Os autores agradecem M. Fiche do Hospital Universitário de Lausanne para fornecer a fotografia mamoplastia, M. Wirth e A. Ayyanan do Swiss Institute for Cancer Research Experimental, Centro Nacional de Competência em Pesquisa de Oncologia Molecular, Faculdade de Ciências da Vida, Ecole Polytechnique Fédérale de Lausanne para a assistência técnica e R. Clarke, da Universidade de Manchester para comentários críticos. A investigação conducente a estes resultados foi recebido apoio de SNF3100A0-112090, Oncosuisse 531.817, eo Innovative Medicines Initiative empresa comum ao abrigo do contrato de concessão não. 115.188, recursos de que são compostas de contribuição financeira do Sétimo Programa da União Europeia Quadro (FP7 / 2007-2013) e Federação Europeia das Associações e Indústrias Farmacêuticas empresas »da contribuição em espécie. O endereço Web de Iniciativa sobre Medicamentos Inovadores é <a href="http://www.imi.europa.eu/" target="_blank">http://www.imi.europa.eu/</a> . &lt;/ P&gt;

Considerações gerais: antes do tempo, o estabelecimento de um protocolo de ética, preparar questionários para os pacientes, e ver a formação do pessoal clínico. Antes de usar o material de cirurgia mamoplastia de redução, obter autorizações, e assegurar o consentimento do paciente. Tecido podem ser infecciosos e precisa de ser tratadas em conformidade. Este protocolo foi aprovado pelo comitê de ética do ISREC - Instituto Suíço de Experimental Cancer Research. 1. Tissue Recovery, Processamento e Bio-banking Para garantir a qualidade da amostra ideal para o bio-banking, uma série de passos são realizados no hospital antes do transporte para o laboratório. No hospital: <ol><li> Obter tecidos da mama humanos sob condições estéreis a partir de amostras de cirurgia mamoplastia de redução e de sangue, se necessário. Tissue precisa ser examinado pelo patologista que vai provar para excluir a presença de lesões malignas. </li><li> Coloque o peito de temitir sob o capô em uma placa de estéril e preparar tesoura, bisturi cirúrgico e pinças que já estão esterilizados com esterilizador a vapor. Faça profundo corte em diferentes locais do tecido mamário utilizando um bisturi para abri-lo. Em tecido mamário humano olhar para fios brancos que consistem em dutos e lóbulos, que são incorporados no tecido adiposo amarelo. Observação: A razão entre o parênquima branco para o tecido adiposo amarelo difere entre as mulheres; Normalmente as mulheres mais jovens têm mais tecido epitelial. </li><li> Escolha pedaços de material branco e mantê-los com uma pinça, então separá-los suavemente do tecido adiposo com uma tesoura. Evite tomar vasos sanguíneos. </li><li> Lavar peças de tecido em solução salina fosfato tamponada (PBS) pH 7,2 para remover o sangue. Transferir o tecido para garrafas de plástico de 250 ml contendo meio F12 (F12 Meio de Eagle Modificado por Dulbecco) sem vermelho de fenol com 2% de penicilina / estreptomicina (incluindo 5.000 unidades / ml de penicilina e 5000 g / ml de estreptomicina) e 1% de umtibiotic / anfotericina B (incluindo 10.000 unidades / ml de penicilina, 10.000 ug / ml de estreptomicina, e 25 ug / ml de anfotericina B). Armazenar tecidos no meio de transporte para o laboratório. </li><li> Prepare pedaços de 3-5 mm de diâmetro para extração de RNA e proteína e flash congelá-los em cryovials com frio (gelo seco) isopentano. Transferi-los para a caixa de gelo seco durante o transporte para o laboratório. </li><li> Coloque pedaços de 3-5 mm no molde e cobri-lo com uma camada de Optimal temperatura de corte (OCT) e, em seguida, coloque o molde sobre o fundo plano da caixa cheia de isopentano frio. Após congelamento total transferir o molde para a caixa de gelo seco. </li><li> Fix 5 pedaços de 3-5 mm de diâmetro em paraformaldeído (PFA) 4% e mais 5 peças em formaldeído (FA) de 4% em PBS a análise histológica RT mais tarde. Dois diferentes fixadores são recomendados como alguns anticorpos trabalham melhor com um ou o outro dos dois. </li></ol> No laboratório: <ol start="8"><li> Fazercument todas as informações sobre a mamoplastia de redução, data da cirurgia e se as amostras são derivados de esquerda versus mama direita. </li><li> Coloque as amostras congeladas a -80 ° C e transferir os tecidos fixados em cassetes de histologia após 2 horas de fixação. Em seguida, colocá-los tanto em PBS ou etanol a 70%, para O armazenamento / N a 4 ° C. No dia seguinte, transferir as cassetes de histologia para instalações de histologia para inclusão em parafina. </li></ol> 2. Tissue Digestão e Hormone Estimulação <ol><li> Transferem-se as garrafas contendo as amostras de tecido para uma câmara de fluxo laminar numa sala de cultura de células P2. Coloque pedaços de tecido sobre a tábua de cortar estéril (29 x 19 cm). Segure o material com uma pinça e raspar o restante gordura e vasos de distância do parênquima mamário (parte branca) com bisturi. Colocar o material branco em PBS numa placa de cultura de 10 centímetros. </li><li> Depois de retirar o material gorduroso de todas as peças e descartá-los de acordo com a norma de seguranças do laboratório, coloque as partes brancas contendo os tecidos epiteliais de volta na tábua de cortar. Usando bisturis opostas cortar o tecido e corte-a em pedaços de até 2 mm de diâmetro. </li><li> Inserir o material triturado em tubos de tamanho adequado: até 1, 2,5 e 10 ml para um 5, 15 ou tubos de 50 ml, respectivamente. Normalmente de 5 ml rendimentos tubo material suficiente para incrustação de parafina e hormonais estímulos, e de 15 ml produz tubos de material suficiente para fluorescência celular ativado (FACS). Tubos maiores são usados ​​para congelar alíquotas. </li><li> Preparar a mistura mestre digestão consistindo em meio (DMEM / F12 sem vermelho de fenol) com 1% de penicilina / estreptomicina / anfotericina B e colagenase A (Clostridiopeptidase A de Clostridium histolyticum). Dose de colagenase de acordo com tempos de digestão. <ol><li> Para 12, 24, e 36 h de incubação, usar concentração final de colagenase de 1,5 mg / ml, 1,0 mg / ml e 0,5 mg / ml, respectivamente. Adicionar mix mestre digestão até 4 vezes davolume do material digerido para assegurar a agitação e o arejamento adequado. NOTA: Dependendo do desenho experimental e a duração da estimulação hormonal, adicionar hormônios durante ou após a etapa de digestão e microestrutura de recuperação enzimática. </li></ol></li><li> Adicionar hormona (s) a uma concentração desejada para testar amostras e o veículo de controlo. Para a estimulação com 17-β-estradiol, e promegestona (R5020) utilizar uma concentração final de 20 nM. NOTA: R5020 é um agonista do receptor de progesterona sintética, que é mais estável do que a progesterona natural em meio. <ol><li> Colocar os tubos sobre o misturador de rolos dentro de uma incubadora a 37 ° C e 40 rpm O / N. Antes de colocar as amostras no misturador, ressuspender-los muito bem para garantir que todo o material distribui ao longo dos tubos de centrífuga. </li></ol></li><li> Como a digestão demora pelo menos 12 horas (O / N), por vezes de estimulação da hormona de menos de 12 horas, adicione os hormônios após a recuperação das microestruturas (deatado na etapa 3). Adicione os hormônios como você voltar a suspender as microestruturas e coloque a suspensão em placas ultra-baixas de fixação. Microestruturas dos tecidos podem ser mantidos em ultra-baixas placas de fixação para até 7 dias em meio de cultura livre de fator de preservação de cerca de 70% de viabilidade. </li><li> Para ARN e análise de proteínas, recolher microestruturas dos tecidos por centrifugação a 400 xg durante 5 min e flash congelamento em tubos de centrífuga. </li></ol> 3. Recuperação de microestruturas de tecidos <ol><li> Centrifuga-se o tubo (s) a 400 xg durante 5 min. Aspirar a gordura a partir do topo do tubo de centrifugação e transferir o sobrenadante do sobrenadante aquoso remanescente, que é enriquecido em fibroblastos, para um novo tubo de centrífuga e centrifuga-se a 1250 xg durante 5 min. NOTA: Como as microestruturas do tecido mamário são muito pegajoso, para todas as etapas subseqüentes colocar o agregado agitando suavemente o tubo. Pipetting não é recomendada devido ao risco de perder o material. </li&gt; <li> Ressuspender as pelotas enriquecidas em microestruturas e em fibroblastos derivados de sobrenadante aquoso em PBS, 2% de Soro Fetal de Vitela (FCS). Subsequentemente girar em ambos os tubos 1250 xg durante 5 min e, em seguida, descartar os sobrenadantes. </li><li> Ressuspender as pelotas em 3-5 ml de tampão de lise de glóbulos vermelhos e transferi-los para tubos limpos. Incubar-los durante 5 min à TA. </li><li> Adicionar o volume de 2x PBS, 2% de FCS e centrifugar para a 1.250 xg durante 5 min. Lavam-se as peletes com PBS, FCS a 2% e centrifugação a 1250 xg durante 5 min. Repita esta etapa duas vezes. </li></ol> 4. As amostras de Citometria de Fluxo <ol><li> Adicionar 1-2 ml de tripsina a 0,25% ao sedimento e misture por pipetagem cima e para baixo com muito cuidado para 2 min com um Pipetman 1.000 ul dissociar células. </li><li> Inibir a actividade da tripsina por adição de 9 ml de PBS, 2% de FCS. Centrifuga-se a 450 xg durante 5 min e ressuspender o sedimento em 10 ml de PBS, 2% de FCS. </li><li> Transferir a suspensão de células para um filtro de células de 40 mm colocada sobre top de um tubo de centrífuga de 50 ml para preparar células individuais. Se alguns microlitros de suspensão de células permanecem na parte superior do filtro, ajudá-los a passar através do filtro por aspiração los a partir do outro lado do filtro com uma pipeta e adicioná-los para as microestruturas filtrados. </li><li> Anticorpos células imunitárias separadas, fibroblastos e células endoteliais a partir de microestruturas com um cocktail de anticorpos anti-CD45, anti-PAF e anti-CD31 e depois separá-los com FACS, com base em pan-epitelial EpCAM marcador CD10 e para separar as células luminais e basais, respectivamente, 10 . </li></ol> 5. As amostras para histologia <ol><li> Depois de se lavar o material mecanicamente e enzimaticamente digerido como descrito acima, a partir de peletes de fixar o material inicial de 1,5 ml em 1 ml de 4% de PFA em PBS à temperatura ambiente durante 30 min. </li><li> Centrifugar a 1.250 xg durante 5 minutos e lava-se com 1 - 2 ml de PBS, duas vezes. </li><li> Centrifuga-se a 16000 xg durante 10 min. Durante este tempo, preparar 1% multiusos de agarose em tampão Tris-Acetato-EDTA (TAE) de tampão e aquecer a solução no forno de microondas até a solução começar a ferver. <ol><li> Remover a solução do forno microondas e agite o frasco suavemente para misturar a solução e voltar a suspender qualquer restantes partículas de agarose. Arrefecer a solução de agarose a 50 ° C e deitar 1 ml num molde da base de parafina dimensionada 31 × 23 × 13,5 milímetros. </li></ol></li><li> Descartar o sobrenadante e colocar os sedimentos em cima da camada solidificada (1-3 mm) de agarose utilizando uma espátula com uma extremidade plana. Cobrir todas elas com uma camada adicional de agarose (40 ° C). </li><li> Após os solidifica de agarose, geralmente dentro de 5 min, colocar os blocos de agarose para as cassetes de histologia para parafina embedment. Junto com as amostras de agarose colocar um papel branco de escritório na cassete com todas as informações sobre o número de mamoplastia redutora, a condição experimental, e data escrita em letras pequenas a lápis; este documento foi escrito resiste todas as etapas de tratamento subsequentes. </li><li> Coloque as cassetes emPBS S / N, ou em etanol a 70% para armazenamento no fim de semana, a 4 ° C. Nós não notei nenhuma diferença entre as duas soluções de armazenamento. Em seguida, transferir cassetes histologia às instalações de histologia para inclusão em parafina. </li></ol> 6. As microestruturas congelamento (Futuro) Cultivar <ol><li> Ressuspender as microestruturas de tecidos em meio consistindo em FCS com 10% de dimetilsulfóxido (DMSO) e congelar em alíquotas de 1 ml criotubos congelação. O número de criotubos obtidas depende da quantidade de matéria-prima obtida a partir de mamoplastia de redução. </li><li> Congelar as criotubos contendo microestruturas dos tecidos utilizando um congelador de taxa controlada com o seguinte programa: 5 minutos a 4 ° habitação C, diminuindo a temperatura de -7 ° C com uma taxa de 6 ° C / min, diminuindo a temperatura a -12 ° C com taxa de 6 ° C / min, 10 min habitação, diminuindo a temperatura a -40 ° C com uma taxa de 6 ° C / min, a diminuição na temperatura de -80 ° C comtaxa de 6 ° C / min. Em seguida, transfira os cryovials a -80 ° C congelador. </li><li> No dia seguinte, a transferência dos criotubos de -80 ° C para o tanque de azoto líquido durante muito tempo de armazenagem. </li></ol>

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Os autores não têm nada a revelar.

A cultura ex vivo descrito aqui fornece microestruturas dos tecidos mamários contendo ductos e lóbulos intactos, juntamente com outros tipos de células encontradas normalmente no peito fêmea humana. No processamento do tecido da mama humana obtidos geralmente de mamoplastias de redução, a remoção de tecido adiposo, a digestão enzimática e mecânica da matriz estromal, e a lise de células vermelhas do sangue enriquece para fragmentos de condutas de leite e unidades lobulares ductais terminais. Digest?...

A correction was made to <a href="/video/52436">An Ex Vivo Model to Study Hormone Action in the Human Breast</a>. There was an error with the author, Marie Shamsheddin&#39;s, name. The author&#39;s name has been corrected to:
Marie Shamseddin
instead of:
Marie Shamsheddin

A correction to the author list has been made for the article <a href="/video/52436">An Ex Vivo Model to Study Hormone Action in the Human Breast</a>.

Erratum: An Ex Vivo Model to Study Hormone Action in the Human Breast

Informações sobre a paisagem mutacional no câncer de mama está aumentando em ritmo acelerado. Menos atenção tem sido dada a fatores sistêmicos que influenciam o desenvolvimento do câncer de mama. A exposição a hormônios reprodutivos tem um grande impacto na progressão da doença 1-3. No entanto, os mecanismos pelos quais as hormonas reprodutivas embatem na mama humano são mal compreendidos. Trabalho com modelos de engenharia genética do rato revelou que envolvem células sinalização intrínseca e paracrine por vários efectores a jusante 4. O conhecimento limitado sobre ação do hormônio no peito humano é largamente atribuível à falta de modelos adequados. A maioria dos trabalhos sobre os mecanismos de receptor de estrogénio (ER) e o receptor de progesterona (PR), a sinalização tem sido realizada com linhas de células positivas do receptor de hormona, cancro da mama, tais como MCF-7 e T47D. Estes foram obtidos a partir de derrame pleural de pacientes com câncer de mama avançado que tinha already recebeu vários tratamentos 5. A importância biológica dos resultados obtidos em tais modelos in vitro simples da mama humano é genes questionáveis ​​e alvo identificados nestes modelos in vitro são diferem dos genes-alvo que são identificados em modelos animais 6. Quando as células epiteliais mamárias humanas primárias são cultivadas in vitro tendem a perder a expressão de receptores hormonais e, portanto, resposta hormonal 7,8. Este problema pode ser circumventd por abordagens 3D sofisticados usando matrigel. Desta forma, C. Clarke e colegas conseguiram estabelecer as células epiteliais da mama que mantiveram a expressão do receptor da hormona e que demonstraram uma resposta proliferativa para a estimulação da progesterona 9. No entanto, importante em dois genes alvo in vivo do receptor da progesterona, Wnt-4 e RANKL, não foram induzidas por estimulação da progesterona no presente sistema 9. Esta abordagem foi tomada recentemente em mais longe com in vitro </em&gt; pré-tratamento hormonal e indução RANKL foi alcançado 10. A ressalva é que matrigel tem atividades que são dependentes do lote, é caro, e exige um projeto experimental que está apto apenas para o número de células pequenas. Com base na descoberta de que em ER vivo e sinalização PR são largamente mediada por interacções parácrinos 11, que argumentaram que as interacções intercelulares precisam de ser mantidas. Outro factor importante que é perdida como tecidos estão dissociados para células únicas para a cultura in vitro são as interacções de células epiteliais com a matriz extracelular; No entanto, estes são fundamentais para diferenciação epitelial e sua perturbação é importante na tumorigênese 12. Com isto em mente, nós estabelecemos um método para isolar microestruturas tecido mamário a partir de material de descarte cirúrgico fresco 13. O parênquima da mama, que consiste de um epitélio de duas camadas com o interior do lúmen e exterior myoepithelicélulas al, é dissecado a partir de tecido adiposo e submetido a dissociação mecânica e enzimática. Após a lavagem e centrifugação, fragmentos de dutos de leite são obtidos que manter interações estreitas com muitas células estromais. Estes micro-estruturas dos tecidos permanecem hormônio responsivo. O modelo foi validado em amostras clínicas 13. Como tal, o presente processo pode ajudar a estudar a acção de hormonas na mama num contexto biológico e clinicamente relevante.

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		<tr height="25">
			<td height="25" style="height:25px;width:335px;">CO2 incubator</td>
			<td style="width:164px;">Hera Cell Heraeus</td>
			<td style="width:119px;">51022391</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Roller Mixer SRT9D</td>
			<td style="width:164px;">Sturt</td>
			<td style="width:119px;">SRT9D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Histology Cassettes</td>
			<td style="width:164px;">Medizintechnik</td>
			<td style="width:119px;">81-0021-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Cell Strainer</td>
			<td style="width:164px;">Falcon</td>
			<td style="width:119px;">352340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Strile Surgical Blade&#160;</td>
			<td style="width:164px;">Aesculap</td>
			<td>BB536&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Scalpel</td>
			<td style="width:164px;">Aesculap</td>
			<td style="width:119px;">BB084R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Forceps</td>
			<td style="width:164px;">Aesculap</td>
			<td style="width:119px;">BD047R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Scissors</td>
			<td style="width:164px;">Aesculap</td>
			<td style="width:119px;">BC374R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Paraffin base mold</td>
			<td style="width:164px;">SAKURA</td>
			<td style="width:119px;">4166</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Optimal Cutting Temperature (OCT) Cryomatrix</td>
			<td style="width:164px;">Thermoscientific</td>
			<td style="width:119px;">6769006</td>
			<td style="width:260px;">Fetal Bovine Serum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Penicillin/Streptomycin&#160;</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:119px;">15070-063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Antibiotic/Antimycotic</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:119px;">15240-062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">DMEM F/12</td>
			<td style="width:164px;">Life Technologies</td>
			<td>11039-021</td>
			<td>Prewarm (37&#176;C)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Collagenase</td>
			<td style="width:164px;">Roche</td>
			<td style="width:119px;">11 088 793 001</td>
			<td>Prewarm (37&#176;C)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Fetal Bovine Serum</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:119px;">10270</td>
			<td>Can be replaced with Fetal Calf Serum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Cell Blood Lysis Buffer</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:119px;">R7757</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:119px;">D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Trypsin-EDTA</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:119px;">15400-054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Agarose</td>
			<td style="width:164px;">Invitrogen</td>
			<td style="width:119px;">16500-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Formaldehyde</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:119px;">F1635</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Paraformaldehyde</td>
			<td style="width:164px;">Roth</td>
			<td style="width:119px;">335</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Isopentane</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:119px;">M32631</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Ethanol</td>
			<td style="width:164px;">Merck</td>
			<td style="width:119px;">1009831000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Cryogenic vials (5.0ml)&#160;</td>
			<td style="width:164px;">VWR, International</td>
			<td style="width:119px;">479-0820</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">Ultra low attachment culture dish</td>
			<td style="width:164px;">Corning, NY 14831</td>
			<td style="width:119px;">3471</td>
			<td></td>
		</tr>
	</tbody>
</table>

an ex vivo model to study hormone action in the human breast

O estudo da ação do hormônio no peito humano tem sido dificultada pela falta de sistemas de modelos adequados. Após a cultura in vitro, células epiteliais mamárias primários tendem a perder a expressão de receptores hormonais. Amplamente receptores hormonais linhas celulares de cancro da mama positivo utilizados são de limitada relevância para a situação in vivo. Aqui, descrevemos um modelo ex vivo para estudar a ação hormonal no peito humano. Amostras frescas de tecido da mama humano a partir de material de descarte cirúrgico, tais como mamoplastias de redução ou mammectomies são mecanicamente e enzimaticamente digerido para obter fragmentos de tecido contendo ductos e lóbulos e vários tipos de células do estroma. Estes micro-estruturas dos tecidos conservados em meio basal sem fatores de crescimento preservar seus contatos intercelulares, a arquitetura do tecido, e permanecem hormônio que responde por vários dias. Eles são processados ​​prontamente para RNA e extração de proteínas, análise histológica ou armazenados em meio de congelamento. Fluorescênciaseparação de células activadas (FACS) pode ser usado para o enriquecimento em populações de células específicas. Este protocolo fornece uma abordagem simples, padrão para estudos translacionais com alta complexidade, amostras humanas variadas.

Para estudar o papel dos estrogênios e progesterona e compreender melhor suas funções moleculares no peito humano, coletamos amostras de tecido fresco de mama humano de pacientes submetidos a mamoplastias de redução (Figura 1), após aprovação do consentimento informado. Nós também obter história médica e reprodutiva do paciente, bem como uma amostra de sangue para determinar os níveis de progesterona no soro no momento da cirurgia. Tecido de mamoplastias de redução frescos é mecanicament...

Watch this Scientific Journal Video about An Ex vivo Model to Study Hormone Action in the Human Breast at JoVE.com

An Ex vivo Model to Study Hormone Action in the Human Breast

Desenvolvemos um novo modelo ex vivo para estudar a acção da hormona no peito humano. Baseia-se em microestruturas dos tecidos isolados de amostras de tecidos mamários cirúrgicos que preservam a arquitetura do tecido, as interações intercelulares, e sinalização parácrina.

Um<em&gt; Ex vivo</em&gt; Modelo para estudar a ação do hormônio no peito humano

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

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Research

JoVE Journal

Medicine

An Ex vivo Model to Study Hormone Action in the Human Breast

11.3K visualizações.  Ecole polytechnique fédérale de Lausanne.  Desenvolvemos um novo modelo ex vivo para estudar a acção da hormona no peito humano. Baseia-se em microestruturas dos tecidos isolados de amostras de tecidos mamários cirúrgicos que preservam a arquitetura do tecido, as interações intercelulares, e sinalização parácrina. 

Vídeo: An Ex vivo Model to Study Hormone Action in the Human Breast

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Biomarcadores em um modelo animal para Revelando correlatos neurais, hematológica e Comportamental do TEPT

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Medicina

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

Humanos Linhas tumor neuroendócrino de células como um modelo tridimensional para o estudo da terapia de tumores humanos Neuroendócrino

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic lesions. Mouse models are an important tool to investigate inflammatory processes in atherogenesis, but these models suffer from the phenotypic and functional differences between the murine and human immune system. In vitro cell experiments are used to specifically evaluate cell type-dependent changes caused by a substance of interest, but culture-dependent variations and the inability to analyze the influence of specific molecules in the context of the inflammatory compound in atherosclerotic lesions limit the impact of the results. In addition, measuring levels of a molecule of interest in human blood helps to further investigate its clinical relevance, but this represents systemic and not local inflammation. Therefore, we here describe a plaque culture model to study human atherosclerotic lesion biology ex vivo. In short, fresh plaques are obtained from patients undergoing endarterectomy or coronary artery bypass grafting and stored in RPMI medium on ice until usage. The specimens are cut into small pieces followed by random distribution into a 48-well plate, containing RPMI medium in addition to a substance of interest such as cytokines or chemokines alone or in combination for defined periods of time. After incubation, the plaque pieces can be shock frozen for mRNA isolation, embedded in Paraffin or OCT for immunohistochemistry staining or smashed and lysed for western blotting. Furthermore, cells may be isolated from the plaque for flow cytometry analysis. In addition, supernatants can be collected for protein measurement by ELISA. In conclusion, the presented ex vivo model opens the possibility to further study inflammatory lesional biology, which may result in identification of novel disease mechanisms and therapeutic targets.

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

Atherosclerosis is a chronic inflammatory process. This manuscript illustrates an easy to use ex vivo model to investigate fresh carotid or coronary artery plaques. The ex vivo model allows for the investigation of potential substances on the inflammatory milieu in human atherosclerotic lesions and results can be analyzed by various methods.

A Human<em&gt; Ex Vivo</em&gt; Placa aterosclerótica modelo para estudar Lesão Biologia

Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Excitotóxico Estimulação do Microslices cérebro como um<em&gt; In vitro</em&gt; Modelo de AVC

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from cirrhosis to cardiovascular diseases). There is no effective treatment and the in vitro tools available do not mimic the in vivo situation rendering the progress of the out of control wound healing process still enigmatic.
To date, 2D and 3D cultures of fibroblasts derived from DD patients are the main experimental models available. Primary cell cultures have many limitations; the fibroblasts derived from DD are altered by the culture conditions, lack cellular context and interactions, which are crucial for the development of fibrosis and weakly represent the derived tissue. Real-time PCR analysis of fibroblasts derived from control and DD samples show that little difference is detectable. 3D cultures of fibroblasts include addition of extracellular matrix that alters the native conditions of these cells. 
As a way to characterize the fibrotic, proliferative properties of these resection specimens we have developed a 3D culture system, using intact human resections of the nodule part of the cord. The system is based on transwell plates with an attached nitrocellulose membrane that allows contact of the tissue with the medium but not with the plastic, thus, preventing the alteration of the tissue. No collagen gel or other extracellular matrix protein substrate is required. The tissue resection specimens maintain their viability and proliferative properties for 7 days. This is the first &#8220;organ&#8221; culture system that allows human resection specimens from DD patients to be grown ex vivo and functionally tested, recapitulating the in vivo situation.

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Dupuytren&#8217;s disease (DD) is a fibroproliferative disease of the palm of the hand. Here, we present a protocol to culture resection specimens from DD in a three-dimensional (3D) culture system. Such short-term culture system allows preservation of the 3D structure and molecular properties of the fibrotic tissue.

Dupuytren Humano do<em&gt; Ex Vivo</em&gt; Cultura para o Estudo da Miofibroblastos e Matriz Extracelular Interações

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from ...

Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little is known about breast cancer cell properties and behaviors within the native microenvironment of human bone tissue.We have developed approaches to track, quantify and modulate human breast cancer cells within the microenvironment of cultured human bone tissue fragments isolated from discarded femoral heads following total hip replacement surgeries. Using breast cancer cells engineered for luciferase and enhanced green fluorescent protein (EGFP) expression, we are able to reproducibly quantitate migration and proliferation patterns using bioluminescence imaging (BLI), track cell interactions within the bone fragments using fluorescence microscopy, and evaluate breast cells after colonization with flow cytometry. The key advantages of this model include: 1) a native, architecturally intact tissue microenvironment that includes relevant human cell types, and 2) direct access to the microenvironment, which facilitates rapid quantitative and qualitative monitoring and perturbation of breast and bone cell properties, behaviors and interactions. A primary limitation, at present, is the finite viability of the tissue fragments, which confines the window of study to short-term culture. Applications of the model system include studying the basic biology of&#160;breast cancer and other bone-seeking malignancies within the metastatic niche, and developing therapeutic strategies to effectively target breast cancer cells in bone tissues.

Protocols are described for studying breast cancer cell migration, proliferation and colonization in a human bone tissue explant model system.

Métodos para a cultura fémur humano explantes de tecido para o Estudo de Câncer de Mama Colonização celular do Niche metastático

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

In Vivo e Ex Vivo Abordagens para o Estudo do cancro do ovário metastático Colonização de Estruturas Láctea natural em Peritoneal adiposo

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

Depleção de células do rato de xenoenxertos de tumor humano Melhora Significativamente Análise Downstream de células alvo

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation

Human small intestinal enteroids are derived from the crypts and when grown in a stem cell niche contain all of the epithelial cell types. The ability to establish human enteroid ex vivo culture systems are important to model intestinal pathophysiology and to study the particular cellular responses involved. In recent years, enteroids from mice and humans are being cultured, passaged, and banked away for future use in several laboratories across the world. This enteroid platform can be used to test the effects of various treatments and drugs and what effects are exerted on different cell types in the intestine. Here, a protocol for establishing primary stem cell-derived small intestinal enteroids derived from neonatal mice and premature human intestine is provided. Moreover, this enteroid culture system was utilized to test the effects of species-specific breast milk. Mouse breast milk can be obtained efficiently using a modified human breast pump and expressed mouse milk can then be used for further research experiments. We now demonstrate the effects of expressed mouse, human, and donor breast milk on the growth and proliferation of enteroids derived from neonatal mice or premature human small intestine.

Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation

This protocol describes how to establish an enteroid culture system from neonatal mouse or premature human intestine as well as an efficient method to collect milk from mice.

O leite materno aumenta o crescimento de Enteroids: um modelo Ex Vivo da proliferação celular

Human small intestinal enteroids are derived from the crypts and when grown in a stem cell niche contain all of the epithelial cell types. The ability to ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Medição de contratilidade isométrica da artéria mesentérica Mouse usando fio miografia

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

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