Este trabalho foi financiado pelo NIH concede R01 CA107469, CA125577 R01 e U01CA154224 para CG Kleer, a Universidade do Centro de Câncer de Apoio de Michigan.

1. Preparação de Materiais e Meios de Cultura <ol><li> Fator de crescimento Thaw reduzida (TFG) Matrigel a 4 ° CO / N. </li><li> Aqueça mídia completas para linha celular necessário a 37 ° C. MCF10A-ATCC especificado media; Meio F-12 de Ham-HME suplementado com 5% de soro fetal bovino, ml, 5 ug / ml de insulina, factor de 1 ug / hidrocortisona de 10 ng / mL de crescimento epidérmico, e 100 ng de toxina da cólera ml /; F-12 media de SUM149-Ham suplementado com FBS a 5%, 2 ug / ml de hidrocortisona e de 5 ug / ml de insulina; e MDA-MB-231-10% de FBS-DMEM </li><li> Precool um slide de câmara de 8 poços no gelo. </li><li> Prepare cultura de tecidos capô e suprimentos. </li></ol> 2. Três Cultura Dimensional em 8 poços Deslize Câmara <ol><li> Casaco de cada poço a 8 poços corrediça com 40 ul de GFR Matrigel. Adicionar o Matrigel no centro da lâmina e, em seguida, espalhada com uma pipeta de ponta P200. Tente espalhar o mais uniformemente possível, evitando bolhas e espalhando que pode levar aformação do menisco. </li><li> Incubar as lâminas a 37 ° C sob 5% de CO 2. Enquanto o Matrigel está solidificando, trypsinize as células, recolher e girar a 150 xg em cultura de tecidos centrifugar por 4 min. </li><li> Contar as células e dilui-se a 12.500 células / mL em meios completos da linha celular respectivos. Adicionar TFG Matrigel a 2% e adicionar 400 ul a cada poço de uma lâmina de câmara de pré-revestidas de Matrigel. </li><li> Incubar as lâminas na incubadora de CO 2. Dar novas mudanças de mídia completas a cada 4 º dia até 15 dias. </li><li> O tratamento com inibidores farmacológicos: Para estudos de inibidor de adicionar os inibidores de moléculas pequenas para o meio completo no momento do plaqueamento seguido por mudanças de meio fresco suplementado com os inibidores de três em três dias, enquanto o crescimento das células em TFG Matrigel. </li><li> O tratamento com a proteína purificada: <ol><li> Placa as células seguintes passos 2,1-2,3. Permitir que as culturas a crescer durante 4 dias, seguido por starvati soropor 16 h. </li><li> No dia 5, tratar as células com uma concentração apropriada de proteína purificada em 0,1% de FBS suplementado com os meios de células em jejum de soro. Dê mudança meio fresco com proteína purificada após 2 dias. </li><li> No dia 10 de substituir a meios condicionados com a mídia HME completos. Permitir que as culturas a crescer durante mais 5 dias. </li></ol></li><li> Coloração por imunofluorescência de estruturas acinares cultivadas em Matrigel: <ol><li> Prepare a 2% de paraformaldeído, 0,5% de Triton X-100 e 100 mM de glicina em 1x PBS, pH 7,4. Preparar tampão de imunofluorescência (IF tampão), que compreende de 1 x PBS contendo 0,1% de albumina de soro bovino, 0,2% de triton X-100, 0,05% de Tween-20. </li><li> Use uma pipeta de ponta P200 para aspirar cuidadosamente a meios condicionados. </li><li> Fixar as estruturas acinares com paraformaldeído a 2% preparada de fresco, à temperatura ambiente (RT) durante 20 min. </li><li> Permeabilizar as células com 0,5% de Triton X-100 durante 10 min a 4 ° C. </li><li> Lava-se a culturas três vezes com 100 mM glicina, 10-15 minutos para cada lavagem. </li><li> Bloquear as células com 10% de soro de cabra em tampão IF, chamado bloco primário, durante 1 hora a RT. </li><li> Incubar as culturas com 20 ng / ml de cabra anti-rato F (ab &#39;) 2 em tampão fragmento bloco primário durante 30 min. Esta etapa é importante para bloquear a espécie de IgG de ratinho imunorreactivas em Matrigel. </li><li> Incubar com anticorpo primário em que a segunda solução de bloqueio (bloco primário 20 ug / ml de cabra anti-rato F (ab &#39;) 2 fragment) O / N a 4 ° C. </li><li> Lavar três vezes com tampão IF por 10-15 min cada um com balanço suave. </li><li> Incube com anticorpos secundários conjugados fluorescentes em tampão IF para 45 min. </li><li> Lavar 3x por 10 minutos cada com tampão IF com balanço suave. </li><li> Montar as lâminas com o reagente anti-fade contendo DAPI para visualizar os núcleos. </li><li> Permitir que as lâminas secar O / N à temperatura ambiente. </li><li> A aquisição de imagens: Adquirir imagens confocal nas milhas da colôniadsection de células cultivadas em cima de Matrigel. Para mais informações adquirir uma série de secções ópticas ao longo de todo o comprimento da estrutura acinar por Z empilhamento. </li></ol></li></ol> 3. Três Cultura Dimensional em 2-bem Câmara Slides para Immunoblotting <ol><li> Precool um slide câmara de 2-bem no gelo. Siga passos 2,1-2,4 intensificação os reagentes para a câmara de 2-bem corrediça por exemplo revestimento com 160 mL de Matrigel e adicionar 1.6 ml de células / Matrigel misturar a cada poço da 2-bem corrediça. </li><li> Colher as estruturas acinares de Matrigel, utilizando o estojo de colheita de células de cultura 3D disponível comercialmente seguindo as instruções do fabricante. </li><li> Diluir a lavagem das células e colheita de células buffers para 1x e pré-resfriamento a 4 ° C. Lavar e deslocamento de Matrigel deve ser levada a cabo em gelo. </li><li> Lavar as estruturas acinares 3x utilizando o tampão de lavagem celular. </li><li> Recolher o ácinos em 15 ml tubos de centrífuga usando bu colheita de célulasffer seguido por 30 min de incubação num agitador rotativo a 4 ° C. Misture bem a suspensão com uma pipeta de 1 ml. Isto vai quebrar o Matrigel em pequenos pedaços e libertar a maior parte das células do tampão de colheita de células. </li><li> Agregar as células a 200 xg numa centrifugadora de cultura. Retire a camada flutuante de hidrogel a partir de células peletizadas com uma pipeta de ponta P200. </li><li> Ressuspender as lisados ​​em tampão de amostra de SDS fornecidos com o kit. Tampão de lise RIPA também pode ser usado. </li></ol> 4. Quantificação de vs maligno Fenótipo benigna da mama <ol><li> Cultivar células em triplicatas para cada conjunto de tratamento seguindo os passos 2,1-2,4. </li><li> Faça imagens de contraste de fase de estruturas acinares de cada poço em 5X ou 10X resolução usando um microscópio de contraste de fase ligada a um shifter de slides e um computador. Tome as imagens movendo o cursor de um quadro para outro e, assim, cobrindo todo o bem do slide compartimentado. </li><li> Contar o número total de estruturas acinareso número de ácinos único plana redonda e do número de estruturas multiacinar ou estruturas esmo crescente falta de organização e aos processos de ramificação que dele emanam. </li><li> Calcule a porcentagem de estruturas acinares plana redondas individuais versus estruturas malignas e enredo como gráfico de colunas. Calcula-se a significância pelo teste t de Student. </li></ol>

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Os autores declaram não haver interesses financeiros concorrentes.

Nós descrevemos aqui a cultura tridimensional de células epiteliais mamárias de uma membrana basal reconstituída e a utilidade deste sistema para diferenciar entre versus maligna fenótipo benigna da mama. Este sistema também pode ser utilizado para cultura de diferentes tipos de células de outros tecidos glandulares e tem sido relatada como tendo sido utilizados com sucesso para a cultura epitelial da próstata, do epitélio brônquico, e as células epiteliais da tiróide, para citar alguns 12-14. A i...

Cultura tridimensional (3D) de células epiteliais de mama na membrana basal reconstituída é um importante sistema modelo para estudar o fenótipo complexo e sinalização associada da mama normal e câncer de mama 2,3. A unidade funcional de mama lobular é a unidade terminal de conduta (TDLU) que consiste de uma pequena conduta que se ramifica em ácinos. Os ácinos são estruturas altamente organizadas, compostas por duas camadas de células, células epiteliais e mioepiteliais, que estão rodeados por uma membrana basal 5. As características mais distintivas de ácinos normais são polarização celular, o apego à membrana basal subjacente e contatos célula-célula especializados. Essa organização complexa é interrompido em carcinomas invasivos. As culturas 2D amplamente utilizados, em que as células são crescidas como monocamadas, não permitem a formação de ácinos. Assim, a cultura em monocamada é falta de fornecimento de um sistema para capturar a intrincada relação entre as células epiteliaisem mama normal e sua desregulamentação em câncer de mama. Culturas epiteliais monotípicas funcionais de células da mama foram desenvolvidas em vários laboratórios de 2,3. Cultura 3D envolve o crescimento de células da mama em Matrigel, que é um extracto solubilizado derivado de Engelbreth-Holm-Swarm células de sarcoma de ratinho e está disponível comercialmente. Outros substratos 3D como o colágeno também são usados. As células da mama podem ser cultivadas em 3D pelo ensaio incorporado 3D, em que as células são cultivadas incorporado em Matrigel 2. Alternativamente, as células podem ser cultivadas por 3D em cima ensaio, também chamado de teste de sobreposição 3D, que é rentável, pois requer menor volume de Matrigel, e facilita o monitoramento do lapso de tempo de formação de colônias por imagem de contraste de fase e é ideal para imagens em situ 2 -3. O 3D no topo ensaio tem sido utilizado para definir a correlação entre o fenótipo acinar e do perfil de expressão do gene 7. Cultura 3D pode ser effectively usado para distinguir células normais e benignos do carcinoma invasivo. Células mamárias normais e benignos formar crescimento preso estruturas polarizadas com um lúmen bem definidas no centro em Matrigel enquanto que as células de carcinoma invasivo crescer prolífica e ao acaso, sem compensação da luz. E6/E7 imortalizada de células epiteliais mamárias humanas e uma linha celular MCF10A, que é uma linha celular imortalizada espontaneamente, isolado a partir de uma paciente de 36 anos de idade com alterações fibrocística, têm sido utilizados com sucesso para recuperar o fenótipo benigna da mama em 3D 3,8 e serviram um sistema-modelo para estudar a função supressora de tumores oncogénicos e de várias moléculas de 8,9. Estas células benignas podem ser modificados para manipular a gene (s) de interesse com a introdução de lentivírus / retrovírus. Se o gene (s) de interesse é um supressor de tumor, knockdown shRNA mediada conduzirá a uma transformação maligna que, quando o gene de interesse é uma sobre-expressão de oncogenes em células benignasdeve mostrar um fenótipo maligno. Em ambos os casos, as estruturas acinares formados em 3D pode captar a transformação maligna. Células individuais benignos banhado em 3D começam a proliferar e formar rodada estruturas esféricas. Por dia 5-8 agregado esférico de células irá diferenciar em uma camada circundante de células polarizadas que está em contacto com o Matrigel e uma massa interna de células polarizadas menos, que carecem de contacto com o Matrigel. Nos próximos 10-15 dias, as células interiores vão começar a morrer por apoptose formando um lúmen claro. As células malignas vai crescer desordenadamente perder sua polaridade. Células altamente malignas geralmente formar estruturas de ramificação. Estas diferenças no fenótipo pode ser capturada por tempo fase lapso de imagem contraste e por in situ imunomarcação com marcadores moleculares relevantes. Os marcadores mais vulgarmente utilizados são alfa 6-integrina, um marcador de polaridade basal, em associação com o marcador da proliferação de fosfo-histona 3 e o apoptotmarcador ic caspase-3. Esta última é importante para determinar se existe a formação de lumen no centro dos ácinos, uma característica de células normais e benignas da mama. Outros polaridade e de contato célula-célula marcadores incluem GM130, laminina, ERM, E-caderina. Alternativamente as linhas de células de cancro da mama podem ser modificados para manipular o gene de interesse. Neste caso, a reversão para um crescimento mais preso fenótipo benigno pode ser usado como um lido para distinguir do fenótipo do cancro. Cultura 3D também pode ser usada com cautela para delinear as vias de sinalização envolvidas na transformação maligna 10-11. Usando o acima mencionado limite lidos, inibidores farmacológicos, os anticorpos de bloqueio ou proteínas recombinantes podem ser usadas se localizar na sinalização molecular conduzindo a uma transformação maligna. Com esforços contínuos de vários laboratórios, é possível extrair as células de 3D para isolar RNA e também preparar lisados ​​por immunoblotting e imunizaçãonoprecipitation. Estas estratégias são descritas em um passo a passo e forma detalhada no protocolo.

<table border="0" cellpadding="0" cellspacing="0" width="585">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Growth Factor Reduced matrigel</td>
			<td style="width:83px;">BD biosciences</td>
			<td style="width:119px;">354230</td>
			<td style="width:167px;">Avoid repeat freeze thaw&#160;</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Lab-Tek glass hamber slides 8-well</td>
			<td style="width:83px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">177402</td>
			<td>Precool on ice&#160;</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Lab-Tek glass chamber slides 2- well&#160;</td>
			<td style="width:83px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">177380</td>
			<td>Precool on ice&#160;</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">goat anti mouse F(ab&#180;)2 fragment</td>
			<td style="width:83px;">Jackson Immunoresearch</td>
			<td style="width:119px;">115-006-006</td>
			<td></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Normal goat serum</td>
			<td style="width:83px;">Jackson Immunoresearch</td>
			<td style="width:119px;">005-000-121</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Fluorescent conjugated Alexa antibodies</td>
			<td style="width:83px;">Molecular probes</td>
			<td style="width:119px;"></td>
			<td>Please look at molecular probes website</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Prolong gold antifade reagent with DAPI</td>
			<td style="width:83px;">Molecular Probes</td>
			<td style="width:119px;">P36935</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">3D Culture Cell Harvesting kit</td>
			<td style="width:83px;">Trevigen</td>
			<td style="width:119px;">3448-020-k</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-Apha-6-integrin</td>
			<td style="width:83px;">Fisher Scientifics&#160;</td>
			<td style="width:119px;">MAB1378</td>
			<td>1:00 dilution for IF</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-GM130</td>
			<td style="width:83px;">BD Biosciences</td>
			<td style="width:119px;">610822</td>
			<td>1:00 dilution for IF</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-phospho-histone 3 (Ser10)FITC Conjugated</td>
			<td style="width:83px;">Fisher Scientifics&#160;</td>
			<td style="width:119px;">16-222</td>
			<td>1:100 dilution for IF</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-Cleaved caspase 3 (Asp 175)</td>
			<td style="width:83px;">Cell Signaling</td>
			<td style="width:119px;">9661s</td>
			<td>1:50 dilution for IF</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-E-cadherin</td>
			<td style="width:83px;">BD Biosciences</td>
			<td style="width:119px;">610181</td>
			<td>1:200 dilution for IF</td>
		</tr>
	</tbody>
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three dimensional cultures: a tool to study normal acinar architecture vs. malignant transformation of breast cells

Carcinomas da mama invasivos são um grupo de tumores epiteliais malignos caracterizada pela invasão de tecidos adjacentes e propensão para metástase. A interação de sinais entre as células cancerosas e seu microambiente exerce uma poderosa influência sobre o crescimento do câncer de mama e comportamento biológico 1. No entanto, a maioria destes sinais a partir da matriz extracelular são perdidas ou a sua relevância é subinvestigada quando as células são cultivadas em cultura de duas dimensões (2D) como uma monocamada. Nos últimos anos, a cultura tridimensional (3D) com uma membrana basal reconstituída tem surgido como um método de escolha para recapitular a arquitectura dos tecidos de células mamárias benignas e malignas. As células cultivadas em 3D reter as pistas importantes da matriz extracelular e fornecem um sistema ex vivo 2,3 fisiologicamente relevante. De notar que há cada vez mais evidências sugerindo que as células se comportam de forma diferente quando cultivada em 3D, em comparação com 4 2D. Cultura 3Dpode ser eficazmente utilizada como um meio para diferenciar o fenótipo maligno do fenótipo benigna da mama e para cimentar a sinalização celular e molecular envolvida 3. Uma das características distintas de células epiteliais benignas é que eles são polarizados de modo a que o citoplasma é apical para o lúmen e o citoplasma basal repousa sobre a membrana basal. Esta polaridade apico-basal é perdida em carcinomas invasivos da mama, que são caracterizadas por desorganização celular e formação da anastomose e ramificação túbulos que esmo infiltrados do estroma circundante. Estas diferenças histopatológicas entre glândula benigna e carcinoma invasivo pode ser reproduzido em 6,7 3D. Usando os Read-outs apropriadas como a quantificação de estruturas individuais acinares redondos, ou a expressão diferencial de marcadores moleculares validados para a proliferação celular, a polaridade e a apoptose, em combinação com outras técnicas de biologia molecular e celular, cultur 3De pode fornecer uma ferramenta importante para compreender melhor as alterações celulares durante a transformação maligna e para delinear a sinalização responsável.

Cultura 3D pode ser efetivamente usado para diferenciar fenótipo maligno das células epiteliais mamárias humanas do peito benigno. Células benignas Solteiro banhado em Matrigel crescer como estruturas esféricas organizados, que podem facilmente ser fotografada como único ácinos plana rodada em um avião usando imagens de contraste de fase. As células malignas único banhado em Matrigel crescer de forma anárquica, mostrar muito elevado índice de refração e são difíceis de imagem em um plano. Como mostrado n...

Watch this Scientific Journal Video about Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells at JoVE.com

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

Três cultura tridimensional de células epiteliais mamárias de uma membrana basal reconstituída é um método útil para recapitular a arquitectura in vivo da mama benigna, e para diferenciar o fenótipo maligno do fenótipo da mama benigna. Importante, este sistema pode ser aplicado para estudar os carcinomas invasivos em outros tecidos.

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 ...

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Medicine

19.8K visualizações.  University of Michigan Comprehensive Cancer Center.  Três cultura tridimensional de células epiteliais mamárias de uma membrana basal reconstituída é um método útil para recapitular a arquitectura in vivo da mama benigna, e para diferenciar o fenótipo maligno do fenótipo da mama benigna. Importante, este sistema pode ser aplicado para estudar os carcinomas invasivos em outros tecidos. 

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

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

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

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

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

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

Medicina

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 ...

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 ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

Injeção Orthotopic de células cancerosas da mama na mamária Fat Pad de Ratos para o Estudo de crescimento tumoral.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling M&#252;ller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.
There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5&#160;has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label M&#252;ller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

Usando vírus adeno-associado como uma ferramenta para o estudo da retina Barreiras na Doença

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Tridimensional (3D) Tumor Esferóide Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

Utilização do ensaio de formação de colónia agar mole para identificar inibidores de tumorigenicidade em células de cancro da mama

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

Tridimensional Cultura alginato-talão de células humanas adenoma pituitário

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including Parkinson's and Huntington's can disrupt it. People with Parkinson's disease, for example, tend to have increased saccadic latencies. Consequently, the quantitative measurement of saccadic eye movements has received considerable attention as a potential biomarker for neurodegenerative conditions. A lot more can be learned about the brain in both health and disease by observing what happens to eye movements when the function of specific brain areas is perturbed. Deep brain stimulation is a surgical intervention used for the management of a range of neurological conditions including Parkinson's disease, in which stimulating electrodes are placed in specific brain areas including several sites in the basal ganglia. Eye movement measurements can then be made with the stimulator systems both off and on and the results compared. With suitable experimental design, this approach can be used to study the pathophysiology of the disease being treated, the mechanism by which DBS exerts it beneficial effects, and even aspects of normal neurophysiology.

This paper describes the use of quantitative measurement of eye movements in conjunction with stimulation of focal areas of the deep brain in order to study physiology, pathophysiology, and the mechanisms of deep brain stimulation.

Usando Saccadometry com Estimulação Cerebral Profunda para estudar normal e patológico função cerebral

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including ...

A Clinical Trial Assessing the Safety, Efficacy, and Delivery of Olive-Oil-Based Three-Chamber Bags for Parenteral Nutrition

Limited evidence exists to precisely estimate efficacy and safety differences between parenteral nutrition (PN) prepared using olive-oil-based three-chamber bags (3CBs) and soybean-oil-based compounded bags (CoBs) in hospitalized adult patients. We designed a multicenter, randomized, prospective, open-label, noninferiority protocol to compare the efficacy, safety, and distribution of olive-oil-based 3CBs and soybean-oil-based CoB formulations in adult Chinese patients requiring PN during surgical intervention. Subjects were randomized to receive either one of the study treatments using an interactive voice or web-based recognition system in accordance with the randomization code. Randomization was further stratified based on the study site and surgical category. Both treatment groups received similar amounts of calories and protein. In addition, the two study treatments contained a similar composition of the amino-acid component. The only difference between the two PN formulations was the lipid constitution. The duration of administration of study treatments was a minimum of 5 days up to a maximum of 14 days after the surgical procedure. The primary efficacy endpoint was serum prealbumin levels on day 5 of the study. Noninferiority was proved if the anti-log of the lower bound of the 95% confidence interval (CI) of the treatment difference was at least 0.80. Other efficacy measures included treatment preparation time; duration to achieve tolerability of oral nutrition; associated infectious complications; length of hospitalization; and laboratory assessment of markers of nutrition, inflammation, metabolism, and oxidative stress. A total of 458 patients were enrolled in the study. The results showed that olive-oil-based 3CBs were non-inferior to soybean-based CoBs, besides being well tolerated. The infection rate was found to be significantly lower in the olive-oil-based 3CB group. Thus, this study may be used as a reference for future research on lipid emulsion and 3CBs.

Here, we present a protocol for a study comparing the efficacy, safety, and delivery of olive-oil-based 3CB and soybean-oil-based CoB formulations in adults requiring parenteral nutrition. The results revealed that olive-oil-based 3CBs is non-inferior and well tolerated compared to soybean formulations.