Name: a hormone-responsive 3d culture model of the human mammary gland epithelium
Uploaded: 2016-02-07T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium at JoVE.com

Agradecemos imensamente as contribuições editoriais pela Cheryl Schaeberle. Esta pesquisa foi apoiada pela Avon Grants # 02-2009-093 e 02-2011-095, e NIEHS / NIH ES 08.314 para AMS. O conteúdo é da exclusiva responsabilidade dos autores e não representam necessariamente as opiniões oficiais do Instituto Nacional de Ciências de Saúde Ambiental ou o National Institutes of Health.

1. Preparação dos Reagentes <ol><li> Dissolve-se o progestagénio sintético promegestona (R5020) e 17-β-estradiol (E2) em etanol para fazer soluções de reserva 10 -3 m. Dissolver prolactina em água desionizada destilada para produzir uma solução stock de 1 mg / ml. Dissolve-se o antiestrogénio ICI 182,780 em DMSO para fazer uma solução estoque de 10 -2 M. Armazenar as soluções à temperatura de -20 ° C durante até 6 meses. </li><li> dextrano carvão (CD) despojado de soro e médio CDFBS: <ol><li> Calor-inactivar soro fetal de bovino (FBS) em um banho de água a 57 ° C durante 30 min. Nota: Use apenas material de vidro lavado com ácido para o restante deste protocolo. </li><li> Calcula-se a quantidade de carvão necessária. Usando 10% mais volume do que a quantidade de soro para compensar o deslocamento provocado pelo carvão, pesar 5% p / v de carvão activado em pó. </li><li> Lavar duas vezes com carvão vegetal água destilada desionizada fria usando o mesmo volume que a quantidade de soro. Para cada lavagem centrifuge a 50 xg durante 2 min para sedimentar o carvão vegetal. Retire a água entre as lavagens. Adicionar 0,5% p / v de Dextrano T-70 para o último sedimento carvão. Re-suspender com água destilada, água desionizada e centrifugar a 100 xg durante 5 min. Aspirar a água. </li><li> Adicionar o volume apropriado de soro para o sedimento e re-suspender o sedimento carvão dextrano no soro. Incubar, enquanto rola (6 RPM), a 37 ºC durante 60 min. Centrifugar a 2000 xg durante 20 min. Se sobrenadante turvo, repita a centrifugação; no entanto, o soro pode não ser completamente claro neste momento, mas serão apagados durante a filtração. </li><li> Filtrar através de um filtro de 0,80 micron e, em seguida, novamente através de um filtro de 0,45 mícron para remover matéria em partículas maiores. Por último, esterilizar-filtro com um filtro de 0,20 micron. Loja filtrada CDFBS em tubos de vidro ou T-25 frascos de cultura a -20 ºC. </li><li> Charcoal Dextran Stripped fetal bovino contendo media (mídia CDFBS): misturar 375 ml de fenol DMEM isento de vermelho e 125 ml de DMEM/ F-12. Remover 37,5 ml e substituí-lo com o mesmo volume de soro fetal bovino carvão dextrano Stripped. Adiciona-se 10 6 U / ml de penicilina e L-glutamina numa concentração final de 2 mM. Os meios de comunicação resultante é de 75% de DMEM, 25% de DMEM / F-12, 7,5% de dextrano a carvão despojado-FBS, 2 mM de L-glutamina, e 10 6 U / ml de penicilina. </li></ol></li><li> Prepare PBS 10X, NaOH 1 N e água desionizada destilada. Prepare um volume suficiente de cada um de acordo com o volume de solução de colágeno necessário. Esterilizar todas as soluções de filtragem. Armazenar a 10x PBS e água desionizada destilada estéril a 4 ° C durante até 1 mês. Não guarde a solução de NaOH 1N. </li><li> Preparar a solução de colagénio a uma concentração final de 1 mg / ml de acordo com o protocolo para &quot;Processo de gelificação alternativo para o colagénio I da cauda de rato&quot; fornecida pelo fabricante. Cada gel é feito de 1,5 ml de solução de colagénio. Por exemplo, preparar a 18,5 ml (0,5 ml para ter em conta perdas durante a pipetagem) de solução de colagénio para preparar12 géis. Utilizar a solução de colagénio imediatamente ou manter em gelo durante até 2-3 horas. </li><li> Carmine Alum corante: <ol><li> Combinar 1 g Carmine e 2,5 g de sulfato de alumínio e potássio em 500 ml de água destilada. Ferver enquanto se agitava durante 20 min. Observe com cuidado para evitar a ferver sobre. Ajustar o volume final de volta para 500 ml com água destilada adicional. </li><li> Adicionar um cristal de timol para a atividade antimicrobiana e cobrir garrafa com folha de alumínio para proteger da luz. Filtrar através de um filtro de papel de grau de laboratório antes de cada utilização. Reutilizar a solução de carmim por até três meses. Armazenar à temperatura ambiente, protegida da luz. </li></ol></li><li> solução de colagenase: dissolver a colagenase em meio DMEM / F12 a uma concentração de 0,125% w / v. Não guarde esta solução. </li></ol> 2. Cultura 3D de T47D As células em Rat cauda colágeno tipo 1 Géis e tratamento hormonal Nota: manter pipetas serológicos estéreis e pontas de pipeta a 4 ° C. <ol><li> Prepare uma suspensão de uma única célula e executar uma contagem de células. culturas de células de divisão T47D antes que eles atinjam 70% de confluência. Centrifuga-se o volume que contém a quantidade de células necessárias. Considere-se que um gel deve conter cerca de 75.000 células T47D. </li><li> Usando uma pipeta a frio, de re-suspender o sedimento de células no volume apropriado de colagénio. Considere-se que cada gel é composta de 1,5 ml de colagénio. Misturar as células e colagénio o cuidado para evitar a introdução de bolhas. </li><li> Para impedir o efeito de electricidade estática na distribuição das células, o escovar fronteiras, superior e inferior da placa de 12 poços com uma escova estática de redução depois de retirar a placa. Para evitar o acúmulo de carga estática durante o contato com a superfície de trabalho da capa, colocar esta placa em cima da outra placa de 12 poços (placa em branco), que também foi escovado com o brush.Gently estática de redução, despeje 1,5 ml da mistura em cada poço da placa de 12 poços. </li><li> Colocar a placa numa incubadora a 37 ° C durante 30 min. Remover the placa da incubadora e coloque-o novamente na capa. </li><li> Usando uma ponta estéril p200, retire cuidadosamente o gel em um suave movimento circular a partir da fronteira do poço. O gel também deve destacar a partir do fundo do poço. </li><li> Diluir hormonas em meio CDFBS a ser adicionado aos poços. Use E2 e R5020 a uma concentração final de 10 -10 M e prolactina num a 10 -7 M concentração final. Como controle, usar a mídia CDFBS sem hormônios adicionados, mas ainda contendo o maior volume de DMSO igual às existências hormonais utilizados. Certifique-se de que cada poço contém 1,5 ml de meio. Coloque as culturas 3D dentro de uma temperatura de 37 ° C, 5% de CO2, 100% tecido humidade cultura incubadora. Manter as culturas por 1 ou 2 semanas, mudando os meios de comunicação a cada 2 dias. </li></ol> 3. Gel de processamento para toda Mounts <ol><li> Remover o meio de cultura e adicionar 1,5 ml de PBS a cada poço. </li><li> Transferir o gel para um slide. Cortar o gel em meio usando uma curvad lâmina cirúrgica. Continuar o processamento com uma metade e guardar a outra metade para análise histológica. </li><li> Transfira a metade gel para toda montar a um slide. Vamos gel seco durante 5 minutos e, em seguida, transferir o slide para um frasco Coplin contendo formol a 10%. Nota: todas as incubações deve ser feito à temperatura ambiente. </li><li> Fix géis em um frasco coplin num agitador O / N. </li><li> Lave as lâminas com PBS duas vezes durante 10 minutos cada em agitador. Transferir as lâminas para 70% de EtOH; incubar durante 1 hora no agitador. lâminas de transferência para Carmine Alum O / N. </li><li> No dia seguinte, desidratar lâminas num agitador em EtOH a 70%, EtOH a 95% e 100% de EtOH, durante 1 h cada. Transferir as lâminas ao xileno duas vezes para 1 hora cada e manter em shaker. Use meio de montagem à base de tolueno para montar os géis e 1,5 mm de espessura Lamelas. </li><li> Examine as peças inteiras sob luz normal (microscópio ou estereoscópio). Para uma análise mais detalhada da forma e lumen usar microscopia confocal e visualizar corante carmim com HeNe 633 a laser nm. </li></ol>4. Processamento de Gel de Processamento de Análise de Histologia <ol><li> Transferir a metade restante do gel para um frasco de vidro de 20 ml contendo 10% de formalina e fixar num agitador de O / N. </li><li> Lavar géis duas vezes com PBS durante 10 min cada, num agitador. géis coloque em cassetes de processamento para inclusão em parafina. </li><li> geles de transferência para 70% de EtOH, incubar durante 1 h e seguir com 95% de EtOH, durante 1 h, para alterar fresco EtOH a 95% durante 1 hora, 100% de EtOH, durante 1 h, e para alterar fresco EtOH a 100% durante 1 hora. Neste ponto, os géis podem ser armazenados em EtOH a 100% O / N à TA ou incubaram-se com xileno, durante 1 h e, em seguida, novamente em xileno fresco durante 1 hr. </li><li> cassetes de transferência de parafina durante 1 hora a 60 ° C na incubadora de vácuo, repetir com parafina fresco mais duas vezes. </li><li> Preencha moldes de plástico com parafina limpo. Abrir a cassete, retirar o gel e colocá-la no molde de plástico contendo parafina. Encha-se com parafina e adicione topo da cassete contendo a etiqueta para cima e deixe bloco legal. </li><li> Remover o Plastic molde e seção do bloco usando um micrótomo. Obter 5 um de espessura para a coloração HE e imunohistoquímica. </li></ol> 5. A extracção de células a partir de células a partir de géis usando colagenase tratamento <ol><li> Remover o gel a partir do poço e transferi-lo para uma placa de Petri contendo PBS. brevemente gel de lavagem. </li><li> Transferir o gel para um tubo de 15 ml contendo 2 ml de colagenase a 0,125%. Pipetar cima e para baixo 4-5 vezes de modo a quebrar o gel em pedaços pequenos. </li><li> Incubar a 37 ° C durante 25 minutos (até que se dissolveu em gel e as células estão em suspensão) </li><li> Adicionar 2 ml de meio de cultura soro suplementado. Coletar células por centrifugação (1000 x g, 5 min), sobrenadante de descarte, as células re-suspender e contar. </li></ol>

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K., Sonnenschein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+cell+proliferation:+evidence+for+negative+control+on+estrogen-sensitive+T47D+human+breast+cancer+cells.">Control of cell proliferation: evidence for negative control on estrogen-sensitive T47D human breast cancer cells.</a> Cancer Res. 46, 2271-2275 (1986).</li><li>Speroni, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormonal+regulation+of+epithelial+organization+in+a+3D+breast+tissue+culture+model.">Hormonal regulation of epithelial organization in a 3D breast tissue culture model.</a> Tissue Eng.Part C Methods. 20, 42-51 (2014).</li><li>Vignon, F., Bardon, S., Chalbos, D., Rochefort, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antiestrogenic+effect+of+R5020,+a+synthetic+progestin+in+human+breast+cancer+cells+in+culture.">Antiestrogenic effect of R5020, a synthetic progestin in human breast cancer cells in culture.</a> J.Clin.Endocrinol.Metab. 56, 1124-1130 (1983).</li></ol>

The authors have nothing to disclose.

Here, we describe a hormone-sensitive 3D culture model to test the action of hormones on breast epithelium. The response to hormones can be assessed at the tissue morphology, cell proliferation and gene expression levels 12. One limitation of this technique is that visualization during the culture period is restricted to light microscopy since the cultures are grown in a plastic bottom plate. The 3D culture system could be adapted to glass bottom plates to allow for live imaging of the cultures 1.
The way in which cells are maintained appears to be critical for retention of hormone-responsive phenotypes. It is important to frequently check for E2 responsiveness in these cells. We recommend performing an E2 dose-response curve on the fourth passage after thawing a new vial, and routinely after every 10 passages thereafter. An E2 concentration of 10 -10 M will typically yield three times the control cell number in 2D culture and two times in 3D culture when compared to those grown in CDFBS medium for 1 week 12. Phenol red-free media should be used for all experiments involving hormone-responsiveness, due to the proven estrogenicity of phenol red 2. In addition, the brands listed in this protocol are current without hormonal activity. If the estrogenic response in 2D or 3D changes, the plastic materials used for tissue culture should be evaluated for leaching of hormonally-active compounds. 
The presence of static electricity during gel preparation may affect cell distribution. This may cause the cells to quickly sink to the bottom of the well during congealing and then grow as a 2D monolayer. In this case, the gel appears mostly empty of cells with them attached directly to the bottom plastic surface. To prevent this effect, it is recommended to brush the borders, top and bottom of the 12-well plate with a static-reducing brush after unwrapping the plate. To avoid static charge buildup during contact with the hood working surface, place this plate on top of another 12-well plate (blank plate) that has also been brushed with the static-reducing brush. These recommendations are especially useful during periods of low ambient humidity. 
As opposed to existing "on top" culture models9, the culture system that we describe here allows for the epithelial cells to form 3D structures which are embedded in a matrix. Furthermore, this culture system enables for the study of the influence of extracellular matrix components, such as collagen fibers, and mechanical properties, such as matrix stiffness, in hormone-instructed morphogenesis of 3D epithelial structures 1,12. 
The hormone-sensitive 3D culture model described herein also allows for the addition of cell types considered of relevance in breast tissue morphogenesis, such as fibroblasts, adipocytes and circulating blood cells 6,8,10. Finally, testing of hormonal influence on the breast epithelium in this 3D culture model is not limited to endogenous hormones alone. The response to environmental estrogenic compounds and other hormonally-active chemicals can also be tested to uncover their effects and those elicited when tested in the presence of the natural, endogenous steroidal and polypeptidic hormones.

Ao contrário das culturas 2D padrão, modelos de cultura de células de substituição 3D permitem o estudo do comportamento das células epiteliais num contexto fisiologicamente relevante, um tecido semelhante a um. culturas 3D da glândula mamária ajudaram a explicar muitos aspectos do desenvolvimento da glândula mamaria e neoplasia. No entanto, a maioria dos modelos de cultura 3D actualmente disponíveis não são adequados para estudar a acção hormonal, porque as linhas de células epiteliais humanos utilizados para a tarefa não possuem a expressão do receptor da hormona 6,7,9. Aqui, descrevemos um modelo de cultura 3D do epitélio mamário humano que é adequado para estudar a ação hormonal 12. Este modelo utiliza a linha disponível comercialmente hormona de resposta epitelial humano da mama celular, T47D 3,11,13, que foram originalmente derivados de um derrame pleural obtido a partir de um paciente do sexo feminino de 54 anos com um carcinoma ductal infiltrante da mama. Nós usamos ratos colagénio de cauda de tipo 1 como uma matriz. Este culto 3Dure modelo é apropriado para o estudo da acção dos três principais hormonas mammotropic (estradiol, promegestona (um análogo da progesterona), e prolactina) em células epiteliais da mama humanas. Morfologia epitelial induzida por hormona pode ser avaliado quantitativamente ao longo do tempo através de análises morfométricas 12. Uma densidade de semeadura adequada permite que essas culturas em 3D para ser mantido por 2 semanas. Por esta altura, o desenvolvimento de estruturas é suficiente para uma avaliação quantitativa da acção da hormona robusta na morfologia epitelial. Os géis podem também ser colhidas a pontos de tempo anteriores para a proliferação celular e analisa a expressão do gene. Além disso, este modelo é apropriado para testar os efeitos de um tratamento hormonal sequencial; por exemplo, após o tratamento com estradiol durante a primeira semana e substituição com outra hormona / combinação de hormonas durante a semana seguinte. O efeito dos compostos estrogénicos e anti-estrogénios, tais como a ICI 182,780, também pode ser stumorreu usando este modelo de cultura 3D 12.

<table><tbody><tr><td>12-well Tissue Culture Plates (Falcon)</td><td>Fisher Scientific</td><td>08-772-29</td><td></td></tr><tr><td>15 ml polystyrene conical Tubes</td><td>Fisher Scientific</td><td>14-959-49D</td><td></td></tr><tr><td>Activated Charcoal</td><td>Sigma</td><td>C-5510</td><td></td></tr><tr><td>Carmine Alum</td><td>Sigma</td><td>C1022-100G</td><td></td></tr><tr><td>Collagenase, Type 3</td><td>Worthington</td><td>S0C11784</td><td></td></tr><tr><td>Confocal Microscope</td><td>Zeiss</td><td>LSM510</td><td>Equiped with HeNe 633nm laser</td></tr><tr><td>Dextran T-70</td><td>Abersham/Pharmacia</td><td>17-0280-01</td><td></td></tr><tr><td>DMEM/F-12, HEPES, no phenol red&amp;nbsp;</td><td>Life Technologies</td><td>11339-021</td><td>Phenol red-free media for hormone use</td></tr><tr><td>DMEM, low glucose, pyruvate, no glutamine, no phenol red</td><td>Life Technologies</td><td>11054-020</td><td>Phenol red-free media for hormone use</td></tr><tr><td>17-&amp;beta;-Estradiol</td><td>EMD Millipore</td><td>3301</td><td>Dissolved in Ethanol</td></tr><tr><td>Ethanol</td><td>Koptec</td><td>V1001</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Hyclone</td><td>SH30070.03</td><td>For use with hormones, must be Charcoal Dextran stripped&amp;nbsp;</td></tr><tr><td>Filters (115 ml)</td><td>Nalgene</td><td>380-0080, 245-0045, 120-0020</td><td>0.88, 0.45, 0.20 micron, respectively&amp;nbsp;</td></tr><tr><td>Formalin, 10%</td><td>Fisher Scientific</td><td>SF93-20</td><td></td></tr><tr><td>L-Glutamine (200 mM)</td><td>Life Technologies</td><td>25030-081</td><td></td></tr><tr><td>ICI 182,780 (fulvestrant)</td><td>Sigma Aldrich</td><td>I4409-25MG</td><td>Dissolved in DMSO</td></tr><tr><td>Microtome&amp;nbsp;</td><td>Leica</td><td>RM2155</td><td></td></tr><tr><td>Tissue embedding media</td><td>McCormick Scientific</td><td>39502004</td><td></td></tr><tr><td>Penicillin</td><td>Sigma</td><td>7794-10MU</td><td>Dissolved in 10 ml of distilled deionized water</td></tr><tr><td>Permount</td><td>Fisher Scientific</td><td>SP15-500</td><td></td></tr><tr><td>Phosphate Buffered Saline pH 7.4</td><td>Sigma Aldrich</td><td>P3813-10PAK</td><td></td></tr><tr><td>Prolactin</td><td>Sigma Aldrich</td><td>L4021-50UG</td><td>Dissolved in distilled deionized water</td></tr><tr><td>Promegestone</td><td>Perkin Elmer</td><td>NLP004005MG</td><td>Dissolved in Ethanol</td></tr><tr><td>Rat-Tail Collagen&amp;nbsp;</td><td>Corning&amp;nbsp;</td><td>354236</td><td>Lots may contain varying concentrations, note accordingly</td></tr><tr><td>Scalpel</td><td>Miltex</td><td>4311</td><td></td></tr><tr><td>Semi-enclosed Benchtop Tissue Processor</td><td>Leica</td><td>TP1020</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>Sigma Aldrich</td><td>S5881</td><td>Prepare 1N NaOH stock</td></tr><tr><td>StaticMaster Anti-static brush&amp;nbsp;</td><td>Amstat</td><td>C3500</td><td></td></tr><tr><td>Stripette Serological Pipettes&amp;nbsp;</td><td>Corning&amp;nbsp;</td><td>4101</td><td></td></tr><tr><td>T-25 flasks</td><td>Corning&amp;nbsp;</td><td>430168</td><td></td></tr><tr><td>Tissue Cassettes</td><td>Fisher Scientific</td><td>15-200-403E</td><td></td></tr><tr><td>Wheaton Vials, Glass, 20 ml</td><td>Fisher Scientific</td><td>03-341-25D&amp;nbsp;</td><td></td></tr><tr><td>Xylene&amp;nbsp;</td><td>VWR</td><td>95057-822</td><td></td></tr></tbody></table>

a hormone-responsive 3d culture model of the human mammary gland epithelium

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

A Figura 1 resume o procedimento para a preparação das culturas em 3D sensível ao hormônio. estruturas epiteliais são observados em peças inteiras de géis cultivadas durante 2 semanas, na presença de E2 sozinho e em combinação com outras hormonas. Apenas as células individuais ou grupos de 2-3 células estão presentes quando não há hormonas são adicionados ao meio de cultura (meio de CDFBS) (Figura 2). Esta condição serve como um controlo negativo. As células em cultura em 3D formam estruturas que variam em tamanho, forma, presença e lúmen. A distribuição das estruturas no gel é tipicamente heterogénea. Como foi mostrado anteriormente, geralmente há mais estruturas ao longo das fronteiras do gel do que o observado no centro 4. Devido a esta heterogeneidade espacial, comparações entre as amostras devem ser limitados a regiões correspondentes em toda amostras. A espécie de colagénio, que é um tipo de derivados influências do fenótipo umqualidade nd das estruturas epiteliais resultantes 5. Rato cauda colagénio de tipo 1, tal como se utiliza aqui, uma vez que foi escolhido semeando em colagénio bovino tipo 1 resultou na formação de grupos de células desorganizados (Figura 3). Uma análise detalhada das estruturas epiteliais pode ser conseguida utilizando microscopia confocal (Figura 4). E2 tratamento resulta em estruturas arredondados e alongada (Figura 4A). A presença de E2 e resultados de prolactina em estruturas de brotamento mais (Figura 4B), enquanto que a presença de E2 e resultados promegestona em estruturas irregulares com projecções celulares reminiscentes da ramificação lateral (Figura 4C). E2 sozinho e E2 além de prolactina engrossar estruturas de tecido, quando medido pelo crescimento na z-direção, e quando comparados com os observados em E2 mais promegestona. A fim de quantificar o número de células após o tratamento com a hormona, as células são extracted a partir do gel utilizando o tratamento com colagenase. Por exemplo, utilizando este método pode-se estudar a forma como o antiestrogénio ICI 182,780 inibe o efeito de E2 sobre a proliferação celular nas culturas 3D (Figura 5). <img alt="figura 1" src="/files/ftp_upload/53098/53098fig1.jpg" /> Figura 1. Processo para a preparação de culturas em 3D sensível à hormona. Resumidamente, uma suspensão de célula única de células T47D é misturado com colagénio de cauda de rato do tipo 1 e verteu-se em poços de uma placa de 12 poços. Os géis são permitidas para coagular durante 30 minutos num banho a 37 ° C / 5% de CO2, incubadora. O meio de cultura é adicionado e os géis são separadas do bem. As culturas podem ser colhidas após 1 ou 2 semanas de acordo com o ponto final. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53098/53098fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> . Figura 2. peças inteiras de culturas 3D após coloração com carmim Alum corante (A) Monte de metade de um gel corado com alúmen carmim de visualizar as estruturas epiteliais, um exemplo de ROI para análise da morfologia está indicado por uma caixa; WM de culturas 3D cultivadas durante 2 semanas em (B) CDFBS media (sem hormonas adicionados) e (C) CDFBS meios suplementados com E2 e prolactina. Barras de escala são de 5.000 mm (A) e 500 m (B &amp; C). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53098/53098fig2large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 3" src="/files/ftp_upload/53098/53098fig3.jpg" /> Figura 3. Comparação de matrizes utilizadas em cultura 3D. (A) Bovine colágeno tipo 1 resulta em aglomerados desorganizados de células, em oposição às estruturas organizadas (B) observado quando se utiliza cauda de rato colágeno tipo bar 1. Escala:. 100 mm <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53098/53098fig3large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 4" src="/files/ftp_upload/53098/53098fig4.jpg" /> Figura 4. imagens confocais de estruturas T47D observadas em géis de colagénio 3-D após 2 semanas. O tratamento com (A) E2, (B) de E2 + prolactina e (C) de E2 + R5020. As setas indicam as projeções citoplasmáticas. Espessura de estruturas epiteliais foi de 24, 40 e 20 uM respectivamente. Barra de escala:. 40 mm <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53098/53098fig4large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> Figura 5. culturas 3D tratados apenas com tanto E2 ou E2 além ICI 182.780 por 1 semana. As contagens de células após a extração de células usando o tratamento colagenase. (A) CDFBS médio, (B) CDFBS meio mais E2 no 1x10 -10 M, (C) ICI 182,780 na 1x10 -8 M mais E2 no 1x10 -10 M e (D) ICI 182.780 em 1x 10-7 M mais E2 a 1x10 -10 M. Bares:. SEM <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53098/53098fig5large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a>

Watch this Scientific Journal Video about A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium at JoVE.com

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

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

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

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

a-hormone-responsive-3d-culture-model-human-mammary-gland

Nanyang Technological University

Research

JoVE Journal

Bioengineering

8.7K visualizações.  Tufts University School of Medicine. We describe a 3D culture model of the human breast epithelium that is suitable to study hormone action.

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

Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction of differentiation of two mouse breast epithelial cell lines, HC11 and EpH4, and their use to study complementary stages of mammary gland development and neoplastic transformation.
The HC11 mouse breast epithelial cell line originated from the mammary gland of a pregnant Balb/c mouse. It differentiates when grown to confluence attached to a plastic Petri dish surface in medium containing fetal calf serum and Hydrocortisone, Insulin and Prolactin (HIP medium). Under these conditions, HC11 cells produce the milk proteins &#946;-casein and whey acidic protein (WAP), similar to lactating mammary epithelial cells, and form rudimentary mammary gland-like structures termed &#34;domes&#34;.
The EpH4 cell line was derived from spontaneously immortalized mouse mammary gland epithelial cells isolated from a pregnant Balb/c mouse. Unlike HC11, EpH4 cells can fully differentiate into spheroids (also called mammospheres) when cultured under three-dimensional (3D) growth conditions in HIP medium. Cells are trypsinized, suspended in a 20% matrix consisting of a mixture of extracellular matrix proteins produced by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, plated on top of a layer of concentrated matrix coating a plastic Petri dish or multiwell plate, and covered with a layer of 10% matrix-containing HIP medium. Under these conditions, EpH4 cells form hollow spheroids that exhibit apical-basal polarity, a hollow lumen, and produce &#946;-casein and WAP.
Using these techniques, our results demonstrated that the intensity of the cadherin/Rac signal is critical for the differentiation of HC11 cells. While Rac1 is necessary for differentiation and low levels of activated RacV12 increase differentiation, high RacV12 levels block differentiation while inducing neoplasia. In contrast, EpH4 cells represent an earlier stage in mammary epithelial differentiation, which is inhibited by even low levels of RacV12.

We describe techniques for differentiation induction of two breast epithelial lines, HC11 and EpH4. While both require fetal calf serum, insulin, and prolactin to produce milk proteins, EpH4 cells can fully differentiate into mammospheres in three-dimensional culture. These complementary models are useful for signal transduction studies of differentiation and neoplasia.

Diferenciação das células Epiteliais do Peito do Camundongo HC11 e EpH4

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction ...

Pesquisa do câncer

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

A Cultura de Tecidos modelo tridimensional para estudar primária da medula óssea humana e suas Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Medicina

Phospholipid Mediator Induced Transformation in Three-Dimensional Cultures

Several models have been developed to study cancer, such as rodent models and established cell lines. Valuable insights into carcinogenesis have been provided by studies using these models. Cell lines have provided an understanding of the deregulation of molecular signaling associated with breast tumorigenesis, while rodent models are widely used to study cellular and molecular characteristics of breast cancer in vivo. The establishment of 3D cultures of breast epithelial and cancerous cells aids in bridging the gap between in vivo and in vitro models by mimicking the in vivo conditions in vitro. This model can be used to understand the deregulation of complex molecular signaling events and the cellular characteristics during breast carcinogenesis. Here, a 3D culture system is modified to study a phospholipid mediator-induced (Platelet Activating Factor, PAF) transformation. Immunomodulators and other secreted molecules play a major role in tumor initiation and progression in the breast. In the present study, 3D acinar cultures of breast epithelial cells are exposed to PAF exhibited transformation characteristics such as loss of polarity and altered cellular characteristics. This 3D culture system will assist in shedding light on genetic and/or epigenetic perturbations induced by various small molecule entities in the tumor microenvironment. Additionally, this system will also provide a platform for the identification of novel as well as known genes that may be involved in the process of transformation.

The present protocol describes the setting up of 3D 'on top' cultures of a non-transformed breast epithelial cell line, MCF10A, that has been modified to study Platelet Activating Factor (PAF) induced transformation. Immuno-fluorescence has been used to assess the transformation and is discussed in detail.

Mediadora fosfolipídida induzida transformação em culturas tridimensionais

Several models have been developed to study cancer, such as rodent models and established cell lines. Valuable insights into carcinogenesis have been ...

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

Três Culturas dimensionais: Uma ferramenta para o estudo normal Acináceo Arquitetura vs transformação maligna das células de mama

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

Quantificação de cancro da mama invasiva das células usando um modelo (3D) tridimensional

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after propagation from primary tissue or stem cells. This method of organoid generation forgoes single-cell differentiation through multiple passages and instead uses differential centrifugation to isolate mammary epithelial organoids from mechanically and enzymatically dissociated tissues. This protocol provides a streamlined technique for rapidly producing small and large epithelial organoids from both mouse and human mammary tissue in addition to techniques for organoid embedding in collagen and basement extracellular matrix. Furthermore, instructions for in-gel fixation and immunofluorescent staining are provided for the purpose of visualizing organoid morphology and density. These methodologies are suitable for myriad downstream analyses, such as co-culturing with immune cells and ex vivo metastasis modeling via collagen invasion assay. These analyses serve to better elucidate cell-cell behavior and create a more complete understanding of interactions within the tumor microenvironment.

This protocol discusses an approach for generating epithelial organoids from primary normal and tumor mammary tissue through differential centrifugation. Furthermore, instructions are included for three-dimensional culturing as well as immunofluorescent imaging of embedded organoids.

Gerando e Imageando Organoides Epitéliares de Camundongos e Humanos a partir de Tecido Mamário Normal e Tumoral Sem Passagem

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

Engenharia tridimensionais tecidos epiteliais incorporado dentro Matriz Extracelular

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Bioengenharia

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

Geração de Organóides Mammary mosaicos por Trypsinização Diferencial

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

Biologia do Desenvolvimento

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

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 cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

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

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

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 ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

Isolamento de células epiteliais mamárias de tridimensional Mixed-célula esferoidal co-cultura

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...