Este trabalho foi apoiado por financiamento do Departamento de Defesa, da próstata Cancer Research Program (W81XWH-12-1-0124), U54CA143876 do Instituto Nacional do Câncer, ea McCooey geniturinário Fundo de Investigação Oncológica Robert. Gostaríamos de agradecer ao Dr. Annarita Lorenzo (Departamento de Patologia) para a prestação de anticorpos VE-caderina, e Dr. Marco Seandel (Departamento de Cirurgia) para a prestação de HUVECs.

1. Cultivar HUVECs em Microslides para Interações Observing CTC-endoteliais <ol><li> Sob a capa de cultura de tecidos, primeiro lave o MicroSlide, a largura do canal de 1 mm com PBS. Suavemente revestimento a microlâmina com 200 mL de 50 ug / ml de fibronectina (dissolvida em PBS) utilizando uma seringa de 1 ml de bloqueio Luer. </li><li> Cobrir a microlâmina com a tampa e mantê-lo dentro do capuz de cultura de tecidos durante 30 min. Distribuição lenta do líquido na microlâmina impede a formação de bolhas no canal. </li><li> Perfundir 200 mL de água morna (37 ° C) HUVEC meio de crescimento (meio M199, 1M de HEPES, 20% FBS, 5 mg / ml de heparina, factor de crescimento de célula endotelial de 100 ug / ml, e L-glutamina) através da microlâmina e incubar 20 min à TA. Durante a perfusão, preparar a suspensão de células HUVEC. </li><li> Lavar HUVECs com PBS e adicionar 0,05% de tripsina-EDTA durante 1-2 minutos à temperatura ambiente. Centrífuga HUVECs em 2 ml de meio de crescimento a 180 xg durante 5 min. </li><li> Medir a concentração de células utilizando um Neubahemocitômetro uer e preparar 10 7 HUVEC células/100 mL meio de crescimento. Em seguida, remover cuidadosamente o meio a partir da entrada da microlâmina com uma pipeta de ponta de 200 ul. </li><li> Trazer o MicroSlide ao nível dos olhos e usando a 1 ml seringa Luer-lock, perfuse gentilmente 200 mL de concentração preparado de HUVECs para o canal. É necessário ter cuidado nesta etapa para evitar a formação de bolhas. Se as bolhas aparecem, manter perfusão por um tempo maior até que as bolhas entrar no canal de saída. </li><li> Colocar um volume igual (~ 80 ul) de meios de HUVEC em tanto à entrada e à saída da microlâmina. Isto evita que o fluxo de células em ambas as direcções. </li><li> Cobrir o corrediça e mantê-lo na incubadora (37 ° C) durante 1,5 horas. </li></ol> 2. Preparação de montagem de câmara de fluxo para Overnight Cultura HUVEC em um MicroSlide <ol><li> Coloque uma seringa estéril de 20 ml, conectores Luer femininos e masculinos, tubulação, e uma bomba de seringa na incubadora por 15 min. </li><li> Para um volume morto mínimo, utilizar o tubo com diâmetro interno de 0,04 polegadas. Diâmetro da tubulação menor evita a formação de bolhas. </li><li> Encher a seringa de 20 ml com água morna (37 ° C) meios de HUVEC (12 ml). Conecte o tubo com os conectores na seringa. Retirar as bolhas. Conecte este conjunto à MicroSlide. </li><li> Terminado o preenchimento da entrada da MicroSlide com a mídia HUVEC. Traga a seringa cheia de 20 ml ligado ao conector do lado da microlâmina. Delicadamente, anexar o conector ao MicroSlide. </li><li> Traga o set-up para a incubadora e conecte-o a bomba de seringa, fixada em 10 taxa de cisalhamento mL / min. Deixar as células S / N na incubadora a 37 ° C. </li><li> No dia seguinte, desmontar o conjunto--se através da remoção do conector ligado à entrada da microlâmina. </li><li> Para regular a expressão de E-selectina em células endoteliais, preparar o meio de crescimento fresco contendo IL-1β a 10 ng / ml em 4 ml de meio. Aspirar a mídia em uma seringa de 10 ml. Remover tque media a partir da entrada da microlâmina e ligar a seringa para a corrediça. </li><li> Definir a bomba de seringa a 10 velocidade de corte ul / min durante 4 horas na incubadora. </li></ol> 3. Preparação de Anti-PSMA (J591-488) marcada células cancerosas da próstata <ol><li> Durante IL-1β incubação de HUVECs, preparar anti-PSMA J591-Alexa488 rotulado células APC. Adicionar 0,05% de tripsina-EDTA para células MDA durante 1 min. Não expor as células a tripsina para tempos mais longos, uma vez que pode afectar os glicopéptidos presente na superfície da célula. Reagente de dissociação de células livres de enzima também pode ser usado em vez de tripsina. Centrifugar a 200 xg durante 5 min. </li><li> Ressuspender MDA sedimento celular em 1 ml de H / H tampão (Hanks sal equilibrada solution/0.1% HSA/10 mM HEPES / 1 mM CaCl2). Adicionar anticorpo anti-PSMA J591-Alexa488 a 20 ug / ml durante 30 minutos à temperatura ambiente num local escuro. Ressuspender as células durante a incubação. </li><li> Depois de 30 minutos, centrifugar a solução de células a 800 xg durante 5 min. Aspitaxa e ressuspender o pellet em 1 ml de tampão de H / H. Contar as células MDA rotulados e trazer a concentração final de 1x10 6 células / ml. Encha uma seringa de 5 ml com J591-488 células MDA rotulados e remover as bolhas. </li></ol> 4. Preparação de Anti-PSMA (J591-488) marcada CTCs Enriched de CaP pacientes <ol><li> Recolha de 7,5 ml de sangue de pacientes com cancro da próstata num tubo de tampa azul (contendo citrato de sódio). Suavemente diluir 1:01 sangue em 0,1% BSA / 1 mM EDTA / PBS. </li><li> Adicionar 5,3 ml de Ficoll-Paque em mais de um tubo cónico de 50 ml. Camada diluído sangue no topo do Ficoll-Paque. Centrifugar a 400 xg durante 30 min. </li><li> Pré-revestimento de todos os tubos e pontas entram em contacto com os CTC com 2% FBS/RPMI-1640 / 1 mM de CaCl 2/4 mM de MgCl2 (tampão de R / S) para evitar a ligação não específica do CTC para as superfícies. Manter 4 ° C Temperatura de mídia e buffers em contacto com CTCs. </li><li> Recolha de células mononucleares de sangue periférico (PBMC) fracçãocontendo CTC a partir da interface de outro tubo cónico de 50 ml contendo 30 ml de tampão de I / S. Centrifugar a 400 xg durante 8 minutos. </li><li> Ressuspender o sedimento e lavar novamente em tampão de R / S a 400 xg durante 8 minutos. Após a centrifugação, ressuspender o sedimento em tampão H / H. Adicionar anti-PSMA J591-488 de anticorpo a 20 ug / ml durante 30 minutos à temperatura ambiente num local escuro. </li><li> Depois de 30 minutos, centrifugar os PBMC contendo J591-488 CTC rotulados a 800 xg durante 5 min. Ressuspender em tampão H / H. Encher uma seringa de 1 ml (pré-revestido com um tampão de R / S) com a amostra. </li></ol> 5. Preparação de microscópio, Bomba de Seringa e montagem de câmara de fluxo <ol><li> Ligue o microscópio invertido e definir a iluminação Kohler em 10X objetiva. Traga a bomba de seringa, ao mesmo nível como a fase da amostra do microscópio e ajustá-la em uma dines / cm 2 a tensão de cisalhamento (~ 10 mL / min). </li><li> Abra o software Zeiss Axiovision. Selecione o objetivo na tela do computador. Criar um novopasta na opção ferramentas do software. </li><li> Abra a opção experimentos inteligentes em Axiovision e ajustar as configurações para gravar 30 vídeos curtos SEC de 30 min. </li><li> Para vídeo fluorescente ao vivo, defina as opções de imagem-12 ms exposição, 2 x 2 bin, 5 Gain. Estes parâmetros ajudar na obtenção de vídeos perto da taxa de quadros de vídeo (~ 23 quadros por segundo) com a câmera Zeiss MRM. </li><li> Para visualizar toda a largura do canal de fluxo em 10 objetiva X na tela do computador, usar um adaptador C-mount (0,63 xf/60 mm Interface). </li><li> Ligue a lâmpada de mercúrio. </li><li> Colocar a seringa e o conector contendo as células sobre a bomba de seringa. </li><li> Traga a IL-1β HUVECs estimuladas da incubadora. Ligue o conector para o canal de entrada cheia do MicroSlide contendo HUVECs. </li><li> Conectar o canal de saída com o conector ligado ao tubo. Coloque o tubo em um prato ou 15 ml tubo cônico para coletar o fluxo através. </li><li> Inicie o infusino meio da microlâmina a 10 ul / min. Observar as interacções entre as células endoteliais e células MDA marcados ou rotulados CTC derivadas de doentes com filtro de 488 nm sobre o microscópio de epifluorescência. </li><li> Comece a gravar o experimento como 30 seg vídeos curtos. Durante a análise de reprodução, medir a velocidade de rolamento. Velocidade de rolamento é medida dividindo-se a distância percorrida pelas células ao longo do tempo. </li></ol> 6. Imunocoloração da microlâmina <ol><li> Remova a mídia da entrada e saída do MicroSlide após perfusão células MDA em IL-1β estimulada pela HUVECs por 10 min. </li><li> Utilizando uma ponta de 200 mL, colocou quente (37 ° C) PBS contendo cálcio e magnésio para dentro da entrada da microlâmina, durante 5 min. Incline a MicroSlide usando sua tampa como um suporte no banco. Manter a microlâmina numa posição inclinada para todas as incubações durante a imunocoloração. </li><li> Fix HUVECs irrigando quente 2% de formaldeído na entrada </li><li&gt; Incubar durante 20 minutos à temperatura ambiente. Lavar duas vezes com PBS durante 5 minutos cada. </li><li> Adicionar Triton X-100 (0,1%) em 5% de BSA em PBS durante 10 min à temperatura ambiente. </li><li> Lavar duas vezes com PBS durante 5 minutos cada. Bloco com 5% BSA em PBS durante 30 min. </li><li> Incubar com anticorpo de cabra primário anti-VE-caderina (1:100) em BSA a 2,5% O / N a 4 ° C. </li><li> No dia seguinte, lava-se duas vezes com PBS durante 5 minutos cada. Adicionar burro secundário anti-cabra 647 anticorpo por 45 min. Lavar duas vezes com PBS durante 5 minutos cada. </li><li> Incubar à temperatura ambiente com anticorpo conjugado J591-Alexa488 humanizado durante 1 hora. </li><li> Lavar duas vezes com PBS durante 5 minutos cada. Adicionar DAPI por 5 min. Lava-se com água destilada durante 5 minutos. </li><li> Adicionar PBS (ou Mowiol para armazenamento a longo prazo). Visualize a MicroSlide sob o microscópio confocal. </li></ol>

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Dr. Bander é o inventor de patentes que são atribuídos a Fundação de Pesquisa de Cornell (&quot;CRF&quot;) para o anticorpo J591 utilizado neste artigo. Dr. Bander é consultor e possui ações em BZL Biologics, a empresa para a qual as patentes foram licenciadas pelo CRF para novas pesquisas e desenvolvimento.

Devido ao baixo número de CTCs entre as células do sangue, é difícil isolar CTCs como uma população pura de células. Para estudar interações CTC / CE, a população rara e impura de CTCs coloca dois desafios principais: a) Identificação de CTCs entre as células do sangue; b) Observação de interações CTC / CE. Para superar a primeira limitação de identificar CTCs próstata entre as células do sangue, aproveitamos o fato de que praticamente todas ...

A metástase é um processo multi-passo complexo que permanece pouco compreendido. O eixo ligando E-selectin/selectin tem mostrado desempenhar um papel importante na metástase de tumores através da promoção de interacções adesivas primárias entre o endotélio vascular e nas células cancerosas 1,2. Endotelial (E)-selectina é uma proteína transmembranar expressa por células endoteliais activadas, enquanto ligando diferente (s) de E-selectina é expressa por células de tumor 3. Numerosas abordagens in vitro têm sido utilizados com sucesso para modelar as interacções ligando E-selectin/selectin entre células tumorais e células endoteliais (ECs) 1. Para estudar essas interações, diferentes sistemas de câmara de fluxo estão sendo empregados para simular sistema vascular sanguíneo. Entre os conjuntos de câmara de fluxo, o fluxo de placas paralelas câmara (PPFC) em conjunto com ECs é rotineiramente utilizado como um modelo in vitro simulando em condições de estresse de cisalhamento in vivo. Nestemétodo, ECs são cultivadas numa placa de 35 mm e depois de atingir uma monocamada, ECs estão ligados ao PPFC e experiências baseadas tensão de cisalhamento são executados. No entanto, PPFC e outros sistemas actuais apresentam muitas limitações para estudar as interacções adesivas entre as células tumorais (CTCs) derivadas de doentes e CEs, circula principalmente, porque CTC são uma população rara de células, verter a partir do tumor primário, que circula entre os milhões de células sanguíneas (1 CTC 10 9 por células do sangue) 4. Assim, ao contrário de oferta ilimitada de linhas de células cultivadas, baixa contagem CTC levar para muito poucos e raros interações CTC / CE, requerendo a largura do canal fluxo adequado para gravar as interações para análise de reprodução. Além disso, uma vez que pacientes CTC derivados são uma população impuro, portanto, um marcador de identificação é necessária para controlar CTC em específico. Para resolver este problema, foi desenvolvido um novo método para identificar o câncer de próstata (CaP) CTCs tirando partido do facto de que praticamente todos estes CTC expressam antigénio de membrana específico da próstata (PSMA) na sua superfície celular 5,6. Neste relatório, foi utilizada a linha de células de câncer de próstata, MDA PCa2b (MDA), para demonstrar a utilidade potencial do nosso novo sistema para estudar as interações de próstata CTC com ECs, eventualmente, para entender o mecanismo de metástases. Nossa metodologia pode ser aplicada para vários experimentos de corte com base em simulação de sistema vivo vascular 7-9. Além de examinar as interações PCa CTC / CE, o atual sistema de câmara de fluxo pode ser facilmente adaptado para a análise de células mononucleares do sangue periférico ou células tumorais interações &#39;com ECs. A facilidade de desmontagem e remontagem da câmara de fluxo, um MicroSlide III (0,1) (doravante referida como MicroSlide), permite a cultura ECs sob perfusão e estimulando ECs com diferentes citocinas para induzir protein expressão. Além disso, em cultura células endoteliais, as proteínas recombinantes, tais como a L-e P-selectina podem ser revestidos na microlâmina e interacções com as células tumorais podem ser observados sob condições de escoamento laminar 10.

<table border="0" cellpadding="0" cellspacing="0" width="744">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Microslide</td>
			<td style="width:192px;">Ibidi</td>
			<td style="width:119px;">80331</td>
			<td style="width:189px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Fibronectin</td>
			<td style="width:192px;">Millipore</td>
			<td style="width:119px;">FC010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Plastic tubing</td>
			<td style="width:192px;">Tygon</td>
			<td style="width:119px;">AAQ04103</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Male luer adapter</td>
			<td style="width:192px;">GlycoTech</td>
			<td style="width:119px;">31-001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Female luer adapter</td>
			<td style="width:192px;">GlycoTech</td>
			<td style="width:119px;">31-001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Syringe pump</td>
			<td style="width:192px;">Chemyx Inc</td>
			<td style="width:119px;">Fusion 100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Luer-lock syringe</td>
			<td style="width:192px;">BD Biosciences</td>
			<td style="width:119px;">309628</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">M199 medium</td>
			<td style="width:192px;">Sigma</td>
			<td style="width:119px;">M7653</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Endothelial Mitogen</td>
			<td style="width:192px;">Biomedical Technologies</td>
			<td style="width:119px;">BT-203</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">HBSS</td>
			<td style="width:192px;">Sigma</td>
			<td style="width:119px;">H9269</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:244px;">Anti-PSMA J591-488</td>
			<td style="width:192px;">Weill Cornell Medical College-Lab of Urologic Oncology</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Interleukin-1 beta</td>
			<td style="width:192px;">Peprotech</td>
			<td style="width:119px;">200-01B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Trypsin</td>
			<td style="width:192px;">Millipore</td>
			<td style="width:119px;">SM-2002-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Heparin</td>
			<td style="width:192px;">Sigma</td>
			<td style="width:119px;">H-3149</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:244px;">HUVECs</td>
			<td style="width:192px;">Weill Cornell Medical College-Department of Surgery</td>
			<td style="width:119px;"></td>
			<td>provided by Marco Seandel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">VE-Cadherin</td>
			<td style="width:192px;">Santa Cruz</td>
			<td style="width:119px;">sc-5648</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">10x objective</td>
			<td style="width:192px;">Zeiss Plan Neofluar</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:244px;">Enzyme free cell dissociation reagent</td>
			<td style="width:192px;">Millipore</td>
			<td style="width:119px;">S-004-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">RPMI-1640</td>
			<td style="width:192px;">Lonza</td>
			<td style="width:119px;">12-702-F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Ficoll-paque plus</td>
			<td style="width:192px;">GE healthcare</td>
			<td style="width:119px;">17-1440-02</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro method to observe e-selectin-mediated interactions between prostate circulating tumor cells derived from patients and human endothelial cells

A metástase é um processo em que as células tumorais derramado a partir do tumor primário intravasate vascular arterial e do sistema linfático, assim, ganhar acesso ao extravasamento e formar um nicho secundário. A extravasão de células de tumor a partir do sistema vascular de sangue podem ser estudados utilizando células endoteliais (ECs) e as células tumorais obtidas a partir de diferentes linhas celulares. Os estudos iniciais foram realizados usando condições estáticas, mas tem sido bem documentado que os CEs se comportar de forma diferente sob condições de fluxo fisiológico. Portanto, diferentes conjuntos de câmara de fluxo estão sendo usados ​​para estudar as interações de células de câncer com ECs. Montagens atuais câmara de fluxo oferecem resultados reproduzíveis, seja através de diferentes linhagens celulares ou fluidos em diferentes condições de estresse de cisalhamento. No entanto, para observar e estudar as interações com células raras, tais como células tumorais (CTCs) circulando, algumas alterações são necessárias a serem feitas para a montagem de câmara de fluxo convencional. CTCssão uma população de células raras entre os milhões de células sanguíneas. Por conseguinte, é difícil a obtenção de uma população pura do CTC. Contaminação de CTCs com diferentes tipos de células normalmente encontradas na circulação é inevitável o uso presente enriquecimento ou técnicas de depleção. No presente relatório, nós descrevemos um método único para rótulo circulando células cancerosas da próstata fluorescente e estudar as suas interacções com os ECs em um sistema de câmara de fluxo auto-montados. Esta técnica pode ser aplicada ainda para observar as interações entre CTCs próstata e qualquer proteína de interesse.

A Figura 1 mostra uma cultura D / N de uma monocamada de células endoteliais na microlâmina. Os extrapolação da Figura 1A mostra que 100% da microlâmina é visível usando objectivo 5X, enquanto 70% é visível usando uma objectiva de 10X (Figura 1B). Para o E-selectina interacções mediadas, as células que rolam nas bordas não são considerados o que torna mais do que 70% da microlâmina disponível para gravação de vídeo e análise de reprodução. Em nossa...

Watch this Scientific Journal Video about In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells at JoVE

In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

O nosso relatório descreve um método único para visualizar e analisar as interações CTC / CE no câncer de próstata em condições de fluxo fisiológicos.

Método in vitro para observar interações E-selectina mediadas entre próstata células tumorais circulantes derivadas de pacientes e células endoteliais humanas

Metastasis is a process in which tumor cells shed from the primary tumor intravasate blood vascular and lymphatic system, thereby, gaining access to ...

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Research

JoVE Journal

Medicine

In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

9.0K visualizações.  Weill Cornell Medical College.  O nosso relatório descreve um método único para visualizar e analisar as interações CTC / CE no câncer de próstata em condições de fluxo fisiológicos. 

Vídeo: In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Isolamento, caracterização e comparativa Diferenciação de células-tronco da polpa dentária Derivado de dentes permanentes usando dois métodos diferentes

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Medicina

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Cultura de Células e Isolamento paciente Derivado de CD133<sup&gt; +</sup&gt; Células-tronco de câncer putativos Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

Isolamento de células-tronco cancerosas em amostras de câncer de próstata humano

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with muscular dystrophy is an invaluable cellular source for in&#160;vitro disease model systems and can be used for drug screening studies. Patient-derived urine cells have been used in successful reprogramming into induced pluripotent stem cells in order to model dystrophic cardiomyopathy1. Addressing the safety concerns of integrating vector systems, we present a protocol using a non-integrating Sendai virus vector for transduction of Yamanaka factors into urine cells collected from patients with muscular dystrophy. This protocol generates fully reprogrammed clones within 2&#8211;3 weeks. The pluripotent cells are vector-free by passage-13. These dystrophic iPSCs can be differentiated into cardiomyocytes and used either to study disease mechanisms or for drug screening.

This protocol entails detailed procedures for isolation of urine derived cells from muscular dystrophy patients; their efficient and rapid reprogramming through Sendai virus transduction.

Geração de pluripotentes induzidas células-tronco de distrofia muscular pacientes: Reprogramação livre de integração eficiente de urina células derivadas

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with ...

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent &#8216;floater&#8217; cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of cells.

Em Enriquecimento vitro de câncer de ovário de iniciação Células Tumorais

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Estudar o cancro do pâncreas Stem características celulares para desenvolver estratégias Novo tratamento

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

Isolamento e propagação de células tumorais circulantes de um cancro do modelo do rato

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

Isolamento e Cultura de Expansão de células endoteliais específicos de tumores

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Circulating tumor cells (CTC) play a critical role in tumor metastasis and have been isolated from patients with several tumor types. Recently, CTCs have been used to generate PDX experimental models of breast and prostate cancer. This manuscript details the method for the generation of prostate cancer PDX models from CTCs developed by our group. Advantages of this method over conventional PDX models include independence from surgical sample collection and generating experimental models at various disease stages. Density gradient centrifugation followed by red blood cell lysis and flow cytometry depletion of CD45 positive mononuclear cells is used to enrich CTCs from peripheral blood samples collected from patients with metastatic disease. The CTCs are then injected into immunocompromised mice; subsequently generated xenografts can be used for functional studies or harvested for molecular characterization. The primary limitation of this method is the negative selection method used for CTC enrichment. Despite this limitation, the generation of PDX models from CTCs provides a novel experimental model to be applied to prostate cancer research.

This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

Geração de paciente com câncer de próstata Derivado modelos de xenotransplante de células tumorais circulantes

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

Depleção de células do rato de xenoenxertos de tumor humano Melhora Significativamente Análise Downstream de células alvo

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...