Este trabalho foi financiado parcialmente por (1) o Departamento de Defesa Breast Cancer Research Programa (US Army Medical Research Aquisição Atividade) prêmio # W81XVVH-10-1-0781 a LAL e VE, e (2) a concessão Susan G. Komen Foundation IR122224446 para Lal e VE. Apoio Patologia e bilheteria tecido foi gentilmente cedido pela Inova Fairfax Departamento de Patologia, Dr. Hassan Nayer, Dr. Gita A. Menezes e Dr. Charles Bechert. Consentimento dos pacientes e obtenção da amostra foi habilmente guiados por Inova Fairfax Hospital coordenadores da pesquisa clínica Holly Gallimore, Heather Huryk, e Emil Kamar.

Tecido mamário humano foi coletado de pacientes inscritos em um estudo de pesquisa, com consentimento informado por escrito, na sequência do Departamento de Defesa, George Mason University, e protocolos aprovados do Sistema de Saúde Inova Institutional Review Board. 1. Prepare Nutrient meio rico com fatores de crescimento e antibióticos <ol><li> Preparar soluções de reserva de insulina, factor de crescimento epidérmico (EGF), sulfato de estreptomicina e sulfato de gentamicina. <ol><li> Reconstituir insulina em água filtrada estéril até uma concentração final de 10 mg / ml. Aspirar 10 ml de reagente do tipo 1 grau a água em uma seringa de 10 ml descartável estéril. Coloque um filtro de 0,22? M de polietersulfona à seringa. Dispense a água filtrada estéril em um tubo cônico de 15 ml estéril. </li><li> Adicionar 10 ml de água filtrada estéril para um frasco de 100 mg de insulina. Misture o conteúdo brevemente em um misturador de vórtice. Mantenha o frasco de insulina no gelo. Dispensar 450 ml da solu estoque de insulinação marcadas, em tubos de microcentrífuga estéreis e armazenar a -20 ° C. Solução estoque de insulina é estável até a data de validade no frasco. </li><li> Preparar a solução stock de factor de crescimento epidérmico (EGF): Adicionar 500 uL de água estéril para 500 ug de EGF (estoque 1 ug / ul). Misture o conteúdo brevemente em um misturador de vórtice. Manter o frasco EGF no gelo. <ol><li> Prepara-se uma solução estoque de trabalho de EGF: Adicionar 100 ul da solução de estoque de EGF para 900 ul meio nutriente (estoque de trabalho será de 0,1 ug / ul). Misture o conteúdo brevemente em um misturador de vórtice. Dispensar 50 ul da solução de EGF trabalhando em estoque rotulados, microtubos estéreis e armazenar a -80 ° C. NOTA: solução de estoque de EGF (1 ug / ul) é estável durante um ano à temperatura de -80 ° C; Solução de trabalho de estoque (0,1 ug / uL) é estável durante 2 meses a -80 ° C. </li></ol></li><li> Pesar 40 mg de sulfato de estreptomicina em uma balança analítica. Colocar o sulfato de estreptomicina intoa estéreis tubo de 15 ml. Adicione 4 ml de meio nutriente. Misture o conteúdo brevemente em um misturador de vórtice. A solução deve ser ligeiramente rosa. Armazenar a 4 ° C, protegida da luz. Solução de sulfato de estreptomicina é estável durante 7 dias. </li><li> Pesar 20 mg de sulfato de gentamicina em uma balança analítica. Coloque o sulfato de gentamicina em um estéril tubo de 15 ml. Adicionar 2 ml de meio nutriente. Misture o conteúdo brevemente em um misturador de vórtice. A solução deve ser amarelas. Armazenar a 4 ° C, protegida da luz. Solução de sulfato de gentamicina é estável durante 7 dias. </li></ol></li><li> Preparar dois lotes de 200 ml de meio nutriente suplementado com EGF humano recombinante (10 ng / ml de concentração final.), Insulina (10 ug / ml de concentração final.), Sulfato de estreptomicina (100 ug / ml de concentração final.) E sulfato de gentamicina (20 ug / ml de concentração final.). <ol><li> Adicionar meio nutriente para 200 ml um balão de filtração a vácuo equipado com um filtro de 0,2 um balão de polietersulfona e um frasco de 250 ml de receber. Adicionar 20 &amp;# 181; l estoque trabalhando EGF, 200 mL de insulina estoque, 2 ml de sulfato de estoque estreptomicina e 400 sulfato de gentamicina estoque ul a 200 ml de meio nutriente. Fixar o frasco de filtração a vácuo. Filtre o meio. Descarte o filtro e rotular o frasco como &quot;meio nutriente rico&quot;. Armazenar a 4 ° C por até 14 dias. </li></ol></li></ol> 2. Aquisição de Tecidos e extrapolação <ol><li> Na sala de cirurgia, manter a técnica estéril após a aquisição do tecido mamário. Coloque o tecido mamário em uma bandeja estéril e cobrir a bandeja com filme plástico estéril. <ol><li> Transportar o tecido na bandeja coberta de Radiologia / Patologia conforme exigido pela sua instituição. Não abra a bandeja. Tempos de transporte variam dentro das instituições. Tecido pode permanecer viável neste bandeja coberta à temperatura ambiente por até 45 minutos após a excisão. No entanto, o prompt de processamento do tecido em meio rico em nutrientes proporciona as condições ideais para a cultura subseqüente organóide. </li></ol></li><li> Dissecção dos tecidos Gross para identificar áreas de CDIS dentro do tecido mamário: Use luvas estéreis, lâminas, bisturis, tecido tintas de marcação, e vinagre durante a dissecção do tecido para manter a esterilidade dos tecidos. <ol><li> Limpar a área de trabalho com etanol a 70% ou 1% de lixívia. Abra as luvas estéreis e colocar a capa luva na superfície de trabalho. Coloque o interior da luva estéril invólucro de face para cima. Coloque as luvas estéreis. <ol><li> Limpar a superfície do recipiente de amostra com etanol a 70%. Retire o filme plástico do recipiente de amostras de tecido e colocar a amostra na embalagem luva. </li></ol></li><li> Mergulhe dois cotonetes com ponta no corante marcação tecido. Aplicar o corante à superfície do tecido por laminagem os esfregaços em toda a superfície do tecido. NOTA: Cada departamento de patologia tem um protocolo de orientação a cor da tintura / tecido padronizado. O tecido é marcada com corante para orientar o tecido em relação à sua posição no paciente. O corante é apagado oupintado sobre o tecido. Não despeje o corante sobre o tecido. Verter o corante pode causar o corante para executar / gotejamento em fendas de tecido confundindo, assim, a orientação das margens cirúrgicas. Orientação tecido fornece ao cirurgião e patologista com marcos anatômicos para a) descrever a amostra de tecido, e b) determinar a localização das margens cirúrgicas em relação ao tumor. </li><li> Pour vinagre branco destilado (5% de ácido acético) para compressas de gaze estéril. Seque o tecido tingido com o vinagre gaze embebida. Descarte a gaze. O vinagre é usado para corrigir a marcação corantes de tecidos. </li><li> Corte o tecido mamário em fatias verticais de aproximadamente 5 mm de espessura. Não corte todo o caminho através do tecido (Figura 2). Observar / palpar o tecido para as áreas de calcificação. Identificar CDIS por sua característica firme, pálida aparência avermelhada cercada por, as fronteiras de borracha que se sentir corajoso (devido a espículas de cálcio). NOTA: Em alguns casos, comedo (espinha-like) áreas podem servistos como pontos brancos, representando material necrótico de CDIS grande diâmetro. </li><li> Cortar áreas de CDIS / calcificada tecido mamário, incluindo uma pequena quantidade de tecido circundante da mama. Colocar o tecido da mama para um tubo de 50 ml estéril contendo 20-30 ml de meio nutriente rico preparado no passo 1. <ol><li> Misturar o tecido e meio invertendo suavemente o tubo várias vezes. Descarte a médio e adicione meio fresco. Colocar o tubo contendo o tecido e forma dentro de um recipiente isolado e transportar o tecido para o laboratório de cultura de tecidos. </li></ol></li></ol></li></ol> 3. Cultura de Tecidos <ol><li> Trabalhando numa câmara de segurança biológica, despeje o tecido e uma pequena quantidade do meio nutriente para uma placa de Petri esterilizada. Usando um bisturi estéril cortar e descartar o tecido fibroso. Cortar o tecido mamário em pedaços (Organóides) cerca de 3 mm 2 (Figura 1C-D). Tente cortar o tecido de modo que cada órgãooid contém pelo menos um segmento de conduta de discernível com estroma circundante. </li><li> Abrir a tampa do frasco de cultura de tecido. Com uma pinça estéril ou pinça, coloque os Organóides para o balão. Fechar a tampa. Descarte a placa de Petri. </li><li> Adicionar 11 ml de meio nutriente rico isento de soro (preparado no passo 1) para o frasco de cultura de tecido. Feche o frasco e agite o frasco de modo que os Organóides e médio estão distribuídas uniformemente por toda a superfície frasco (Figura 1E-F). </li><li> Incubar o balão a 37 ° C numa atmosfera humidificada com 5,0% de CO2 durante 2 dias. Não mova o frasco durante este tempo. </li><li> No dia 2 de pós incubação, retirar o balão da incubadora para verificar se há potencial de contaminação bacteriana / fúngica. Evite repente, movimentos bruscos, ou roda do balão. Colocar o balão num palco de microscópio invertido. <ol><li> Observar a forma de bactérias, leveduras e / ou fungos. Se nenhuma contaminação é observado, devolver o frasco para a incubadora para um suplementodia itional. Se a contaminação for observado, descartar o balão eo seu conteúdo em um recipiente adequado. </li></ol></li><li> No dia 3 pós incubação, substituir o meio condicionado com meio fresco. <ol><li> Colocar 11 ml de meio em um tubo estéril a 37 ° C durante 20-30 min. Retirar o frasco de cultura de tecido a partir do incubador. Sem perturbar os Organóides, remover e descartar o meio no frasco, utilizando uma pipeta serológica estéril. </li><li> Usando uma pipeta serológica estéril novo, adicionar 11 ml de meio fresco pré-aquecido. Muito rodar cuidadosamente o frasco para distribuir o meio através da superfície do balão. </li><li> Incubar o balão a 37 ° C numa atmosfera humidificada com 5,0% CO2. </li></ol></li></ol> 4. Manutenção da estabeleceram colônias Celulares organóide / epiteliais <ol><li> Prepare meio fresco a cada 2 semanas e substituir os meios de comunicação no frasco de cultura de tecidos 3 vezes por semana. <ol><li> Colocar 11 ml do meio num recipiente estéril a 37° C por 20-30 min. Retirar o balão da incubadora. Com uma pipeta sorológica estéril, retire e descarte o meio condicionado a partir do balão, tomando cuidado para não perturbar os Organóides. </li><li> Usando uma pipeta serológica estéril novo, adicionar 11 ml de meio fresco pré-aquecido. Muito rodar cuidadosamente o frasco para distribuir o meio através da superfície do balão. Incubar o balão a 37 ° C numa atmosfera humidificada com 5,0% CO2. </li></ol></li><li> Após 10-14 dias em cultura, remover quaisquer fragmentos de tecido do balão de cultura que não são aderentes. <ol><li> As células e / ou Organóides do balão para a propagação em novos frascos de cultura Periodicamente colheita, para o transplante de xenoenxerto, ou para fenotípica e / ou análise molecular. </li><li> Retirar o frasco de cultura de tecido a partir do incubador. Pulverizar o balão com etanol a 70%. Limpe o excesso de etanol no frasco com uma toalha de papel que foi pulverizada com etanol 70%. </li><li> Propagação organóide: &lt;ol&gt; <li> Abra a tampa do frasco e coloque a tampa virada para cima na cabine de segurança biológica. Sob visualização microscópica, localizar o organóide (s) a ser colhida. Use uma pinça ou pinça estéril para pick-up um organóide. </li><li> Para propagar o organóide em uma nova garrafa de cultura, coloque o organóide no novo frasco. Adicionar 11 ml, meio quente fresco. Incubar a 37 ° C, numa atmosfera humidificada com 5% de CO2, como descrito nas etapas 3.3 - 3.6.3. Substituir o meio de cultura de células na cultura de tecidos balão de três vezes por semana. </li></ol></li><li> Células de colheita: <ol><li> Sob vixualization microscópico direto, raspe e células de aspirado e mammospheres utilizando uma pipeta de 1000 mL com pontas de pipetas esterilizadas descartáveis. Aspirar as células e o meio circundante. Dispensar a células / meio para um tubo de microcentrífuga estéril. </li></ol></li><li> Rodar as células brevemente em um mini-centrifugar a 12100 xg durante 5 seg. Remova e descarte o meio. &lt;ol&gt; <li> Para análise do DNA, congelar imediatamente o sedimento de células em gelo seco, num pequeno volume de meio (10 ul), com a armazenagem a longo prazo a -80 ° C. </li><li> Para análise proteômica, lisar as células em 10 ml de 8 M de ureia para a espectrometria de massa ou de tampão tris-glicina 2x SDS por Western Blotting / reversa microarrays de proteínas de fase. Alternativamente, girar células em uma citocentrífuga fazer esfregaços de células para análise imuno-histoquímica. </li></ol></li><li> Após a colheita de células a partir de um frasco, retire e descarte o meio restante. Adicionar 11 ml de nutrientes fresco meio quente, rica. Incubar o balão a 37 ° C, numa atmosfera humidificada com 5% de CO2. </li></ol></li></ol>

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O sistema de cultura aqui descrito constitui um novo modelo para a geração de células mamárias neoplásicas vivos pré-invasivas para estudos básicos e Pesquisa Translacional. No passado, a pré-maligno de progressão do cancro da mama tem sido tipicamente estudada usando três métodos diferentes. O primeiro método é histopatológico e análise genética de amostras microdissecadas humanos congelados ou fixados 12-14. O segundo método utiliza modelos de mouse que contêm nódulos alveolares (HAN) les...

A proliferação de células epiteliais dentro dos limites de ductos mamários e alvéolos (carcinoma ductal in situ) é reconhecido como um precursor obrigatório para ductal invasivo e carcinoma lobular da mama. No entanto, os mecanismos moleculares e dinâmica de populações de células que permitem a progressão para o câncer invasivo são mal compreendidos. Elucidar os mecanismos de sobrevivência utilizadas pelas células do carcinoma da mama pré-invasivas, ou qualquer tumor pré-invasivo, pode revelar estratégias terapêuticas para matar, ou mesmo impedindo, neoplasias pré-invasivas 1. No entanto, métodos de baixo custo simples para estudar funcionalmente lesões pré-invasivas humanos têm faltado. Embora a cultura in vitro em monocamada de linhas celulares transformadas é estabelecido um método de laboratório, o genótipo e o fenótipo destas linhas de células imortalizadas não recapitular o estatuto molecular de células de tumores humanos primários 2. Além disso, mesmo a linha não-tumorigenic células MCF-10A, que recapitulates 3-D arquitetura da glândula mamária, não representam adequadamente o fenótipo funcional e as características moleculares do pré-invasivo lesão peito de um paciente individual 3,4. Para determinar se as células-tronco neoplásicas semelhantes capazes de invasão existiu dentro do carcinoma ductal in situ (CDIS) população de células, desenvolvemos uma metodologia de recolha e cultura estéril tecido mamário humano no momento da cirurgia (Figura 1) 5. O nosso ex vivo da mama sistema de cultura organï¿½de não depende de interrupção enzimática tecido, extracto de membrana basal da matriz, ou o esgotamento de fibroblastos, para o isolamento e propagação de células formadoras de mammosphere de fresco humano de mama ductal tecido de carcinoma de 6-8. O nosso novo sistema baseia-se no princípio da célula de fluxo / migração 5. Os ductos mamários discerníveis, e estroma circundante estão submersos num volume mínimo de meio nutriente livre de soro (e apenasnough a cobrir os fragmentos de condutas) para maximizar a troca de gás, com a superfície de corte da conduta exposta ao meio de cultura, mas em qualquer orientação específica no balão (Figura 1E-F). Este sistema de cultura permite que as células migrem para fora da conduta e para dentro / para o balão de estroma e cultura autólogo. O meio nutriente, suplementado apenas com o Factor de Crescimento Epidérmico (EGF), insulina, e antibióticos, suporta o crescimento de populações de células mistas que emanam do organï¿½de. O balão de cultura de tecidos tem um, re-selável tampa removível que permite que os Organóides e / ou células a serem colhidas, sem perturbar a totalidade do balão ou vizinhos Organóides, enquanto se mantém um ambiente estéril humidificada.

<table border="0" cellpadding="0" cellspacing="0" width="1024">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Name of Reagent/ Equipment</td>
			<td style="width:187px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:365px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Ethanol</td>
			<td style="width:187px;">Fisher</td>
			<td style="width:119px;">A405-P</td>
			<td>prepare a 70% solution in Type 1 reagent grade water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">18 M&#937;-cm water, sterile filtered</td>
			<td style="width:187px;"></td>
			<td style="width:119px;"></td>
			<td>sterile filtered, Type 1 reagent grade water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10cc plastic disposable syringe, sterile</td>
			<td style="width:187px;">BD</td>
			<td style="width:119px;">305482</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.2&#181;m polyethersulfone (PES) syringe filter, sterile</td>
			<td style="width:187px;">Thermo Scientific</td>
			<td style="width:119px;">194-2520</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">15ml polypropylene conical tubes, sterile</td>
			<td style="width:187px;">Fisher</td>
			<td>14-959-49B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">50ml polypropylene conical tubes, sterile</td>
			<td style="width:187px;">Fisher</td>
			<td>05-539-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">1.5ml low retention microcentrifuge tubes,sterile</td>
			<td style="width:187px;">Fisher</td>
			<td style="width:119px;">02-681-331</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">nutrient medium, DMEM-F12/HEPES</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:119px;">11330-032</td>
			<td>with L-glutamine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insulin, human recombinant</td>
			<td style="width:187px;">Roche</td>
			<td style="width:119px;">11376497001</td>
			<td>10mg/ml stock</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Epidermal Growth Factor (EGF), human recombinant</td>
			<td style="width:187px;">Millipore</td>
			<td style="width:119px;">GF144</td>
			<td>100&#181;g/ml stock</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptomycin sulfate</td>
			<td style="width:187px;">Sigma-Aldrich</td>
			<td style="width:119px;">S1567</td>
			<td>10mg/ml stock</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gentamicin sulfate</td>
			<td style="width:187px;">Sigma-Aldrich</td>
			<td style="width:119px;">G19114</td>
			<td>10mg/ml stock</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Filtration flask and filter top, sterile</td>
			<td style="width:187px;">Millipore</td>
			<td>SCGPU02RE</td>
			<td>0.22&#181;m PES membrane</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">25ml sterile, disposable pipettes</td>
			<td style="width:187px;">Fisher</td>
			<td>4489</td>
			<td>paper-plastic wrapped</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10ml sterile, disposable pipettes</td>
			<td style="width:187px;">Fisher</td>
			<td style="width:119px;">4488</td>
			<td>paper-plastic wrapped</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:353px;">Tissue marking dyes (black, blue, red, green, yellow and orange)</td>
			<td style="width:187px;">CDI</td>
			<td style="width:119px;">MD2000</td>
			<td style="width:365px;">after opening use only with single-use, sterile cotton tipped applicators, or use once and discard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Cotton tipped applicators, sterile</td>
			<td style="width:187px;">Fisher</td>
			<td>23-400-115</td>
			<td>single use only</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Gauze pads, 10x10cm, sterile</td>
			<td style="width:187px;">Fisher</td>
			<td style="width:119px;">2187</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plastic transfer pipettes, sterile, disposable</td>
			<td style="width:187px;">Samco</td>
			<td>202-20S</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;">Vinegar, white distilled</td>
			<td style="width:187px;">household use</td>
			<td></td>
			<td style="width:365px;">5% acetic acid; after opening use only with sterile pipettes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">#10 scalpels, sterile, disposable&#160;</td>
			<td style="width:187px;">Thermo Scientific</td>
			<td>31-200-32&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">petri dish, sterile</td>
			<td style="width:187px;">Fisher</td>
			<td>FB0875713A</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">TPP 115cm2 flask, with removable lid</td>
			<td style="width:187px;">MidSci</td>
			<td style="width:119px;">90652</td>
			<td>screw cap with filter</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:353px;">CO2 incubator</td>
			<td style="width:187px;">Fisher</td>
			<td>13-998-074</td>
			<td>5% CO2, 37 oC, humidified chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">inverted light microscope</td>
			<td style="width:187px;">Olypmus</td>
			<td>IX51</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">8M urea</td>
			<td style="width:187px;">Fisher</td>
			<td style="width:119px;">BP169-500</td>
			<td>optional, for mass spectrophotometric analysis of cultured cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">2X SDS tris-glycine buffer</td>
			<td style="width:187px;">Life Technologies</td>
			<td>LC2676</td>
			<td>optional, for proteomic analysis of cultured cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Cytocentrifuge</td>
			<td style="width:187px;">Thermo Scientific</td>
			<td>A78300003</td>
			<td>optional, for preparing cell smears</td>
		</tr>
	</tbody>
</table>

non-enzymatic, serum-free tissue culture of pre-invasive breast lesions for spontaneous generation of mammospheres

Mama carcinoma ductal in situ (CDIS), por definição, é a proliferação de células epiteliais neoplásicas dentro dos limites do ducto mamário, sem ultrapassar a membrana basal colágeno. Enquanto DCIS é um precursor não-obrigatório para os cânceres de mama invasivos, os mecanismos moleculares e as populações de células que permitem a progressão para o câncer invasivo não são totalmente conhecidos. Para determinar se as células progenitoras, capazes de invasão existia dentro da população de células DCIS, desenvolvemos uma metodologia de recolha e cultura estéril tecido mamário humano no momento da cirurgia, sem interrupção enzimática do tecido. Tecido mamário estéril contendo segmentos ductal é colhida a partir de tecido mamário extirpado cirurgicamente após exame anatomopatológico de rotina. Tissue contendo DCIS é colocado em meio rico em nutrientes, contendo antibióticos, soro livre, e transportadas para o laboratório de cultura de tecidos. O tecido mamário é mais dissected para isolar as áreas calcificadas. Vários pedaços de tecido da mama (Organóides) são colocados num volume mínimo de meio isento de soro num frasco com uma tampa removível e cultivadas numa incubadora de CO 2 humidificado. Populações de células epiteliais e de fibroblastos emergir da organï¿½de após 10 - 14 dias. Mammospheres formam espontaneamente e em torno da monocamada de células epiteliais. Populações de células específicas podem ser colhidas directamente a partir do frasco, sem perturbar as células vizinhas. Nosso sistema de cultura de tecidos não-enzimática revela de forma confiável, as células progenitoras invasivos citogeneticamente anormais de CDIS humanos frescos.

Fluxo de trabalho para a aquisição e cultura carcinoma ductal de mama estéril em tecido situ Esterilidade tecido mamário é mantida desde a sala de cirurgia para o laboratório de cultura de células via pequenas alterações no fluxo de trabalho típico de patologia do hospital (Figura 1). O tecido é transportado num recipiente esterilizado com um revestimento de película de plástico, que permite a avaliação radiológica, mantendo a esterilidade do t...

Watch this Scientific Journal Video about Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres at JoVE.com

Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres

Cultura primária de células de tecidos intactos utilizando Organóides proporciona um sistema modelo que imita o microambiente de multi-celular, in vivo. Foi desenvolvido um modelo de cultura livre de soro primário tecido epitelial da mama que perpetua linhagens de cultura de células mistas e exposições diferenciadas morfologia, sem rompimento do tecido enzimática. Organóides mama permanecer viáveis ​​por&gt; 6 meses.

Não enzimática, Soro livre de Cultura de Tecidos de lesões mamárias pré-invasivas para a geração espontânea de Mammospheres

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without ...

non-enzymatic-serum-free-tissue-culture-pre-invasive-breast-lesions

Research

JoVE Journal

Biology

12.5K visualizações.  George Mason University.  Cultura primária de células de tecidos intactos utilizando Organóides proporciona um sistema modelo que imita o microambiente de multi-celular, in vivo. Foi desenvolvido um modelo de cultura livre de soro primário tecido epitelial da mama que perpetua linhagens de cultura de células mistas e exposições diferenciadas morfologia, sem rompimento do tecido enzimática. Organóides mama permanecer viáveis ​​por> 6 meses. 

Vídeo: Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres 1. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro and in vivo 2. The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.

HC11 lactogenic differentiation can be characterized by the formation of domed structures referred to as mammospheres. The structures can be enumerated by phase contrast microscopy to aid in quantifying lactogenic differentiation.

Rato células epiteliais mamárias forma Mammospheres durante a diferenciação lactogênica

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of ...

Biologia

Generation of Single-Cell Suspensions from Mouse Neural Tissue

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a repertoire of cell surface molecules, or molecular determinants, through which it can be identified and characterized. Currently, robust cell identification and separation technologies require single-cell preparations to be generated while simultaneously limiting cell death and destruction of characteristic surface protein. The gentleMACS Dissociator, when used in combination with trypsin or papain-based dissociation kits, can effectively and gently dissociate brain tissue while preserving antigen epitopes and limiting cell loss. Standardized preparation of single-cell suspensions is achieved using C Tubes and optimized, preset gentleMACS Programs. Once generated, single-cell suspensions can be treated with monoclonal conjugates like Anti-Prominin-1 MicroBeads, which identify neural progenitors, or purified further using Myelin Removal Beads.

Dissociating cells from specific tissue types requires specific parameters for tissue aggitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS Dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on nerual tissue is explained.

Geração de Single-Cell Tissue Suspensões de rato Neural

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a ...

Preparation of Pooled Human Platelet Lysate (pHPL) as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for mesenchymal stromal cells (MSCs), fibroblasts and endothelial colony-forming cells with platelets activated by thrombin3-5 or lysed by freeze/thaw cycles6-14 before the platelet releasate is added to the cell culture medium. The trophic effect of platelet derived growth factors has already been tested in several trials for tissue engineering and regenerative therapy.1,15-17 Varying efficiency is considered to be at least in part due to individually divergent concentrations of growth factors18,19 and a current lack of standardized protocols for platelet preparation.15,16 This protocol presents a practicable procedure to generate a pool of human platelet lysate (pHPL) derived from routinely produced platelet rich plasma (PRP) of forty to fifty single blood donations. By several freeze/thaw cycles the platelet membranes are damaged and growth factors are efficiently released into the plasma. Finally, the platelet fragments are removed by centrifugation to avoid extensive aggregate formation and deplete potential antigens. The implementation of pHPL into standard culture protocols represents a promising tool for further development of cell therapeutics propagated in an animal protein-free system.

Preparation of Pooled Human Platelet Lysate pHPL as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Human platelet lysate is a rich source of growth factors and a potent supplement in cell culture. This protocol presents the process of preparing a large pool of human platelet lysate by starting from platelet rich plasma, performing several freeze-thaw cycles and depleting the platelet fragments.

Preparação de pool de plaquetas Lysate Humanos (pHPL) como um suplemento eficiente para animais de soro livre de culturas de células estaminais humanas

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for ...

Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address the requirements (e.g., mechanics and vascularity) of more intricate organs and tissues. Electrospinning is a popular technique to create fibrous scaffolds that mimic the architecture and size scale of the native extracellular matrix. These fibrous scaffolds are also useful as cell culture substrates since the fibers can be used to direct cellular behavior, including stem cell differentiation (see extensive reviews by Mauck et al. and Sill et al. for more information). In this article, we describe the general process of electrospinning polymers and as an example, electrospin a reactive hyaluronic acid capable of crosslinking with light exposure (see Ifkovits et al. for a review on photocrosslinkable materials). We also introduce further processing capabilities such as photopatterning and multi-polymer scaffold formation. Photopatterning can be used to create scaffolds with channels and multi-scale porosity to increase cellular infiltration and tissue distribution. Multi-polymer scaffolds are useful to better tune the properties (mechanics and degradation) of a scaffold, including tailored porosity for cellular infiltration. Furthermore, these techniques can be extended to include a wide array of polymers and reactive macromers to create complex scaffolds that provide the cues necessary for the development of successful tissue engineered constructs.

The process of electrospinning polymers for tissue engineering and cell culture is addressed in this article. Specifically, the electrospinning of photoreactive macromers with additional processing capabilities of photopatterning and multi-polymer electrospinning is described.

Eletrofiação Andaimes Polymer fibrosos para Engenharia de Tecidos e Cultura de Células

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address ...

Long-term Culture of Human Breast Cancer Specimens and Their Analysis Using Optical Projection Tomography

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later distally, is a major factor in patient prognosis. Experimental systems of breast cancer rely on cell lines usually derived from primary tumours or pleural effusions. Two major obstacles hinder this research: (i) some known sub-types of breast cancers (notably poor prognosis luminal B tumours) are not represented within current line collections; (ii) the influence of the tumour microenvironment is not usually taken into account.
We demonstrate a technique to culture primary breast cancer specimens of all sub-types. This is achieved by using three-dimensional (3D) culture system in which small pieces of tumour are embedded in soft rat collagen I cushions. Within 2-3 weeks, the tumour cells spread into the collagen and form various structures similar to those observed in human tumours1. Viable adipocytes, epithelial cells and fibroblasts within the original core were evident on histology. Malignant epithelial cells with squamoid morphology were demonstrated invading into the surrounding collagen. Nuclear pleomorphism was evident within these cells, along with mitotic figures and apoptotic bodies.
We have employed Optical Projection Tomography (OPT), a 3D imaging technology, in order to quantify the extent of tumour spread in culture. We have used OPT to measure the bulk volume of the tumour culture, a parameter routinely measured during the neo-adjuvant treatment of breast cancer patients to assess response to drug therapy. 
Here, we present an opportunity to culture human breast tumours without sub-type bias and quantify the spread of those ex vivo. This method could be used in the future to quantify drug sensitivity in original tumour. This may provide a more predictive model than currently used cell lines.

We have developed a collagen-based in vitro assay which promotes proliferation and invasion from samples of all breast cancer subtypes. Optical Projection Tomography, a three dimensional microscopy technique was utilised to visualise and quantify tumour expansion. This assay may be used to quantify drug response of individual tumour samples.

Longo prazo Cultura de Amostras de mama Humano do Câncer e sua análise por meio de tomografia óptica de Projeção

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

Rato Desenvolvimento Embrionário em todo um sistema de cultura de embriões sem soro

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

A Tissue Culture Model of Estrogen-producing Primary Bovine Granulosa Cells

Ovarian granulosa cells (GC) are the major source of estradiol synthesis. Induced by the preovulatory luteinizing hormone (LH) surge, cells of the theca and, in particular, of the granulosa cell layer profoundly change their morphological, physiological, and molecular characteristics and form the progesterone-producing corpus luteum that is responsible for maintaining pregnancy. Cell culture models are essential tools to study the underlying regulatory mechanisms involved in the folliculo-luteal transformation. The presented protocol focuses on the isolation procedure and cryopreservation of bovine GC from small- to medium-sized follicles (&#60; 6 mm). With this technique, a nearly pure population of GC can be obtained. The cryopreservation procedure greatly facilitates time management of the cell culture work independent of a direct primary tissue (ovaries) supply. This protocol describes a serum-free cell culture model that mimics the estradiol-active status of bovine GC. Important conditions that are essential for a successful steroid-active cell culture are discussed throughout the protocol. It is demonstrated that increasing the plating density of the cells induces a specific response as indicated by an altered gene expression profile and hormone production. Furthermore, this model provides a basis for further studies on GC differentiation and other applications.

A long-term culture model of bovine granulosa cells under serum-free conditions is described. This model allows researchers to study the effects of diverse factors and conditions as different plating densities on the characteristics of estrogen-producing bovine granulosa cells.

Um modelo de cultura de tecidos de produção de estrogênio primário Granulosa bovina células

Ovarian granulosa cells (GC) are the major source of estradiol synthesis. Induced by the preovulatory luteinizing hormone (LH) surge, cells of the theca ...

Tissue Collection of Bats for -Omics Analyses and Primary Cell Culture

As high-throughput sequencing technologies advance, standardized methods for high quality tissue acquisition and preservation allow for the extension of these methods to non-model organisms. A series of protocols to optimize tissue collection from bats has been developed for a series of high-throughput sequencing approaches. Outlined here are protocols for the capture of bats, desired demographics to be collected for each bat, and optimized methods to minimize stress on a bat during tissue collection. Specifically outlined are methods for collecting and treating tissue to obtain (i) DNA for high molecular weight genomic analyses, (ii) RNA for tissue-specific transcriptomes, and (iii) proteins for proteomic-level analyses. Lastly, also outlined is a method to avoid lethal sampling by creating viable primary cell cultures from wing clips. A central motivation of these methods is to maximize the amount of potential molecular and morphological data for each bat and suggest optimal ways to preserve tissues so they retain their value as new methods develop in the future. This standardization has become particularly important as initiatives to sequence chromosome-level, error-free genomes of species across the world have emerged, in which multiple scientific parties are spearheading the sequencing of different taxonomic groups. The protocols outlined herein define the ideal tissue collection and tissue preservation methods for Bat1K, the consortium that is sequencing the genomes of every species of bat.

This is a protocol for the optimal tissue preparation for genomic, transcriptomic, and proteomic analyses of bats caught in the wild. It includes protocols for bat capture and dissection, tissue preservation, and cell culturing of bat tissue.

Coleção de tecidos de morcegos para análises de Omics e cultura de células primárias

As high-throughput sequencing technologies advance, standardized methods for high quality tissue acquisition and preservation allow for the extension of ...

Mucociliary Epithelial Organoids from Xenopus Embryonic Cells: Generation, Culture and High-Resolution Live Imaging

Mucociliary epithelium provides the first line of defense by removing foreign particles through the action of mucus production and cilia-mediated clearance. Many clinically relevant defects in the mucociliary epithelium are inferred as they occur deep within the body. Here, we introduce a tractable 3D model for mucociliary epithelium generated from multipotent progenitors that were microsurgically isolated from Xenopus laevis embryos. The mucociliary epithelial organoids are covered with newly generated epithelium from deep ectoderm cells and later decorated with distinct patterned multiciliated cells, secretory cells, and mucus-producing goblet cells that are indistinguishable from the native epidermis within 24 h. The full sequences of dynamic cell transitions from mesenchymal to epithelial that emerge on the apical surface of organoids can be tracked by high-resolution live imaging. These in vitro cultured, self-organizing mucociliary epithelial organoids offer distinct advantages in studying the biology of mucociliary epithelium with high-efficiency in generation, defined culture conditions, control over number and size, and direct access for live imaging during the regeneration of the differentiated epithelium.

We describe a simple protocol to develop mucociliary epithelial organoids from deep ectoderm cells isolated from Xenopus laevis embryos. The multipotent progenitors regenerate epithelial goblet cell precursors and allow live tracking of the initiation and progression of the cell transitions on the surface of organoids.

Organoides epiteliais mucociliais de células embrionárias xenopus: geração, cultura e imagem ao vivo de alta resolução

Mucociliary epithelium provides the first line of defense by removing foreign particles through the action of mucus production and cilia-mediated ...

Bovine Ovarian Cortex Tissue Culture

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors from somatic cells and cumulus cell-oocyte communication. Little is known about the ovarian microenvironment and how the cytokines and steroids produced in the surrounding milieu affect follicle progression or arrest. In vitro culture of ovarian cortex enables follicles to develop in a normalized environment that remains supported by adjacent stroma. Our objective was to determine the effect of nutritional Stair-Step diet on the ovarian microenvironment (follicle development, steroid, and cytokine production) through in vitro culture of bovine ovarian cortex. To accomplish this, ovarian cortical pieces were removed from heifers undergoing two different nutritionally developed schemes prior to puberty: Control (traditional nutrition development) and Stair-Step (feeding and restriction during development) that were cut into approximately 0.5-1 mm3 pieces. These pieces were subsequently passed through a series of washes and positioned on a tissue culture insert that is set into a well containing Waymouth&#39;s culture medium. Ovarian cortex was cultured for 7 days with daily culture media changes. Histological sectioning was performed to determine follicle stage changes before and after the culture to determine effects of nutrition and impact of culture without additional treatment. Cortex culture medium was pooled over days to measure steroids, steroid metabolites, and cytokines. There were tendencies for increased steroid hormones in ovarian microenvironment that allowed for follicle progression in the Stair-Step versus Control ovarian cortex cultures. The ovarian cortex culture technique allows for a better understanding of the ovarian microenvironment, and how alterations in endocrine secretion may affect follicle progression and growth from both in vivo and in vitro treatments. This culture method may also prove beneficial for testing potential therapeutics that may improve follicle progression in women to promote fertility.

In vitro culture of bovine ovarian cortex and the effect of nutritional Stair-step diet on ovarian microenvironment is presented. Ovarian cortex pieces were cultured for seven days and steroids, cytokines, and follicle stages were evaluated. The Stair-Step diet treatment had increased steroidogenesis resulting in follicle progression in culture.

Cultura do tecido do córtex ovariano bovino

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors ...