efeitos terapêuticos e cardiotóxicos de drogas na medicina cardiovascular. Já foi aplicado em diversas abordagens experimentais 2,12,13,15. Aqui, damos uma descrição detalhada passo a passo da preparação do tecido humano e fornecemos soluções para problemas de cultivo frequentemente encontrados.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose Low melting point</td>
			<td>Roth</td>
			<td>6351.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Bay-K8644</td>
			<td>Cayman Chemical</td>
			<td>19988</td>
			<td></td>
		</tr>
		<tr>
			<td>BDM (2,3-Butanedione monoxime)</td>
			<td>Sigma</td>
			<td>B0753-1kg</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2*H2O</td>
			<td>Merck</td>
			<td>2382.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Calciseptine</td>
			<td>Alomone Labs</td>
			<td>SPC-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose*H2O</td>
			<td>AppliChem</td>
			<td>A3730.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>H2O</td>
			<td>BBraun</td>
			<td>3703452</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>AppliChem</td>
			<td>A1069.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Histoacryl</td>
			<td>BBraun</td>
			<td>1050052</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol 100%</td>
			<td>SAV LP GmbH</td>
			<td>UN1219</td>
			<td></td>
		</tr>
		<tr>
			<td>ITS-X-supplement</td>
			<td>Gibco</td>
			<td>5150056</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Merck</td>
			<td>1.04933.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium 199</td>
			<td>Gibco</td>
			<td>31150-022</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2*6H2O</td>
			<td>AppliChem</td>
			<td>A1036.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S5886-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4*H2O</td>
			<td>Merck</td>
			<td>1.06346.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Nifedipine</td>
			<td>Sigma</td>
			<td>N7634-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin / streptomycin x100</td>
			<td>Sigma</td>
			<td>P0781-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>AppliChem</td>
			<td>A1108.0100</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cabinet</td>
			<td>Thermo Scientific</td>
			<td>KS15</td>
			<td></td>
		</tr>
		<tr>
			<td>Frigomix waterpump and cooling + BBraun Thermomix BM</td>
			<td>BBraun</td>
			<td></td>
			<td>In-house made combination of cooling and heating solution.</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Binder</td>
			<td>CB240</td>
			<td></td>
		</tr>
		<tr>
			<td>MyoDish bioreactor system</td>
			<td>InVitroSys GmbH</td>
			<td>MyoDish 1</td>
			<td>Myodish cultute system</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica</td>
			<td>VT1200s</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath 37 degrees</td>
			<td>Haake</td>
			<td>SWB25</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath 80 degrees</td>
			<td>Daglef Patz KG</td>
			<td>7070</td>
			<td></td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 mL plastic single-use beaker</td>
			<td>Sarstedt</td>
			<td>75.562.105</td>
			<td></td>
		</tr>
		<tr>
			<td>Filtration unit, Steritop Quick Release</td>
			<td>Millipore</td>
			<td>S2GPT05RE</td>
			<td></td>
		</tr>
		<tr>
			<td>Needles 0.9 x 70 mm 20G</td>
			<td>BBraun</td>
			<td>4665791</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic triangles</td>
			<td></td>
			<td></td>
			<td>In-house made</td>
		</tr>
		<tr>
			<td>Razor Derby premium</td>
			<td>Derby Tokai</td>
			<td>B072HJCFK6</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor Gillette Silver Blue</td>
			<td>Gillette</td>
			<td>7393560010170</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel disposable</td>
			<td>Feather</td>
			<td>02.001.30.020</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 10 mL Luer tip BD Discardit</td>
			<td>BBraun</td>
			<td>309110</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Dish 10 cm</td>
			<td>Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Dish 3.5 cm</td>
			<td>Falcon</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubes 50 mL</td>
			<td>Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of human myocardial tissue for long-term cultivation

O cultivo de cardiomiócitos tem visto um grande número de desenvolvimentos, desde o cultivo de células bidimensionais (2D) até organoides derivados do iPSC. Em 2019, foi demonstrada uma forma ex vivo de cultivar fatias de miocárdio obtidas a partir de amostras de coração humano, ao mesmo tempo em que se aproximava da condição in vivo de contração do miocárdio. Estas amostras são originárias principalmente de transplantes cardíacos ou colocações de dispositivos de assistência à esquerda. Utilizando um vibratome e um sistema de cultivo especialmente desenvolvido, fatias de 300 μm de espessura são colocadas entre um fio fixo e um fio de mola, permitindo um cultivo estável e reprodutível por várias semanas. Durante o cultivo, as fatias são continuamente estimuladas de acordo com as configurações individuais. Contrações podem ser exibidas e registradas em tempo real, e agentes farmacológicos podem ser prontamente aplicados. Os protocolos de estimulação definidos pelo usuário podem ser agendados e realizados para avaliar parâmetros vitais de contração, como potencialização pós-pausa, limiar de estimulação, relação força-freqüência e período refratário. Além disso, o sistema permite uma configuração variável de pré e pós-carga para um cultivo mais fisiológico.
Aqui, apresentamos um guia passo-a-passo sobre como gerar um cultivo bem-sucedido a longo prazo de fatias de miocárdio ventricular esquerdo humano, utilizando uma solução comercial de cultivo biomimético.

A contração das fatias do miocárdio foi exibida na tela do computador após a inserção da câmara de cultivo em seu conector correspondente (Figura 3). A contração das fatias do miocárdio humano começou imediatamente após a estimulação. As fatias hipercontratadas por 5-10 min. Isso foi visível como um aumento das forças diastólicas, causadas por uma contratura tônica de frações de tecido danificadas. Esse processo foi revertido para diferentes graus dentro de 1-1,5 h. Após...

Watch this Scientific Journal Video about Preparation of Human Myocardial Tissue for Long-Term Cultivation at JoVE.com

Preparation of Human Myocardial Tissue for Long-Term Cultivation

Apresentamos um protocolo para o cultivo ex vivo de tecido miocárdio ventricular humano. Permite uma análise detalhada da força de contração e cinética, bem como a aplicação de carga pré e posterior para imitar o ambiente fisiológico in vivo mais de perto.

Preparação do tecido miocárdio humano para cultivo a longo prazo

Cardiomyocyte cultivation has seen a vast number of developments, ranging from two-dimensional (2D) cell cultivation to iPSC derived organoids. In 2019, ...

Usando este protocolo, fatias de tecido ventricular esquerdo humano podem ser cultivadas ex vivo em uma câmara biomimética. A aplicação de pré e pós-carga melhora a semelhança do ambiente fisiológico. Esta técnica permite o registro contínuo de contração tecidual.Os protocolos de estimulação definidos pelo usuário permitem a avaliação de parâmetros vitais de contração, como potencialização pós-pausa, limiar de estimulação, relação força-freqüência e período refratário. O cultivo a longo prazo de fatias de miocárdio, utilizando essa configuração, abrirá caminho para futuras pesquisas ex vivo, facilitando essa triagem para efeitos terapêuticos e cardiotóxicos na medicina cardiovascular. Para começar, submergir as câmaras de cultivo e os eletrodos de grafite em um litro de uma solução isopropanol de 10% e agitar durante a noite.No dia seguinte, transfira as câmaras para uma solução de isopropanol 100% por 3 minutos e, em seguida, permita que as câmaras e eletrodos de grafite sequem sob um capô de fluxo laminar. Conecte uma placa de circuito a cada câmara de acordo com as posições disponíveis no roqueiro. Coloque dois eletrodos de grafite na placa do circuito de acordo com as instruções do fabricante e coloque uma tampa de placa de Petri de 35 milímetros na câmara para evitar infecções.Em seguida, encha a bandeja de corte de até 90 a 95% com tampão de corte. Transfira a amostra de tecido para uma placa de Petri de 100 milímetros cheia de tampão de corte frio, e coloque a placa em uma placa de resfriamento a 4 graus Celsius. Remova a trabecula do endocárdio segurando o endoárdio com pinças e use uma tesoura para cortar aproximadamente 3 milímetros de tecido endocárdio.Fixar a amostra de tecido cortado, lado endocárdio até um patch de borracha de 2 por 2 centímetros usando quatro agulhas de calibre 0,9 por 70 milímetros de calibre 20 que são fixadas em uma posição quadrada. Certifique-se de que a borda diagonal de cada ponta da agulha está apontando para dentro, melhorando a fixação e evitando danos ao miocárdio. Usando um bisturi, corte todo o excesso de tecido fora dos quatro quadrados de agulha.Usando pinças, coloque a amostra aparada em um pedaço de tecido estéril por 10 segundos para remover o excesso de tampão de corte. Em seguida, remova a seringa agarose do banho de água e submerse a amostra em agarose. Deixe a agarose solidificar por cinco minutos em uma placa de resfriamento.O lado endocárdio da amostra deve ser visível na agarose. Usando pinças, coloque o lado epicárido da amostra contida em agarose em cima da área colada e pressione suavemente a amostra contendo agarose do topo com uma ferramenta sem cortar ou danificar a agarose. Deixe a cola se solidificar por 1 minuto.Defina a amplitude de vibração para 1 milímetro, velocidade inicial de corte para 0,07 milímetros por segundo, e espessura da fatia para 300 mícrons. Depois de conectar o sistema de cultivo a um computador, inicie o programa de software correspondente. Defina a velocidade do roqueiro para 60 rpm e predefina os parâmetros de estimulação.Para fatias cardíacas humanas, defina a estimulação padrão para impulsos bifásicos com 50 miliamperes ou corrente composta por 3 milissegundos de corrente positiva, 1 milissegundo pausa, e um pulso de 3 milissegundos de corrente invertida a uma taxa de ritmo de 30 batidas por minuto ou bpm, em seguida, verifique os indicadores de eletrodos do software e verifique se os eletrodos das câmaras de cultivo estão funcionando corretamente. Usando pinças, separe a ágarose do tecido. Evite tocar o tecido e manuseá-lo cuidadosamente, pois qualquer dano ao tecido reduzirá a taxa de sucesso do cultivo.Para anexar dois triângulos plásticos a uma amostra, coloque 1 microliter de cola em uma tampa estéril da placa de Petri. Use uma pinça ligada para pegar um triângulo plástico autoclaved. Mergulhe rapidamente a borda frontal do triângulo na cola e cole o triângulo na amostra perpendicular ao alinhamento cardiomioceste.Repita para o outro triângulo. Usando um bisturi, corte o tecido que exceda a largura do triângulo e coloque a fatia com os dois triângulos montados de volta na bandeja de corte contendo tampão de corte. Remova a câmara de cultivo de preenchimento médio da incubadora.Selecione uma fatia preparada e insira-a na câmara conectando um triângulo a cada pino. Ajuste a distância entre os pinos de montagem até o tamanho da amostra. Certifique-se de que a amostra está submersa no meio.Depois de colocar o prato no roqueiro, diminua a pré-carga girando o parafuso de ajuste no sentido anti-horário até que a linha de base do gráfico correspondente na tela do computador não mude mais, em seguida, aumente cuidadosamente a pré-carga ou tensão girando o parafuso de ajuste no sentido horário. Para câmaras com alta rigidez, continue até que a linha de base correspondente no gráfico tenha aumentado em 1.000 para 1.200 unidades correspondentes a uma pré-carga milinovto. Depois de preparar o meio de cultivo fresco, pré-aqueça o meio em um banho de água ou incubadora de ar quente a 37 graus Celsius por 30 a 45 minutos.Retire o meio da câmara, deixando aproximadamente 0,8 mililitros na câmara, em seguida, adicione 1,6 mililitros de médio fresco para a mesma câmara, fazendo um volume total de médio a 2,4 mililitros por câmara. Por fim, coloque a tampa da câmara de volta e coloque a câmara de cultivo em sua respectiva posição. A leitura da contração de cinco fatias de miocárdio durante uma estimulação típica consistiu em quatro seções distintas de potencialização pós-pausa, limiar de estimulação, relação força-freqüência e período refratário.Em comparação com o controle, a força de contração das fatias tratadas com antagonistas de cálcio, nifedipina e calciseptina, foi diminuída em 10 minutos. Em contraste, o agonista do canal de cálcio fechado de voltagem, Bay-K8644, aumentou a força de contração. A potencialização pós-pausa do controle e das fatias tratadas foi avaliada para a liberação intracelular de cálcio do ânticulo sarcoplasmático.A fatia de controle não mostrou nenhuma mudança. Na presença de calciseptina e nifedipina, a inibição dos canais de cálcio do tipo L levou à potencialização da primeira contração após uma pausa de 50 segundos, refletindo uma maior contribuição relativa da liberação intracelular de cálcio à contratilidade total. O efeito oposto foi observado com Bay-K8644, que estimula a entrada de cálcio extracelular através dos canais de cálcio do tipo L.Na análise da relação de frequência de força, não foi observada alteração na fatia de controle. O tratamento da calciseptina não alterou a capacidade da fatia de seguir os estímulos após o aumento da frequência de estimulação ao comparar os dados pré e pós-tratamento. Em comparação com a calciseptina, a nifeipina impediu um aumento da contração a taxas de ritmo mais altas e reduziu a taxa máxima de captura em 80 bpm.Com Bay-K8644, observou-se aumento da força de contração em frequências de estimulação muito baixas. No entanto, em frequências superiores a 50 bpm, a força de contração foi menor do que durante a condição pré-tratamento. O tecido excisado deve ser rapidamente transferido para 4 graus de cardioplegia.Após o corte, triângulos plásticos devem ser anexados perpendicularmente à direção da fibra. A pré-carga não deve exceder 1500 mililitros. A cultura de fatias biomiméticas pode ajudar os pesquisadores a utilizar manipulações genéticas, bem como terapias baseadas em células para estudar a regeneração e a reparação no miocárdio humano adulto.

preparation-of-human-myocardial-tissue-for-long-term-cultivation

Research

JoVE Journal

Medicine

Corneal Donor Tissue Preparation for Endothelial Keratoplasty

Over the past ten years, corneal transplantation surgical techniques have undergone revolutionary changes1,2. Since its inception, traditional full thickness corneal transplantation has been the treatment to restore sight in those limited by corneal disease. Some disadvantages to this approach include a high degree of post-operative astigmatism, lack of predictable refractive outcome, and disturbance to the ocular surface. The development of Descemet's stripping endothelial keratoplasty (DSEK), transplanting only the posterior corneal stroma, Descemet's membrane, and endothelium, has dramatically changed treatment of corneal endothelial disease. DSEK is performed through a smaller incision; this technique avoids 'open sky' surgery with its risk of hemorrhage or expulsion, decreases the incidence of postoperative wound dehiscence, reduces unpredictable refractive outcomes, and may decrease the rate of transplant rejection3-6.
Initially, cornea donor posterior lamellar dissection for DSEK was performed manually1 resulting in variable graft thickness and damage to the delicate corneal endothelial tissue during tissue processing. Automated lamellar dissection (Descemet's stripping automated endothelial keratoplasty, DSAEK) was developed to address these issues. Automated dissection utilizes the same technology as LASIK corneal flap creation with a mechanical microkeratome blade that helps to create uniform and thin tissue grafts for DSAEK surgery with minimal corneal endothelial cell loss in tissue processing.
Eye banks have been providing full thickness corneas for surgical transplantation for many years. In 2006, eye banks began to develop methodologies for supplying precut corneal tissue for endothelial keratoplasty. With the input of corneal surgeons, eye banks have developed thorough protocols to safely and effectively prepare posterior lamellar tissue for DSAEK surgery. This can be performed preoperatively at the eye bank. Research shows no significant difference in terms of the quality of the tissue7 or patient outcomes8,9 using eye bank precut tissue versus surgeon-prepared tissue for DSAEK surgery. For most corneal surgeons, the availability of precut DSAEK corneal tissue saves time and money10, and reduces the stress of performing the donor corneal dissection in the operating room. In part because of the ability of the eye banks to provide high quality posterior lamellar corneal in a timely manner, DSAEK has become the standard of care for surgical management of corneal endothelial disease.
The procedure that we are describing is the preparation of the posterior lamellar cornea at the eye bank for transplantation in DSAEK surgery (Figure 1).

Endothelial corneal transplantation is a surgical technique for treatment of posterior corneal diseases. Mechanical microkeratome dissection to prepare tissue results in thinner, more symmetric grafts with less endothelial cell loss and improved outcomes. Dissections can be performed at the eye bank prior to corneal transplantation surgery.

Preparação do tecido da córnea Doadores de ceratoplastia endotelial

Over the past ten years, corneal transplantation surgical techniques have undergone revolutionary changes1,2. Since its inception, traditional full ...

Medicina

Working with Human Tissues for Translational Cancer Research

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human specimens, precious samples must be handled with the greatest care in order to ensure integrity of organs, tissues, and cells to the highest degree. Unfortunately, tissue processing by definition requires extraction of tissues from the host, a change which can cause great cellular stress and have major repercussions on subsequent analyses. These stresses could result in the specimen being no longer representative of the site from which it was retrieved. Therefore, a strict protocol must be adhered to while processing these specimens to ensure representativeness. The desired assay(s) must also be taken into consideration in order to ensure that an optimal technique is used for sample processing. Outlined here is a protocol for tissue retrieval, processing and various analyses which may be performed on processed tissue in order to maximize downstream production from limited tissue samples.

Translational cancer research is dependent on extraction of human tissues. Much work has gone into optimizing extraction methods for ex vivo analysis. Here, we describe tissue processing methods allowing for maximal data output from limited samples.

Trabalhando com tecidos humanos para Translational Cancer Research

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human ...

Long-Term Catheterization of the Intestinal Lymph Trunk and Collection of Lymph in Neonatal Pigs

Catheterization of the intestinal lymph trunk in neonatal pigs is a technique allowing for the long-term collection of large quantities of intestinal (central) efferent lymph. Importantly, the collection of central lymph from the intestine enables researchers to study both the mechanisms and lipid constitutes associated with lipid metabolism, intestinal inflammation and cancer metastasis, as well as cells involved in immune function and immunosurveillance. A ventral mid-line surgical approach permits excellent surgical exposure to the cranial abdomen and relatively easy access to the intestinal lymph trunk vessel that lies near the pancreas and the right ventral segment of the portal vein underneath the visceral aspect of the right liver lobe. The vessel is meticulously dissected and released from the surrounding fascia and then dilated with sutures allowing for insertion and subsequent securing of the catheter into the vessel. The catheter is exteriorized and approximately 1 L/24 hr of lymph is collected over a 7 day period. While this technique enables the collection of large quantities of central lymph over an extended period of time, the success depends on careful surgical dissection, tissue handling and close attention to proper surgical technique. This is particularly important with surgeries in young animals as the lymph vessels can easily tear, potentially leading to surgical and experimental failure. The video demonstrates an excellent surgical technique for the collection of intestinal lymph.

We present a surgical procedure to catheterize the intestinal lymph trunk in neonatal pigs to collect large quantities of lipid metabolism components from efferent lymph.

Long-Term Cateterismo do tronco linfa intestinal e Recolha de linfa no Neonatais Pigs

Catheterization of the intestinal lymph trunk in neonatal pigs is a technique allowing for the long-term collection of large quantities of intestinal ...

Surgical Placement of Catheters for Long-term Cardiovascular Exercise Testing in Swine

This protocol describes the surgical procedure to chronically instrument swine and the procedure to exercise swine on a motor-driven treadmill. Early cardiopulmonary dysfunction is difficult to diagnose, particularly in animal models, as cardiopulmonary function is often measured invasively, requiring anesthesia. As many anesthetic agents are cardiodepressive, subtle changes in cardiovascular function may be masked. In contrast, chronic instrumentation allows for measurement of cardiopulmonary function in the awake state, so that measurements can be obtained under quiet resting conditions, without the effects of anesthesia and acute surgical trauma. Furthermore, when animals are properly trained, measurements can also be obtained during graded treadmill exercise.
Flow probes are placed around the aorta or pulmonary artery for measurement of cardiac output and around the left anterior descending coronary artery for measurement of coronary blood flow. Fluid-filled catheters are implanted in the aorta, pulmonary artery, left atrium, left ventricle and right ventricle for pressure measurement and blood sampling. In addition, a 20 G&#160;catheter is positioned in the anterior interventricular vein to allow coronary venous blood sampling.
After a week of recovery, swine are placed on a motor-driven treadmill, the catheters are connected to pressure and flow meters, and swine are subjected to a five-stage progressive exercise protocol, with each stage lasting 3 min. Hemodynamic signals are continuously recorded and blood samples are taken during the last 30 sec of each exercise stage.
The major advantage of studying chronically instrumented animals is that it allows serial assessment of cardiopulmonary function, not only at rest but also during physical stress such as exercise. Moreover, cardiopulmonary function can be assessed repeatedly during disease development and during chronic treatment, thereby increasing statistical power and hence limiting the number of animals required for a study.

Here we present a protocol to assess cardiopulmonary function in awake swine, at rest and during graded treadmill exercise. Chronic instrumentation allows for repeated hemodynamic measurements uninfluenced by cardiodepressive anesthetic agents.

Cirurgia de colocação de cateteres para Cardiovascular Testes exercício de longo prazo em Suínos

This protocol describes the surgical procedure to chronically instrument swine and the procedure to exercise swine on a motor-driven treadmill. Early ...

Long-term Blood Pressure Measurement in Freely Moving Mice Using Telemetry

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. Indeed, research focusing on the discovery of new potential therapeutic targets using genetically altered mice requires a reliable, long-term assessment of the systemic arterial pressure variation. Currently, the gold standard for obtaining long-term measurements of blood pressure in ambulatory mice uses implantable radio-transmitters, which require&#160;artery cannulation. This technique eliminates the need for tethering, restraining, or anesthetizing the animals which introduce stress and artifacts during data sampling. However, arterial blood pressure monitoring in mice via catheterization can be rather challenging due to the small size of the arteries. Here we present a step-by-step guide to illustrate the crucial key passages for a successful subcutaneous implantation of radio-transmitters and carotid artery cannulation in mice. We also include examples of long-term blood pressure activity taken from freely moving mice after a period of post-surgery recovery. Following this procedure will allow reliable direct blood pressure recordings from multiple animals simultaneously.

The goal of this protocol is to assess systemic blood pressure in conscious freely moving mice using implantable radio-telemetry devices.

Longo prazo medição de pressão arterial em ratos movimentando-se livremente utilizar telemetria

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. ...

3D Whole-heart Myocardial Tissue Analysis

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other biologicals that enhance angio- or vasculogenesis into the infarct border zone (IBZ), tissue perfusion is improved, and the myocardium can be protected from further damage. For maximum therapeutic effect, it is hypothesized that the regenerative substance is best delivered to the IBZ. This requires accurate injections and has led to the development of new injection techniques. To validate these new techniques, we have designed a validation protocol based on myocardial tissue analysis. This protocol includes whole-heart myocardial tissue processing that enables detailed two-dimensional (2D) and three-dimensional (3D) analysis of the cardiac anatomy and intramyocardial injections. In a pig, myocardial infarction was created by a 90-min occlusion of the left anterior descending coronary artery. Four weeks later, a mixture of a hydrogel with superparamagnetic iron oxide particles (SPIOs) and fluorescent beads was injected in the IBZ using a minimally-invasive endocardial approach. 1 h after the injection procedure, the pig was euthanized, and the heart was excised and embedded in agarose (agar). After the solidification of the agar, magnetic resonance imaging (MRI), slicing of the heart, and fluorescence imaging were performed. After image post-processing, 3D analysis was performed to assess the IBZ targeting accuracy. This protocol provides a structured and reproducible method for the assessment of the targeting accuracy of intramyocardial injections into the IBZ. The protocol can be easily used when the processing of scar tissue and/or validation of the injection accuracy of the whole heart is desired.

This protocol describes a novel method for the 3D comparison of whole-heart myocardial tissue with MRI. This is designed for the accurate assessment of intramyocardial injections in the infarct border zone of a chronic porcine model of myocardial infarction.

Análise Tecidos do Miocárdio Whole-coração 3D

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other ...

Improvement of a Closed Chest Porcine Myocardial Infarction Model by Standardization of Tissue and Blood Sampling Procedures

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved drug to prevent or reduce myocardial I/R injury. The study and understanding of the pathophysiological mechanisms of myocardial I/R injury is essential to develop successful treatments. Large animal experiments are an important step in translational methods. The porcine model of acute myocardial infarction has been established and described by ourselves and others. We aimed to further improve the value of the model by focusing in detail on the sampling techniques for use in future experiments. Furthermore, we emphasize small but important steps that can affect the quality of the final results. To mimic the clinical situation of myocardial I/R injury, a percutaneous coronary intervention (PCI) catheter was inserted into the left anterior descending coronary artery (LAD) of an anesthetized pig. &#176;&#176;&#176;This model mimics acute myocardial infarction and PCI treatment in humans with the possibility of accurately determining the area at risk as well as the necrotic- and viable ischemic tissue. Here the model was used to investigate the effect of a bicyclic peptide inhibitor of FXIIa. The model can also be modified to allow longer reperfusion times to study later effects of myocardial infarction.

Here we demonstrate a protocol to standardize sampling procedures of an established porcine model of acute myocardial infarction in order to increase its translational value in the understanding of the pathophysiology of myocardial ischemia/reperfusion injury and to test novel drug candidates.

Aprimoramento do modelo de suínos do miocárdio peito fechado pela padronização do tecido e os procedimentos de recolha de amostras de sangue

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved ...

Analyzing Long-Term Electrocardiography Recordings to Detect Arrhythmias in Mice

Arrhythmias are common, affecting millions of patients worldwide. Current treatment strategies are associated with significant side effects and remain ineffective in many patients. To improve patient care, novel and innovative therapeutic concepts causally targeting arrhythmia mechanisms are needed. To study the complex pathophysiology of arrhythmias, suitable animal models are necessary, and mice have been proven to be ideal model species to evaluate the genetic impact on arrhythmias, to investigate fundamental molecular and cellular mechanisms, and to identify potential therapeutic targets.
Implantable telemetry devices are among the most powerful tools available to study electrophysiology in mice, allowing continuous ECG recording over a period of several months in freely moving, awake mice. However, due to the huge number of data points (&gt;1 million QRS complexes per day), analysis of telemetry data remains challenging. This article describes a step-by-step approach to analyze ECGs and to detect arrhythmias in long-term telemetry recordings using the software, Ponemah, with its analysis modules, ECG Pro and Data Insights, developed by Data Sciences International (DSI). To analyze basic ECG parameters, such as heart rate, P wave duration, PR interval, QRS interval, or QT duration, an automated attribute analysis was performed using Ponemah to identify P, Q, and T waves within individually adjusted windows around detected R waves. 
Results were then manually reviewed, allowing adjustment of individual annotations. The output from the attribute-based analysis and the pattern recognition analysis was then used by the Data Insights module to detect arrhythmias. This module allows an automatic screening for individually defined arrhythmias within the recording, followed by a manual review of suspected arrhythmia episodes. The article briefly discusses challenges in recording and detecting ECG signals, suggests strategies to improve data quality, and provides representative recordings of arrhythmias detected in mice using the approach described above.

Here we present a step-by-step protocol for a semiautomated approach to analyze murine long-term electrocardiography (ECG) data for basic ECG parameters and common arrhythmias. Data are obtained by implantable telemetry transmitters in living and awake mice and analyzed using Ponemah and its analysis modules.

Analisando gravações de eletrocardiografia de longo prazo para detectar arritmias em camundongos

Arrhythmias are common, affecting millions of patients worldwide. Current treatment strategies are associated with significant side effects and remain ...

Technique for Obtaining Mesenchymal Stem Cell from Adipose Tissue and Stromal Vascular Fraction Characterization in Long-Term Cryopreservation

Human mesenchymal stem cells derived from adipose tissue have become increasingly attractive as they show appropriate features and are an accessible source for regenerative clinical applications. Different protocols have been used to obtain adipose-derived stem cells. This article describes different steps of an improved time-saving protocol to obtain a more significant amount of ADSC, showing how to cryopreserve and thaw ADSC to obtain viable cells for culture expansion. One hundred milliliters of lipoaspirate were collected, using a 26 cm three-hole and 3 mm caliber syringe liposuction, from the abdominal area of nine patients who subsequently underwent elective abdominoplasty. The stem cells isolation was carried out with a series of washes with Dulbecco&#39;s Phosphate Buffered Saline (DPBS) solution supplemented with calcium and the use of collagenase. Stromal Vascular Fraction (SVF) cells were cryopreserved, and their viability was checked by immunophenotyping. The SVF cellular yield was 15.7 x 105 cells/mL, ranging between 6.1-26.2 cells/mL. Adherent SVF cells reached confluence after an average of 7.5 (&#177;4.5) days, with an average cellular yield of 12.3 (&#177; 5.7) x 105 cells/mL. The viability of thawed SVF after 8 months, 1 year, and 2 years ranged between 23.06%-72.34% with an average of 47.7% (&#177;24.64) with the lowest viability correlating with cases of two-year freezing. The use of DPBS solution supplemented with calcium and bag resting times for fat precipitation with a shorter time of collagenase digestion resulted in an increased stem cell final cellular yield. The detailed procedure for obtaining high yields of viable stem cells was more efficient regarding time and cellular yield than the techniques from previous studies. Even after a long period of cryopreservation, viable ADSC cells were found in the SVF.

The present protocol describes an improved methodology for ADSC isolation resulting in a tremendous cellular yield with time gain compared to the literature. This study also provides a straightforward method for obtaining a relatively large number of viable cells after long-term cryopreservation.

Técnica para obtenção de células-tronco mesenquimais a partir da caracterização do tecido adiposo e da fração vascular estromal em criopreservação a longo prazo

Human mesenchymal stem cells derived from adipose tissue have become increasingly attractive as they show appropriate features and are an accessible ...

Long-Term Continuous Measurement of Renal Blood Flow in Conscious Rats

The kidneys play a crucial role in maintaining the homeostasis of body fluids. The regulation of renal blood flow (RBF) is essential to the vital functions of filtration and metabolism in kidney function. Many acute studies have been carried out in anesthetized animals to measure RBF under various conditions to determine mechanisms responsible for the regulation of kidney perfusion. However, for technical reasons, it has not been possible to measure RBF continuously (24 h/day) in unrestrained unanesthetized rats over prolonged periods. These methods allow the continuous determination of RBF over many weeks while also simultaneously recording blood pressure (BP) with implanted catheters (fluid-filled or by telemetry). RBF monitoring is carried out with rats placed in a circular servo-controlled rat cage that enables the unrestrained movement of the rat throughout the study. At the same time, the tangling of cables from the flow probe and arterial catheters is prevented. Rats are first instrumented with an ultrasonic flow probe placement on the left renal artery and an arterial catheter implanted in the right femoral artery. These are routed subcutaneously to the nape of the neck, and connected to the flowmeter and pressure transducer, respectively, to measure RBF and BP. Following surgical implantation, rats are immediately placed in the cage to recover for at least one week and stabilize the ultrasonic probe recordings. Urine collection is also feasible in this system. The surgical and post-surgical procedures for continuous monitoring are demonstrated in this protocol.

The present protocol describes a long-term continuous measurement of renal blood flow in conscious rats and simultaneously recording blood pressure with implanted catheters (fluid-filled or by telemetry).