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>

<ol>
	<li>Barnes, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+single+cells+to+tissues:+interactions+between+the+matrix+and+human+breast+cells+in+real+time.">From single cells to tissues: interactions between the matrix and human breast cells in real time.</a> PLoS ONE. 9, e93325 (2014).</li><li>Berthois, Y., Katzenellenbogen, J. A., Katzenellenbogen, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenol+red+in+tissue+culture+is+a+weak+estrogen:+implications+concerning+the+study+of+estrogen+responsive+cells+in+culture.">Phenol red in tissue culture is a weak estrogen: implications concerning the study of estrogen responsive cells in culture.</a> Proc.Nat.Acad.Sci.USA. 83, 2496-2500 (1986).</li><li>Chalbos, D., Vignon, F., Keydar, I., 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=Estrogens+stimulate+cell+proliferation+and+induce+secretory+proteins+in+a+human+breast+cancer+cell+line+(T47D).">Estrogens stimulate cell proliferation and induce secretory proteins in a human breast cancer cell line (T47D).</a> J.Clin.Endocrinol.Metab. 55, 276-283 (1982).</li><li>Dhimolea, E., Maffini, M. V., Soto, A. M., 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=The+role+of+collagen+reorganization+on+mammary+epithelial+morphogenesis+in+a+3D+culture+model.">The role of collagen reorganization on mammary epithelial morphogenesis in a 3D culture model.</a> Biomaterials. 31, 3622-3630 (2010).</li><li>Dhimolea, E., Soto, A. M., 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=Breast+epithelial+tissue+morphology+is+affected+in+3D+cultures+by+species-specific+collagen-based+extracellular+matrix.">Breast epithelial tissue morphology is affected in 3D cultures by species-specific collagen-based extracellular matrix.</a> J.Biomed.Mat.Res.A. 100, 2905-2912 (2012).</li><li>Krause, S., 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=Dual+regulation+of+breast+tubulogenesis+using+extracellular+matrix+composition+and+stromal+cells.">Dual regulation of breast tubulogenesis using extracellular matrix composition and stromal cells.</a> Tissue Eng Part A. 18, 520-532 (2012).</li><li>Krause, S., Maffini, M. V., Soto, A. M., 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=A+novel+3D+in+vitro.+culture+model+to+study+stromal-epithelial+interactions+in+the+mammary+gland.">A novel 3D in vitro. culture model to study stromal-epithelial interactions in the mammary gland.</a> Tissue Eng.Part C Methods. 14, 261-271 (2008).</li><li>Krause, S., Maffini, M. V., Soto, A. M., 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=The+microenvironment+determines+the+breast+cancer+cells'+phenotype:+organization+of+MCF7+cells+in+3D+cultures.">The microenvironment determines the breast cancer cells' phenotype: organization of MCF7 cells in 3D cultures.</a> BMC Cancer. 10, 263-275 (2010).</li><li>Lee, G. Y., Kenny, P. A., Lee, E. H., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+culture+models+of+normal+and+malignant+breast+epithelial+cells.">Three-dimensional culture models of normal and malignant breast epithelial cells.</a> Nat.Methods. 4, 359-365 (2007).</li><li>Martinson, H. A., Jindal, S., Durand-Rougely, C., Borges, V. F., Schedin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing-like+immune+program+facilitates+postpartum+mammary+gland+involution+and+tumor+progression.">Wound healing-like immune program facilitates postpartum mammary gland involution and tumor progression.</a> Int.J.Cancer. , (2014).</li><li>Soto, A. M., Murai, J. T., Siiteri, P. 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.</...

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 border="0" cellpadding="0" cellspacing="0" width="1344">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">12-well Tissue Culture Plates (Falcon)</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:171px;">08-772-29</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">15 ml polystyrene conical Tubes</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:171px;">14-959-49D</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Activated Charcoal</td>
			<td style="width:249px;">Sigma</td>
			<td style="width:171px;">C-5510</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Carmine Alum</td>
			<td style="width:249px;">Sigma</td>
			<td style="width:171px;">C1022-100G</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Collagenase, Type 3</td>
			<td style="width:249px;">Worthington</td>
			<td style="width:171px;">S0C11784</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Confocal Microscope</td>
			<td style="width:249px;">Zeiss</td>
			<td style="width:171px;">LSM510</td>
			<td style="width:488px;">Equiped with HeNe 633nm laser</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Dextran T-70</td>
			<td style="width:249px;">Abersham/Pharmacia</td>
			<td style="width:171px;">17-0280-01</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">DMEM/F-12, HEPES, no phenol red&#160;</td>
			<td style="width:249px;">Life Technologies</td>
			<td style="width:171px;">11339-021</td>
			<td style="width:488px;">Phenol red-free media for hormone use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:22px;width:436px;">DMEM, low glucose, pyruvate, no glutamine, no phenol red</td>
			<td style="width:249px;">Life Technologies</td>
			<td style="width:171px;">11054-020</td>
			<td style="width:488px;">Phenol red-free media for hormone use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">17-&#946;-Estradiol</td>
			<td style="width:249px;">EMD Millipore</td>
			<td style="width:171px;">3301</td>
			<td style="width:488px;">Dissolved in Ethanol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Ethanol</td>
			<td style="width:249px;">Koptec</td>
			<td style="width:171px;">V1001</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Fetal Bovine Serum</td>
			<td style="width:249px;">Hyclone</td>
			<td style="width:171px;">SH30070.03</td>
			<td style="width:488px;">For use with hormones, must be Charcoal Dextran stripped&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:436px;">Filters (115ml)</td>
			<td style="width:249px;">Nalgene</td>
			<td style="width:171px;">380-0080, 245-0045, 120-0020</td>
			<td style="width:488px;">0.88, 0.45, 0.20 micron, respectively&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Formalin, 10%</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:171px;">SF93-20</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">L-Glutamine (200 mM)</td>
			<td style="width:249px;">Life Technologies</td>
			<td style="width:171px;">25030-081</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">ICI 182,780 (fulvestrant)</td>
			<td style="width:249px;">Sigma Aldrich</td>
			<td style="width:171px;">I4409-25MG</td>
			<td style="width:488px;">Dissolved in DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Microtome&#160;</td>
			<td style="width:249px;">Leica</td>
			<td style="width:171px;">RM2155</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Tissue embedding media</td>
			<td style="width:249px;">McCormick Scientific</td>
			<td style="width:171px;">39502004</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Penicillin</td>
			<td style="width:249px;">Sigma</td>
			<td style="width:171px;">7794-10MU</td>
			<td style="width:488px;">Dissolved in 10 ml of distilled deionized water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Permount</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:171px;">SP15-500</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Phosphate Buffered Saline pH 7.4</td>
			<td style="width:249px;">Sigma Aldrich</td>
			<td style="width:171px;">P3813-10PAK</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Prolactin</td>
			<td style="width:249px;">Sigma Aldrich</td>
			<td style="width:171px;">L4021-50UG</td>
			<td style="width:488px;">Dissolved in distilled deionized water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Promegestone</td>
			<td style="width:249px;">Perkin Elmer</td>
			<td style="width:171px;">NLP004005MG</td>
			<td style="width:488px;">Dissolved in Ethanol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Rat-Tail Collagen&#160;</td>
			<td style="width:249px;">Corning&#160;</td>
			<td style="width:171px;">354236</td>
			<td style="width:488px;">Lots may contain varying concentrations, note accordingly</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Scalpel</td>
			<td style="width:249px;">Miltex</td>
			<td style="width:171px;">4311</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Semi-enclosed Benchtop Tissue Processor</td>
			<td style="width:249px;">Leica</td>
			<td style="width:171px;">TP1020</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Sodium Hydroxide</td>
			<td style="width:249px;">Sigma Aldrich</td>
			<td style="width:171px;">S5881</td>
			<td style="width:488px;">Prepare 1N NaOH stock</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">StaticMaster Anti-static brush&#160;</td>
			<td style="width:249px;">Amstat</td>
			<td style="width:171px;">C3500</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Stripette Serological Pipettes&#160;</td>
			<td style="width:249px;">Corning&#160;</td>
			<td style="width:171px;">4101</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">T-25 flasks</td>
			<td style="width:249px;">Corning&#160;</td>
			<td style="width:171px;">430168</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Tissue Cassettes</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:171px;">15-200-403E</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Wheaton Vials, Glass, 20mL</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:171px;">03-341-25D&#160;</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Xylene&#160;</td>
			<td style="width:249px;">VWR</td>
			<td style="width:171px;">95057-822</td>
			<td style="width:488px;"></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. <p class="jove_co...

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

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

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

Uma simples gota de suspensão Protocolo Cultura de Células para a geração de esferóides 3D

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

Bioengenharia

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

Sistemas Célula de rotação de cultura para cultura de células humanas: células trofoblásticas Humanos como um modelo

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Engenharia de Tecidos de um 3D Humano<em&gt; In vitro</em&gt; Sistema de Teste de Tumor

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

Produção, Caracterização e usos potenciais de uma mucosa esofágica Modelo 3D Human Tissue-engenharia

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Preparação de 3D colágeno Géis e microcanais para o estudo das interações 3D<em&gt; In Vivo</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Sintonizável hidrogéis a partir de Pulmonar Matriz Extracelular para Cultura Celular 3D

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Intraductal Delivery to the Rabbit Mammary Gland

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and adverse effects1,2,3,4,5,6,7. However, several challenges remain before these treatments can be applied more widely. The development and validation of intraductal therapeutics in an appropriate animal model facilitate the development of intraductal therapeutic strategies for patients. While the mouse mammary gland has been widely used as a model system of mammary development and tumorigenesis, the anatomy is distinct from the human gland. A larger animal model, such as the rabbit, may serve as a better model for mammary gland structure and intraductal therapeutic development. In contrast to mice, in which ten ductal trees are spatially distributed along the body axis, each terminating in a separate teat, the rabbit mammary gland more closely resembles the human gland, with multiple overlapping ductal systems that exit through separate openings in one teat. Here, we present minimally invasive methods for the delivery of reagents directly into the rabbit mammary duct and for visualization of the delivery itself with high-resolution ultrasound imaging.

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

Entrega Intraductal ao Coelho glândula mamária

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

High Resolution 3D Imaging of the Human Pancreas Neuro-insular Network

Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological sections are generally limited to &#60;20 &#181;m as formalin fixed paraffin section on glass slides or &#60;500 &#181;m for free-floating fixed sections. Therefore, extensive efforts are required for serial sectioning and large-scale image reconstruction methods to recreate 3D for samples &#62;500 &#181;m using traditional methods. In addition, light scatters from macromolecules within tissues, particularly lipids, prevents imaging to a depth &#62;150 &#181;m with most confocal microscopes. To reduce light scatter and to allow for deep tissue imaging using simple confocal microscopy, various optical clearing methods have been developed that are relevant for rodent and human tissue samples fixed by immersion. Several methods are related and use protein crosslinking with acrylamide and tissue clearing with sodium dodecyl sulfate (SDS). Other optical clearing techniques used various solvents though each modification had various advantages and disadvantages. Here, an optimized passive optical clearing method is described for studies of the human pancreas innervation and specifically for interrogation of the innervation of human islets.

Here, we present a protocol to image human pancreas sections in three dimensions (3D) using optimized passive clearing methods. This manuscript demonstrates these procedures for passive optical clearing followed by multiple immunofluorescence staining to identify key elements of the autonomic and sensory neural networks innervating human islets.

Imagens 3D de alta resolução da rede Neuro-insular pâncreas humano

Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological ...

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low cell retention and poor survival within the target zone. However, during the in vitro construction process, a solution for maintaining stem cell bioactivity and increasing the cell amount within the cell sheet is urgently needed. Here, this protocol presents a method for constructing a multilayered cell sheet with favorable stem cell bioactivity and optimal operability. Decellularized porcine pericardium (DPP) is prepared by phospholipase A2 (PLA2) decellularization method as the cell sheet scaffold, and rat bone marrow mesenchymal stem cells (BMSCs) are isolated and expanded as the seeded cells. The temporary multilayered cell sheet structure is constructed by using RAD16-I peptide hydrogel. Finally, the cell sheet is cultured with a dynamic perfusion system to stabilize the three-dimensional (3D) structure, and the cell sheet could be obtained following a 48-hour culture in vitro. This protocol provides an efficient and feasible method for constructing a multilayered stem cell sheet, and the cell sheet could be developed as a favorable stem cell therapy product in the future.

This article provides an efficient and feasible method for constructing multilayered stem cell sheets with favorable stem cell property.

Construção de uma folha de células-tronco mesenquimais em várias camadas com um sistema de cultura dinâmica 3D

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...

Growth and Characterization of Irradiated Organoids from Mammary Glands

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

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

Crescimento e caracterização de organóides irradiados de glândulas mamárias

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

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

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