Este trabalho é apoiado financeiramente pela Fundação Nacional Suíço (NRP programa 64, conceder nenhuma 4064-131232).

1. Preparação do sistema de perfusão <ol><li> Configure o sistema de perfusão que consiste em um banho de água, uma câmara de perfusão, duas colunas para a oxigenação, duas bombas peristálticas, duas armadilhas bolha, dois geradores de fluxo e um sensor de pressão (Figura 1). Ligação desses componentes com as secções de tubagem composta de materiais de silicone e cloreto de polivinilo de acordo com o esquema da Figura 2. Finalmente, existem dois circuitos representando o circuito fetal e materna, respectivamente. </li><li> Ligue o banho de água, os aquecedores de fluxo eo aquecimento para a câmara de perfusão. A temperatura deve ser de 37 ° C. </li><li> Aquecer o meio de perfusão (NCTC-135 meio de cultura de tecidos diluídas 1:2 com tampão de Earle (6,8 g / L de cloreto de sódio, 0,4 g / L de cloreto de potássio, 0,14 g / L de fosfato monossódico, 0,2 g / L de sulfato de magnésio, 0,2 g / L de cloreto de cálcio, 2 g / L de glucose) suplementado com glucose (1 g / L), dextrano 40 (10 g / L), albumina do soro bovino (10 g/ L), heparina de sódio (2.500 UI / L), amoxicilina (250 mg / L) e bicarbonato de sódio (2,2 g / L), pH 7,4), em banho de água. </li><li> Consecutivamente enxaguar os sistemas arteriais de o circuito fetal e materno com um) 200 ml de água destilada, b) 50 mL de hidróxido de sódio a 1%, c) de 1% de ácido fosfórico e d) novamente 200 ml de água destilada (taxa de fluxo: 15 - 20 ml / min). </li><li> Ligue a cânula fetal (ø 1,2 mm; agulha romba deve ser anexado a um conjunto de infusão agulha alada modificada) para o tubo arterial fetal. </li><li> Enxaguar os sistemas arteriais de o circuito fetal e materno com um meio de perfusão até que todos os tubos contêm meio (caudal: 15-20 mL / min.) Durante esta etapa de encher os retentores de bolhas e remover todas as bolhas de jusante da armadilha. Em seguida, parar nas bombas. É muito importante que os tubos arteriais aferentes são sempre livre de bolhas, caso contrário, após punção especialmente os vasos fetais finas podem se romper. </li><li> Ligue o fluxo de gás. O circuito maternal é oxigenado wipo 5% de dióxido de carbono e 95% de ar sintético e o circuito fetal, com 5% de dióxido de carbono e 95% de azoto. </li><li> Iniciar a gravação do sensor de pressão. </li></ol> 2. Canulação da Placenta <ol><li> Obter placentas intacto de gestações complicadas prazo após cesariana primária. Consentimento por escrito tem de ser dada (foi obtido no caso de nossos estudos) pelas mães antes do parto e que o estudo tem de ser aprovado pelo comitê de ética local (foi o caso em nossos estudos). Primeiro controle visual deve ser feito por parteiras para garantir uma placenta saudável e intacta. </li><li> Canulação da placenta é um passo crítico! Durante a perfusão a cada pequena ruptura no tecido pode conduzir a uma fuga entre a circulação materna e fetal. A placenta tem de ser obtida dentro de 30 min após o parto. </li><li> Seleccione um dos cotilédones intactos a zona marginal da placenta sem perturbações visíveis no lado materno. Na placa coriônica,amarrar ambos os ramos associados da artéria umbilical e veia a montante para o lado de punção mais tarde (em direção ao cordão umbilical) usando material de sutura cirúrgica. Faça sempre dois nós. </li><li> Canular a artéria fetal do primeiro. As artérias da placenta fetal são sempre menores e mais finas do que as veias. </li><li> Fazer uma sutura ao redor da artéria fetal, mas não amarrá-lo imediatamente. Mantenha o recipiente com uma pinça, corte o vaso com cuidado e colocar a pequena cânula (ø 1,2 mm) na artéria. Em seguida, amarre-se a sutura (dois nós). </li><li> Continuar com a veia fetal da mesma maneira, mas com um maior cânula (Ø 1,5-1,8 mm; agulha romba deve ser ligado a um conjunto de infusão de agulha com abas modificado). </li><li> Ligar a bomba fetal (2 mL / min.) Se não houver fugas e sangue visível emana da cânula da fetal, lentamente aumentar o fluxo de 4 ml / min. Observe a pressão na artéria fetal, não deve exceder os 70 mm Hg. Se o fluido vaza na fetal ou companheirornal cânula corrigi-los com outro sutura. </li><li> Coloque a placenta no suporte do tecido com o lado fetal e puxe a membrana placentária e tecido sobre os pontos. No final, o cotilédone perfundido deve estar no meio do furo no suporte do tecido. </li><li> Estabilizar a parte em que a membrana tem apenas a placenta com uma membrana de silicone (Ø 1 mm) ou, alternativamente, duas peças de parafilme. </li><li> Monte o suporte do tecido completo, aperte os parafusos e cortar o tecido pendendo. Por favor, note que o cânulas venosa e arterial não fiquem presos, mas em vez disso estava nos pequenos canais de suporte do tecido. </li><li> Gire o suporte do tecido de cabeça para baixo, colocá-lo na câmara de perfusão e adicionar a tampa. Agora, o lado materno deve estar no topo. Verifique sempre se o circuito fetal ainda está intacto eo meio está fluindo para fora do tubo de veia fetal. </li><li> Ligar a bomba materna (12 ml / min.) Introduzir os três contundente cânulas (Ø 0,8 mm) foi no the extremidade do tubo arterial materno para o espaço intervilositário penetrando a placa decidual. Para retornar o perfusato no circuito materna colocar um tubo de drenagem venosa, que também está ligado com a bomba materno para a posição mais baixa na parte superior da câmara de perfusão. </li><li> Ligação a cânula na veia fetal ao tubo veia fetal. </li></ol> 3. Executar o pré-and Experimental Fase de Perfusão <ol><li> Para permitir que o tecido para se recuperar a partir do período isquémico após o parto e para expulsar o sangue no espaço intervilositário, uma abertura pré-fase de 20 minutos é necessária. Isso significa que a veia materna e fetal não estão levando de volta para o reservatório arterial contendo o meio de perfusão. Recolher o fluxo venoso fetal e materna em uma garrafa e descartá-lo após a pré-fase. </li><li> Para avaliar a integridade da perfusão realizar outra pré-fase de 20 minutos, mas em um circuito fechado. Usar dois reservatórios separados com o meio de perfusãopara o circuito fetal e maternal e fechar os circuitos, levando o fluxo venoso fetal de volta no reservatório fetal e maternal fluxo venoso de volta no reservatório maternal. </li><li> Para o experimento principal perfusão preparar dois frascos com 120 ml de meio de perfusão (um para a mãe e para o reservatório de um feto). Adicionar o radiomarcado 14 C-antipirina (4 nCi / ml, serve como controlo positivo; CUIDADO: substância radioactiva) e xenobióticos ou nanopartículas marcado por fluorescência que se pretende analisar para o reservatório materna. Misturar bem no perfusato materna. </li><li> Iniciar o experimento trocando o meio de perfusão puro com os dois balões preparados (reservatórios fetais e maternas). Feche os circuitos, levando o fetal venoso saída de volta no reservatório fetal e maternal fluxo venoso de volta no reservatório maternal. </li><li> Continuar a perfusão durante 6 horas e recolher amostras regularmente. Sempre ressuspender o meio no fetal e maternareservatório antes da retirada. </li><li> Controlar a pressão na artéria fetal (não deve exceder os 70 mm Hg), em ambos os circuitos de pH (deve estar numa gama fisiológica 7,2-7,4), e o volume de ambos os reservatórios (perda de volume do feto não deve ser superior a 4 ml / h) durante a perfusão . Se necessário ajustar o pH usando quer o ácido clorídrico ou hidróxido de sódio. </li><li> Se a perda de volume no reservatório excede fetal de 4 ml / hr existe uma fuga no tecido e tem de se parar a perfusão. A taxa de perfusão durante 6 horas, sem vazamento de sucesso é de cerca de 15-20%. </li><li> Pare a perfusão após 6 horas. Apague as bombas, banhos de água, aquecedores de fluxo e fluxo de gás. </li><li> Retire a placenta do detentor do tecido, corte o cotilédones perfusão (mais brilhante do que o tecido não perfundidada) e pesá-lo. </li><li> Colher amostras de não perfundidada (parte da placenta que foi cortado no início, já poderia ser tomado durante a pré-fase) do tecido e perfusão (cada uma cerca de 1 g) e armazená-los a -20 ° Caté homogeneização ou em azoto líquido, para posterior análise. Fix outra amostra de tecido em formalina 4% para avaliação histopatológica. As amostras devem incluir todas as camadas da placenta. </li><li> Limpar os tubos após perfusão, por lavagem, sucessivamente, os sistemas arteriais de o circuito fetal e materno com um) 200 ml de água destilada, b) 50 mL de hidróxido de sódio a 1%, c) 50 ml de ácido fosfórico a 1% e d) novamente 200 ml de água destilada (caudal: 15-20 mL / min.) </li></ol> O procedimento de funcionamento de todo o experimento perfusão placenta é ilustrado na Figura 3. 4. Analisando as amostras <ol><li> Centrifugar as amostras de perfusato durante 10 minutos a 800 xg antes da análise para remover os eritrócitos residuais. Leve o sobrenadante para a análise posterior. As amostras podem ser deixadas durante a noite a 4 ° C. Para a análise da produção de hCG, a leptina e as amostras podem ser armazenadas a -20 ° C. </li><li> Para avaliar a permeabilidade daplacenta analisar o C-antipirina 14 por cintilação líquida. Misturar 300 ul das amostras fetais e maternas com 3 ml de cocktail de cintilação e medida durante 5 minutos num contador beta. </li><li> Para avaliar a transferência das nanopartículas fluorescentes ou xenobióticos de interesse ler a fluorescência a 485 nm de excitação e de emissão de 528 nm em um leitor de microplacas (comprimentos de onda são indicadas para a análise do rótulo verde amarelo que foi usado para as nanopartículas). </li><li> Para determinar a viabilidade do tecido placentário durante a perfusão medida do consumo de glucose e produção de lactato no circuito fetal e materno com um sistema automático de gás do sangue. Além disso, avaliar a produção das hormonas choriongonadotropin placentária humana (hCG) e leptina nas amostras de tecido homogeneizado e os perfusatos por ensaio imunoenzimático (ELISA). </li></ol>

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Os autores declaram que não têm interesses financeiros concorrentes.

Sob a perfusão de recirculação dupla mostrou aqui, existem várias outras configurações experimentais possíveis, dependendo da questão que tem de ser respondida. Perfusões placentários Particularmente abertos são usados ​​para avaliar a depuração da droga a uma concentração de estado estacionário 3. A perfusão de recirculação set-up também pode ser aplicado para confirmar o transporte ativo de substâncias endógenas ou exógenas. Por esta abordagem, a mesma concentração do xenobióti...

A placenta é um órgão complexo que é responsável pela troca de oxigénio e dióxido de carbono, de nutrientes e de produtos de resíduos e, ao mesmo tempo capaz de manter os dois circuitos de sangue da mãe e para o feto em crescimento separados uns dos outros. Além disso, evita a rejeição da criança pelo sistema imune materno e segrega hormonas para manter a gravidez. A barreira celular é formada pelas células citotrofoblasto que fundem e formam uma verdadeira sincício sem membranas celulares laterais 4,5. Toda a placenta é organizado em várias cotilédones, que contêm uma árvore vilosidades fetal e representam uma unidade funcional da placenta. O estudo da função de barreira da placenta foi intensificada, com a descoberta dos malformações induzidas talidomida na década de 1960. Por razões óbvias, os estudos de translocação com mulheres grávidas não pode ser executada. Consequentemente, vários modelos alternativos foram desenvolvidos 6,7 </sup&gt;. O modelo relevante mais promissora, e provavelmente mais clínica é o modelo de perfusão ex-vivo de placenta humana desenvolvido por Panigel e colaboradores 2,3. Muitas mulheres estão expostas a diferentes xenobióticos, tais como drogas ou poluentes ambientais durante a gravidez 8. Para alguns medicamentos, que já foram administradas regularmente durante a gravidez, em estudos in vivo, pode ser realizada por comparação da concentração de sangue materno com aquele no sangue do cordão umbilical. No entanto, geralmente há apenas informações limitadas sobre a farmacocinética e-dinâmica no feto e da teratogenicidade destas substâncias. Por exemplo opiáceos como a heroína facilmente atravessar a barreira placentária e pode levar à restrição de crescimento intra-uterino, parto prematuro ou aborto espontâneo 9,10. Assim, em caso de abstinência desaparecidos durante a gravidez recomenda-se uma terapia de substituição com metadona. O exmodelo de perfusão in vivo de placenta humana revelou que a transferência de metadona na circulação fetal é negligenciável 11, o que se correlaciona bem com o sangue materno para cabo rácio de concentração no sangue calculados após entrega 12. A nanotecnologia é um campo em crescimento, especialmente em medicina. Assim, sob a natural fina (&lt;2,5 m de diâmetro) e partículas ultrafinas (&lt;0,1 m de diâmetro) em fumaça de incêndios florestais, erupções vulcânicas e poeira do deserto, a exposição a nanomateriais artificiais (pelo menos uma dimensão &lt;0,1 mícron 13 ) está a aumentar. Isso levantou questões sobre o potencial toxicológico de nanomateriais. Apesar de não haver perigo humano poderia ser provado, no entanto, não são os principais estudos experimentais indicam que as nanopartículas de engenharia pode provocar respostas biológicas adversas que levam a resultados toxicológicos 14. Recentemente, alguns estudos indicam que a exposição pré-natal aopoluição do ar está ligada a uma maior necessidade respiratório e inflamação das vias aéreas em recém-nascidos e crianças 15,16. Além disso, pequenas nanopartículas podem ser utilizados como veículos de fármacos para tratar especificamente tanto o feto ou a mãe. Portanto, torna-se evidente que os estudos extensivos de xenobióticos distintos ou nanomateriais e sua capacidade de atravessar a barreira placentária são obrigatórios. Um panorama atual sobre os estudos atuais sobre a permeabilidade placentária para nanomateriais é resumido em Menezes et al. 2.011 17 e Buerki-Thurnherr et al. 2.012 7. A dupla ex vivo modelo de perfusão de recirculação de placenta humana proporciona um sistema fiável e controlada para o estudo do transporte placentário de vários compostos endógenos e exógenos 3,11,12,18,19 e uma ampla gama de outras funções da placenta como mecanismos responsáveis ​​pela desenvolvimento de estados patológicos como a pré-eclâmpsia &lt;sup&gt; 20-22. Neste protocolo, focadas principalmente no set up, manuseio e método que permitem o estudo de acumulação, os efeitos e as taxas de translocação de um amplo conjunto de xenobióticos ou nanopartículas.

<table border="1">
	<tbody>
 <tr>
 <td>Name of the Reagent</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>NCTC-135 medium</td>
 <td>ICN Biomedicals, Inc.</td>
 <td>10-911-22C</td>
 <td>could be replaced by Medium 199 from Sigma (M3769)</td>
 </tr>
 <tr>
 <td>Sodium chloride (NaCl)</td>
 <td>Sigma-Aldrich, Fluka</td>
 <td>71381</td>
 <td></td>
 </tr>
 <tr>
 <td>Potassium chloride (KCl)</td>
 <td>Hospital pharmacy</td>
 <td></td>
 <td>also possible: Sigma (P9541)</td>
 </tr>
 <tr>
 <td>Monosodium phosphate (NaH2PO4 &middot; H2O)</td>
 <td>Merck</td>
 <td>106346</td>
 <td></td>
 </tr>
 <tr>
 <td>Magnesium sulfate (MgSO4 &middot; H2O)</td>
 <td>Sigma-Aldrich, Fluka</td>
 <td>63139</td>
 <td></td>
 </tr>
 <tr>
 <td>Calcium chloride (CaCl, anhydrous)</td>
 <td>Merck</td>
 <td>102388</td>
 <td></td>
 </tr>
 <tr>
 <td>D(+) Glucose (anhydrous)</td>
 <td>Sigma-Aldrich, Fluka</td>
 <td>49138</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium bicarbonate (NaHCO3)</td>
 <td>Merck</td>
 <td>106329</td>
 <td></td>
 </tr>
 <tr>
 <td>Dextran from Leuconostoc spp.</td>
 <td>Sigma-Aldrich</td>
 <td>31389</td>
 <td></td>
 </tr>
 <tr>
 <td>Bovine serum albumin (BSA)</td>
 <td>Applichem</td>
 <td>A1391</td>
 <td></td>
 </tr>
 <tr>
 <td>Amoxicilline (Clamoxyl)</td>
 <td>GlaxoSmithKline AG</td>
 <td>2021101A</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium heparin</td>
 <td>B. Braun Medical AG</td>
 <td>3511014</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium hydoxide (NaOH) pellets</td>
 <td>Merck</td>
 <td>106498</td>
 <td>CAUTION: corrosive</td>
 </tr>
 <tr>
 <td>Ortho-phosphoric acid 85% (H3PO4)</td>
 <td>Merck</td>
 <td>100573</td>
 <td>CAUTION: corrosive</td>
 </tr>
 <tr>
 <td>Maternal gas mixture: 95% synthetic air, 5% CO2</td>
 <td>PanGas AG</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Fetal gas mixture: 95% N2, 5% CO2</td>
 <td>PanGas AG</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Antipyrine (N-methyl-14C)</td>
 <td>American Radiolabeled Chemicals, Inc.</td>
 <td>ARC 0108-50 &mu;Ci</td>
 <td>CAUTION: radioactive material (specific activity: 55mCi/mmol)</td>
 </tr>
 <tr>
 <td>Scintillation cocktail (IrgaSafe Plus)</td>
 <td>Zinsser Analytic GmbH</td>
 <td>1003100</td>
 <td></td>
 </tr>
 <tr>
 <td>Polystyrene particles 80 nm</td>
 <td>Polyscience, Inc.</td>
 <td>17150</td>
 <td></td>
 </tr>
 <tr>
 <td>Polystyrene particles 500 nm</td>
 <td>Polyscience, Inc.</td>
 <td>17152</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT</td>
 </tr>
 <tr>
 <td>Water bath</td>
 <td>VWR</td>
 <td>462-7001</td>
 <td></td>
 </tr>
 <tr>
 <td>Thermostat</td>
 <td>IKA-Werke GmbH &amp; Co. KG</td>
 <td>3164000</td>
 <td></td>
 </tr>
 <tr>
 <td>Peristaltic pumps</td>
 <td>Ismatec</td>
 <td>ISM 833</td>
 <td></td>
 </tr>
 <tr>
 <td>Bubble traps (glass)</td>
 <td>UNI-GLAS Laborbedarf</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Flow heater</td>
 <td>UNI-GLAS Laborbedarf</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Pressure sensor + Software for analyses</td>
 <td>MSR Electronics GmbH</td>
 <td>145B5</td>
 <td></td>
 </tr>
 <tr>
 <td>Notebook</td>
 <td>Hewlett Packard</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Miniature gas exchange oxygenator</td>
 <td>Living Systems Instrumentation</td>
 <td>LSI-OXR</td>
 <td></td>
 </tr>
 <tr>
 <td>Tygon Tube (ID: 1.6 mm; OD: 4.8 mm)</td>
 <td>Ismatec</td>
 <td>MF0028</td>
 <td></td>
 </tr>
 <tr>
 <td>Tubes for pumps (PharMed BPT; ID: 1.52 mm)</td>
 <td>Ismatec</td>
 <td>SC0744</td>
 <td></td>
 </tr>
 <tr>
 <td>Blunt cannulae (&Oslash; 0.8 mm)</td>
 <td>Polymed Medical Center</td>
 <td>03.592.81</td>
 <td></td>
 </tr>
 <tr>
 <td>Blunt cannulae (&Oslash; 1.2 mm)</td>
 <td>Polymed Medical Center</td>
 <td>03.592.90</td>
 <td></td>
 </tr>
 <tr>
 <td>Blunt cannulae (&Oslash; 1.5 mm)</td>
 <td>Polymed Medical Center</td>
 <td>03.592.94</td>
 <td></td>
 </tr>
 <tr>
 <td>Blunt cannulae (&Oslash; 1.8 mm)</td>
 <td>Polymed Medical Center</td>
 <td>03.952.82</td>
 <td></td>
 </tr>
 <tr>
 <td>Parafilm</td>
 <td>VWR</td>
 <td>291-1212</td>
 <td></td>
 </tr>
 <tr>
 <td>Perfusion chamber with tissue holder (plexiglass)</td>
 <td>Internal technical department</td>
 <td></td>
 <td>Similar equipment is available from Hemotek Limited, UK</td>
 </tr>
 <tr>
 <td>Surgical suture material (PremiCron)</td>
 <td>B. Braun Medical AG</td>
 <td>C0026005</td>
 <td></td>
 </tr>
 <tr>
 <td>Winged Needle Infusion Set (21G Butterfly)</td>
 <td>Hospira, Inc.</td>
 <td>ASN 2102</td>
 <td></td>
 </tr>
 <tr>
 <td>Multidirectional stopcock (Discofix C-3)</td>
 <td>B. Braun Medical AG</td>
 <td>16494C</td>
 <td></td>
 </tr>
 <tr>
 <td>Surgical scissors</td>
 <td>B. Braun Medical AG</td>
 <td>BC304R</td>
 <td></td>
 </tr>
 <tr>
 <td>Dissecting scissors</td>
 <td>B. Braun Medical AG</td>
 <td>BC162R</td>
 <td></td>
 </tr>
 <tr>
 <td>Needle holder</td>
 <td>B. Braun Medical AG</td>
 <td>BM200R</td>
 <td></td>
 </tr>
 <tr>
 <td>Dissecting forceps</td>
 <td>B. Braun Medical AG </td>
 <td>BD215R</td>
 <td></td>
 </tr>
 <tr>
 <td>Automated blood gas system </td>
 <td>Radiometer Medical ApS</td>
 <td>ABL800 FLEX</td>
 <td></td>
 </tr>
 <tr>
 <td>Multi-mode microplate reader</td>
 <td>BioTek</td>
 <td>Synergy HT</td>
 <td></td>
 </tr>
 <tr>
 <td>Liquid scintillation analyzer</td>
 <td>GMI, Inc.</td>
 <td>Packard Tri-Carb 2200</td>
 <td></td>
 </tr>
 <tr>
 <td>Scintillation tubes 5.5 ml</td>
 <td>Zinsser Analytic GmbH</td>
 <td>3020001</td>
 <td></td>
 </tr>
 <tr>
 <td>Tissue Homogenizer</td>
 <td>OMNI, Inc.</td>
 <td>TH-220</td>
 <td></td>
 </tr>
 <tr>
 <td>pH meter + electrode</td>
 <td>VWR</td>
 <td>662-2779 </td>
 <td></td>
 </tr>
	 </tbody>
 </table>

determination of the transport rate of xenobiotics and nanomaterials across the placenta using the ex vivo human placental perfusion model

Décadas atrás, a placenta humana foi pensado para ser uma barreira impenetrável entre a mãe eo feto. No entanto, a descoberta de defeitos congénitos, induzidos por talidomida e muitos estudos posteriores depois provado o oposto. Hoje vários xenobióticos nocivos, como nicotina, heroína, metadona ou drogas, bem como poluentes ambientais foram descritos para superar essa barreira. Com o crescente uso da nanotecnologia, a placenta é susceptível de entrar em contacto com novas nanopartículas seja acidental ou intencionalmente através da exposição no caso de potenciais aplicações nanomedical. Os dados de experiências em animais não podem ser extrapolados para seres humanos porque a placenta é o órgão mais específico da espécie de mamífero 1. Portanto, o ex vivo dupla recirculação perfusão placentária humana, desenvolvido pela Panigel et ai. Em 1967 2 e continuamente modificado por Schneider et ai. Em 1972 3, pode servir como um excelente modelo tó estudar a transferência de partículas ou xenobióticos. Aqui, vamos nos concentrar na dupla ex vivo recirculação protocolo de perfusão da placenta humana e seu desenvolvimento para obter resultados reproduzíveis. O placentas foram obtidas após consentimento das mães de gestações complicadas prazo submetidas ao parto cesariana. Os vasos fetais e maternos de um dos cotilédones intactos foram canulados e perfundidos pelo menos por cinco horas. Como um modelo de partículas partículas de poliestireno marcadas com fluorescência, com tamanhos entre 80 e 500 nm de diâmetro foram adicionados ao circuito materna. As partículas de 80 nm eram capazes de atravessar a barreira placentária e proporcionar um perfeito exemplo para uma substância que é transferido através da placenta para o feto, enquanto as partículas de 500 nm foram retidos no tecido placentário ou circuito materna. O modelo de perfusão ex vivo da placenta humana é um dos poucos modelos que fornecem informações confiáveis ​​sobreo comportamento de transporte de xenobióticos em uma barreira importante tecido que proporciona dados relevantes preditivos e clínicas.

A figura 4A mostra os perfis de perfusão de pequenas partículas de poliestireno (80 nm), que foram transportados através da placenta maiores em comparação com partículas de poliestireno (500 nm), que não foram transferidos para o compartimento fetal. Cada ponto de dados representa a concentração média de partícula para um determinado ponto de pelo menos 3 experiências independentes do tempo. Para poliestireno nanopartículas a transferência placentária é dependente do tamanho 19.</su...

Watch this Scientific Journal Video about Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model at JoVE.com

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

O<em&gt; Ex vivo</em&gt; Modelo de perfusão de recirculação dupla placenta humana pode ser utilizada para investigar a transferência de xenobióticos e nanopartículas pela placenta humana. Neste protocolo de vídeo que descreve o equipamento e as técnicas necessárias para uma boa realização de uma perfusão placenta.

Determinação da taxa de transporte de xenobióticos e nanomateriais através da placenta usando o<em&gt; Ex vivo</em&gt; Modelo de perfusão da placenta humana

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

determination-transport-rate-xenobiotics-nanomaterials-across

Research

JoVE Journal

Bioengineering

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

17.2K visualizações.  University Hospital Zurich. O Ex vivo Modelo de perfusão de recirculação dupla placenta humana pode ser utilizada para investigar a transferência de xenobióticos e nanopartículas pela placenta humana. Neste protocolo de vídeo que descreve o equipamento e as técnicas necessárias para uma boa realização de uma perfusão placenta.

Vídeo: Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

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 ...

Bioengenharia

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

Hematopoietic stem cells require a unique microenvironment in order to sustain blood cell formation1; the bone marrow (BM) is a complex three-dimensional (3D) tissue wherein hematopoiesis is regulated by spatially organized cellular microenvironments termed niches2-4. The organization of the BM niches is critical for the function or dysfunction of normal or malignant BM5. Therefore a better understanding of the in vivo microenvironment using an ex vivo mimicry would help us elucidate the molecular, cellular and microenvironmental determinants of leukemogenesis6.
Currently, hematopoietic cells are cultured in vitro in two-dimensional (2D) tissue culture flasks/well-plates7 requiring either co-culture with allogenic or xenogenic stromal cells or addition of exogenous cytokines8. These conditions are artificial and differ from the in vivo microenvironment in that they lack the 3D cellular niches and expose the cells to abnormally high cytokine concentrations which can result in differentiation and loss of pluripotency9,10.
Herein, we present a novel 3D bone marrow culture system that simulates the in vivo 3D growth environment and supports multilineage hematopoiesis in the absence of exogenous growth factors. The highly porous scaffold used in this system made of polyurethane (PU), facilitates high-density cell growth across a higher specific surface area than the conventional monolayer culture in 2D11. Our work has indicated that this model supported the growth of human cord blood (CB) mononuclear cells (MNC)12 and primary leukemic cells in the absence of exogenous cytokines. This novel 3D mimicry provides a viable platform for the development of a human experimental model to study hematopoiesis and to explore novel treatments for leukemia.

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

A 3D culture system for hematopoiesis is described using human cord blood and leukemic bone marrow cells. The method is based on the use of a porous synthetic polyurethane scaffold coated with extracellular matrix proteins. This scaffold is adaptable to accommodate a wide range of cells.

Ex vivo Mimetismo de normal e anormal Hematopoese Humanos

Hematopoietic stem cells require a unique microenvironment in order to sustain blood cell formation1; the bone marrow (BM) is a complex three-dimensional ...

Anatomical Reconstructions of the Human Cardiac Venous System using Contrast-computed Tomography of Perfusion-fixed Specimens

A detailed understanding of the complexity and relative variability within the human cardiac venous system is crucial for the development of cardiac devices that require access to these vessels. For example, cardiac venous anatomy is known to be one of the key limitations for the proper delivery of cardiac resynchronization therapy (CRT)1 Therefore, the development of a database of anatomical parameters for human cardiac venous systems can aid in the design of CRT delivery devices to overcome such a limitation. In this research project, the anatomical parameters were obtained from 3D reconstructions of the venous system using contrast-computed tomography (CT) imaging and modeling software (Materialise, Leuven, Belgium). The following parameters were assessed for each vein: arc length, tortuousity, branching angle, distance to the coronary sinus ostium, and vessel diameter.
	CRT is a potential treatment for patients with electromechanical dyssynchrony. Approximately 10-20% of heart failure patients may benefit from CRT2. Electromechanical dyssynchrony implies that parts of the myocardium activate and contract earlier or later than the normal conduction pathway of the heart. In CRT, dyssynchronous areas of the myocardium are treated with electrical stimulation. CRT pacing typically involves pacing leads that stimulate the right atrium (RA), right ventricle (RV), and left ventricle (LV) to produce more resynchronized rhythms. The LV lead is typically implanted within a cardiac vein, with the aim to overlay it within the site of latest myocardial activation. 
	We believe that the models obtained and the analyses thereof will promote the anatomical education for patients, students, clinicians, and medical device designers. The methodologies employed here can also be utilized to study other anatomical features of our human heart specimens, such as the coronary arteries. To further encourage the educational value of this research, we have shared the venous models on our free access website: <a href="http://www.vhlab.umn.edu/atlas" target="_blank">www.vhlab.umn.edu/atlas</a>.

The objective of this research is to recreate and then access the anatomy of the human cardiac venous system using 3D reconstructions generated from contrast-computed tomography scans.

Reconstruções anatômicas do sistema venoso cardíaco humano usando contraste Tomografia computadorizada de Amostras de perfusão-fixados

A detailed  understanding of the complexity and relative variability within the human  cardiac venous system is crucial for the development of cardiac ...

The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits

The foreign body reaction occurs when a synthetic surface is introduced to the body. It is characterized by adsorption of blood proteins and the subsequent attachment and activation of platelets, monocyte/macrophage adhesion, and inflammatory cell signaling events, leading to post-procedural complications. The Chandler Loop Apparatus is an experimental system that allows researchers to study the molecular and cellular interactions that occur when large volumes of blood are perfused over polymeric conduits. To that end, this apparatus has been used as an ex vivo model allowing the assessment of the anti-inflammatory properties of various polymer surface modifications. Our laboratory has shown that blood conduits, covalently modified via photoactivation chemistry with recombinant CD47, can confer biocompatibility to polymeric surfaces. Appending CD47 to polymeric surfaces could be an effective means to promote the efficacy of polymeric blood conduits. Herein is the methodology detailing the photoactivation chemistry used to append recombinant CD47 to clinically relevant polymeric blood conduits and the use of the Chandler Loop as an ex vivo experimental model to examine blood interactions with the CD47 modified and control conduits.

The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits

Blood exposure to polymeric blood conduits initiates the foreign body reaction that has been implicated in clinical complications. Here, the Chandler Loop Apparatus, an experimental tool mimicking blood perfusion through these conduits, is described. Appendage of recombinant CD47 results in decreased evidence of the foreign body reaction on these conduits.

O uso do<em&gt; Ex Vivo</em&gt; Chandler Circuito Aparelho para avaliar a biocompatibilidade de Modificados Condutas sangue Poliméricos

The foreign body reaction occurs when a synthetic surface is introduced to the body. It is characterized by adsorption of blood proteins and the ...

Determination of Zeta Potential via Nanoparticle Translocation Velocities through a Tunable Nanopore: Using DNA-modified Particles as an Example

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and nanoparticles. Tunable resistive pulse sensing (TRPS) is a relatively recent adaptation to RPS that incorporates a tunable pore that can be altered in real time. Here, we use TRPS to monitor the translocation times of DNA-modified nanoparticles as they traverse the tunable pore membrane as a function of DNA concentration and structure (i.e., single-stranded to double-stranded DNA).
TRPS is based on two Ag/AgCl electrodes, separated by an elastomeric pore membrane that establishes a stable ionic current upon an applied electric field. Unlike various optical-based particle characterization technologies, TRPS can characterize individual particles amongst a sample population, allowing for multimodal samples to be analyzed with ease. Here, we demonstrate zeta potential measurements via particle translocation velocities of known standards and apply these to sample analyte translocation times, thus resulting in measuring the zeta potential of those analytes.
As well as acquiring mean zeta potential values, the samples are all measured using a particle-by-particle perspective exhibiting more information on a given sample through sample population distributions, for example. Of such, this method demonstrates potential within sensing applications for both medical and environmental fields.

Here we use a polyurethane tunable nanopore integrated into a resistive pulse sensing technique to characterize nanoparticles surface chemistry via the measurement of particle translocation velocities, which can be used to determine the zeta potential of individual nanoparticles.

Determinação do Potencial Zeta de nanopartículas de translocação através de velocidades por meio de um Tunable Nanopore: Utilizando partículas modificadas-ADN como um Exemplo

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and ...

An Experimental and Finite Element Protocol to Investigate the Transport of Neutral and Charged Solutes across Articular Cartilage

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular cartilage impairs its load-bearing function substantially as it experiences tremendous chemical degradation, i.e. proteoglycan loss and collagen fibril disruption. One promising way to investigate chemical damage mechanisms during OA is to expose the cartilage specimens to an external solute and monitor the diffusion of the molecules. The degree of cartilage damage (i.e. concentration and configuration of essential macromolecules) is associated with collisional energy loss of external solutes while moving across articular cartilage creates different diffusion characteristics compared to healthy cartilage. In this study, we introduce a protocol, which consists of several steps and is based on previously developed experimental micro-Computed Tomography (micro-CT) and finite element modeling. The transport of charged and uncharged iodinated molecules is first recorded using micro-CT, which is followed by applying biphasic-solute and multiphasic finite element models to obtain diffusion coefficients and fixed charge densities across cartilage zones.

We propose a protocol to investigate the transport of charged and uncharged molecules across articular cartilage with the aid of recently developed experimental and numerical methods.

Um Experimental e Finite Element protocolo para investigar o transporte de solutos neutras e carregadas em toda a cartilagem articular

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular ...

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential to establish an ex vivo model that can evaluate SIM performance accurately, for developing high-performance SIMs. In our previous study, we developed a new ex vivo model that can be used to evaluate the performance of various SIMs in detail by applying constant tension to the specimen&#39;s ends. We also confirmed that the proposed new ex vivo model allows accurate submucosal elevation height (SEH) measurement under uniform conditions and detailed comparisons of the performances of various types of SIMs. Here, we describe the new ex vivo model and explain the detailed setup methodology of this model. Since all parts of the new model were easy to obtain, the setup of the new model could be completed quickly. SEH of various SIMs could be measured more accurately by using the new model. The critical factor that determines SIM performance can be identified using the new model. SIM development speed will drastically increase after the factor has been identified.

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

We developed a new ex vivo model that applies constant tension to the porcine gastric specimen. This development made it possible to evaluate the performance (the height and duration of the submucosal elevation) of various SIMs accurately.&#160; The detailed setup methodology of this new model is explained.

Um novo modelo Ex Vivo para a avaliação de desempenho de Material de injeção submucosa endoscópica

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential ...

Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to the placenta while minimizing fetal and maternal side effects remains challenging. Targeted nanoparticle carriers provide new opportunities to treat placental disorders. We recently demonstrated that a synthetic placental chondroitin sulfate A binding peptide (plCSA-BP) could be used to guide nanoparticles to deliver drugs to the placenta. In this protocol, we describe in detail a system for assessing the efficiency of drug delivery to the placenta by plCSA-BP that employs three separate methods used in combination: in vivo imaging, high-frequency ultrasound (HFUS), and high-performance liquid chromatography (HPLC). Using in vivo imaging, plCSA-BP-guided nanoparticles were visualized in the placentas of live animals, while HFUS and HPLC demonstrated that plCSA-BP-conjugated nanoparticles efficiently and specifically delivered methotrexate to the placenta. Thus, a combination of these methods can be used as an effective tool for the targeted delivery of drugs to the placenta and development of new treatment strategies for several pregnancy complications.

We describe a system that utilizes three methods to evaluate the safety and effectiveness of placenta-targeted drug delivery: in vivo imaging to monitor nanoparticle accumulation, high-frequency ultrasound to monitor placental and fetal development, and HPLC to quantify drug delivery to tissue.

Avaliação global da eficácia e segurança de entrega de droga Placenta-alvo usando três métodos complementares

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to ...

Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically been studied in vivo in animal models showing characteristic patterns of cell migration. However, in vivo models have several limitations, including intercellular mediators that are difficult to access and analyze, as well as the inability to directly analyze human neutrophils. Because of these limitations, there is a need for an in vitro platform that studies swarming with human neutrophils and provides easy access to the molecular signals generated during swarming. Here, a multistep microstamping process is used to generate a bioparticle microarray that stimulates swarming by mimicking an in vivo infection. The bioparticle microarray induces neutrophils to swarm in a controlled and stable manner. On the microarray, neutrophils increase in speed and form stable swarms around bioparticle clusters. Additionally, supernatant generated by the neutrophils was analyzed and 16 proteins were discovered to have been differentially expressed over the course of swarming. This in vitro swarming platform facilitates direct analysis of neutrophil migration and protein release in a reproducible, spatially controlled manner.

This protocol generates bioparticle microarrays that provide spatially controlled neutrophil swarming. It provides easy access to the mediators that neutrophils release during migration and allows for quantitative imaging analysis.

Micromatrizes de biopartículas para análise quimiotática e molecular de swarming de nêutrons humanos in vitro

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically ...

Biofunctionalization of Magnetic Nanomaterials

Magnetic nanomaterials have received great attention in different biomedical applications. Biofunctionalizing these nanomaterials with specific targeting agents is a crucial aspect to enhance their efficacy in diagnostics and treatments while minimizing the side effects. The benefit of magnetic nanomaterials compared to non-magnetic ones is their ability to respond to magnetic fields in a contact-free manner and over large distances. This allows to guide or accumulate them, while they can also be monitored. Recently, magnetic nanowires (NWs) with unique features were developed for biomedical applications. The large magnetic moment of these NWs enables a more efficient remote control of their movement by a magnetic field. This has been utilized with great success in cancer treatment, drug delivery, cell tracing, stem cell differentiation or magnetic resonance imaging. In addition, the NW fabrication by template-assisted electrochemical deposition provides a versatile method with tight control over the NW properties. Especially iron NWs and iron-iron oxide (core-shell) NWs are suitable for biomedical applications, due to their high magnetization and low toxicity.
In this work, we provide a method to biofunctionalize iron/iron oxide NWs with specific antibodies directed against a specific cell surface marker that is overexpressed in a large number of cancer cells. Since the method utilizes the properties of the iron oxide surface, it is also applicable to superparamagnetic iron oxide nanoparticles. The NWs are first coated with 3-aminopropyl-tri-ethoxy-silane (APTES) acting as a linker, which the antibodies are covalently attached to. The APTES coating and the antibody biofunctionalization are proven by electron energy loss spectroscopy (EELS) and zeta potential measurements. In addition, the antigenicity of the antibodies on the NWs is tested by using immunoprecipitation and western blot. The specific targeting of the biofunctionalized NWs and their biocompatibility are studied by confocal microscopy and a cell viability assay.

In this work, we provide a protocol to biofunctionalize magnetic nanomaterials with antibodies for specific cell targeting. As examples, we utilize iron nanowires to target cancer cells.

Biofuncionalização de Nanomateriais Magnéticos

Magnetic nanomaterials have received great attention in different biomedical applications. Biofunctionalizing these nanomaterials with specific targeting ...