Name: a brain tumor/organotypic slice co-culture system for studying tumor microenvironment and targeted drug therapies
Uploaded: 2015-11-07T14:00:00
Description: Watch this Scientific Journal Video about A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies at JoVE.com

This work is supported by grants from the NIH (P01CA142536 to RAS, T32CA009361 to DPY) and the Pediatric Low Grade Astrocytoma foundation.

Declaração de Ética: O procedimento a seguir envolvendo seres animais foram realizados de acordo com os Institutos Nacionais de Saúde e orientações foram aprovadas pelo Comitê de Dana-Farber Cancer Institutional Animal Care e Use. Todos os seres humanos trabalho foram revisadas pelos Comitês Conselho de Revisão Institucional do Hospital Brigham and Women e Dana-Farber Cancer Institute, e pela Universidade de Stanford para o uso apropriado, que informaram o consentimento foi obtido de todos os indivíduos, quand...

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Este protocolo descreve como células tumorais do cérebro pode ser marcado por fluorescência e plaqueadas sobre uma secção sagital do cérebro de um rato e, em seguida, P6 monitorizada durante uma semana em cultura. Este ensaio fatia de co-cultura do tumor cerebral / organotípica pode ser utilizado para determinar o efeito do microambiente regional sobre o número de células tumorais e pode também ser usado como um sistema para medir a eficácia de novos tratamentos com drogas sobre o crescimento de tumores human...

Uma pesquisa recente do cancro tem feito avanços significativos na identificação de mutações genéticas, alterações moleculares e os possíveis tratamentos para uma variedade de tumores cerebrais. Apesar deste progresso, tumores cerebrais continuam a ser uma das principais causas de mortalidade relacionada ao câncer em adultos e crianças. Fatores limitantes na investigação tumor cerebral incluem a disponibilidade restrita de amostras de pacientes primários e linhas celulares ea dificuldade em replicar o microambiente único e heterogêneo cérebro em sistemas experimentais acessíveis. Para muitos tumores cerebrais as condições necessárias para manter as células tumorais ao longo do tempo ainda não são conhecidos. Mesmo para os tumores cerebrais que podem ser cultivadas em suspensão de células como neuroesferas, condições de cultura podem afectar as células tumorais 1,2. Com efeito, a adição de factor de crescimento de fibroblastos básico ou factor de crescimento epidérmico para encorajar a proliferação e diferenciação inibem pode alterar a expressão genética 1. Outros métodos para o crescimento de células de tumor, taiscomo a propagação do tumor em ratinhos através de xenoenxerto ortotópico ou subcutânea de células tumorais são ensaios valiosos, mas estão limitados por factores tais como o tempo de desenvolvimento de tumor (especialmente para tumores de baixo grau), o custo, e o número de células de tumor que pode ser injectado e estudou . Assim, os métodos atuais para células tumorais do cérebro humano em crescimento são inadequadas para a manutenção de certos tipos de tumores, e muitas vezes oferecem ambientes artificiais que não fazer estreitamente in vivo ambientes tumorais imitar. Tipos distintos de tumores cerebrais em crianças crescem em locais altamente especializados no cérebro [3, 4] e esta é susceptível de reflectir os requisitos do microambiente distintos para o crescimento do tumor [5]. Este protocolo descreve um novo sistema em que as células que são difíceis de propagar em condições de cultura normais, pode ser cultivada em um micro-ambiente que imita cérebro organotípicas em condições de crescimento tumoral in vivo. Neste ensaio quantitativo, marcado por fluorescênciacélulas tumorais do cérebro são banhados no rato do cérebro fatias organotípicas juvenis e monitorados ao longo do tempo. Este ensaio pode ser utilizado para investigar o efeito do microambiente no crescimento do tumor, e para testar novas terapias de droga em um microambiente cérebro clinicamente relevante.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>HEPES</td>
			<td>Invitrogen&#160;</td>
			<td>17504044</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Invitrogen</td>
			<td>17502048</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>Pennicillin Streptomycin</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Life Technologies</td>
			<td>14185-052</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>B-27</td>
			<td>Life Technologies</td>
			<td>17504-044</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>N2</td>
			<td>Life Technologies</td>
			<td>17502-048</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Life Technologies</td>
			<td>35050061</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td rowspan="2">Neurobasal-A- Medium minus phenol red</td>
			<td rowspan="2">Invitrogen&#160;</td>
			<td rowspan="2">12349015</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>Low Melting Point Agarose</td>
			<td>Promega</td>
			<td>V2111</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>Slice Culture Inserts&#160;</td>
			<td>Milipore</td>
			<td>PICM0RG50</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>laminin</td>
			<td>Invitrogen</td>
			<td>23017015</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>Cm-DiI&#160;</td>
			<td>Invitrogen&#160;</td>
			<td>V22888</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>EDU (Labeling and Detection)&#160;</td>
			<td>Life Technologies</td>
			<td>c10337</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>Microspheres&#160;</td>
			<td>Life Technologies</td>
			<td>F-21010</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>Vibratome&#160;</td>
			<td>Leica</td>
			<td>N/A</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>Confocal Microscope&#160;</td>
			<td>Nikon Eclipse Ni C2si</td>
			<td>N/A</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>Image J software&#160;</td>
			<td>N/A</td>
			<td>N/A</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>5mm Cover Glasses&#160;</td>
			<td>Fisher Scientific</td>
			<td>64-0700 (CS-5R)</td>
			<td colspan="3"></td>
		</tr>
	</tbody>
</table>

a brain tumor/organotypic slice co-culture system for studying tumor microenvironment and targeted drug therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Esta seção exemplifica o tipo de resultados que se espera de utilizar o tumor cerebral / organotípico fatia co-cultura para investigar preferências microambiente regional, bem como para testar novas terapias. Mostra-se que o ensaio é concebido para reproduzir o microambiente para tumores cerebrais, como a organização do tecido e o estado proliferativo de fatia é mantida (Figura 3). Nós também demonstram que um aumento do número de células tumorais na fatia ao longo do tempo pode ser parcialm...

Watch this Scientific Journal Video about A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies at JoVE.com

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

Um tumor cerebral / Slice organotípicas Co-cultura Sistema de Estudos de Tumor Microenvironment e alvejado droga Terapias

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