Agradecemos ao Dr. Robert A. Weinberg (Whitehead Institute for Biomedical Research, Cambridge) para apoio generoso e supervisão deste trabalho e Kieran Mr. Mellody (University of Manchester, Manchester) para a edição crítica deste manuscrito. Este projecto foi apoiado pela Research UK (CR-UK) conceder número C147/A6058 (AO)

1. Isolamento da principal cultura de fibroblastos humanos normais mamárias Procedimentos experimentais para isolar principal cultura de fibroblastos humanos normais mamárias são descritas na fig. 1A. <ol><li> Prepare a célula tampão dissociação, como descrito anteriormente 12: Médio Modified Dulbecco Águia (DMEM) com 10% de soro fetal bovino (FCS), a penicilina-estreptomicina (200 unidades / ml), colagenase tipo I (1 mg / ml) e hialuronidase ( 125 unidades / ml). </li><li> Lave o tecido mamário dissecada a partir de uma mamoplastia de redução (~ 0,5 grama) várias vezes em tampão fosfato salino (PBS). Picar o tecido em fragmentos pequenos (&lt;1,5 mm 3) com lâminas de barbear estéril. </li><li> Transferir os fragmentos de tecido em um tubo cônico de 15 ml contendo uma quantidade adequada de células acima dissociação buffer (10 ml por 0,5 grama de tecido) e vortex por 1 min em velocidade máxima. </li><li> Digerir os fragmentos de tecido na célula dissociação tampão f18/12 h ou a 37 ° C com agitação lenta. * Nota que haverá ainda muitos pedaços de fragmentos de tecido não digerida restante no tubo. </li><li> Incubar o tubo por 5 min à temperatura ambiente sem agitação. Transferência resultante do estroma celular enriquecido sobrenadante para um novo tubo cônico com uma pipeta de 5 ml sorológicos. </li><li> Centrífuga a fração estromal por 5 min a 250 X g e ressuspender o sedimento celular em PBS. Centrifugar novamente por 5 min a 250 X g e ressuspender o pellet celular em DMEM suplementado com 10% SFB. Cultura as células em DMEM contendo 10% SFB em um prato de 15 centímetros de Petri a 37 ° C e dióxido de carbono de 5%. </li><li> Propagar as células até confluentes. Pode levar 80-10 dias. Armazenar as células a -80 ° C, utilizando meio de congelamento (10% dimetil sulfóxido e FCS de 20% em DMEM). Prepare 5 frascos congelados como um estoque original. Descongelar um frasco e expandir as células para preparar 5 frascos para a secundária de ações contendo fibroblastos várias passagens dentro de 5 a populaçãoduplicações (PDs) para os experimentos subseqüentes. Estes procedimentos minimizar seleção clonal e estresse cultura que poderia ocorrer durante a cultura de tecidos estendidos. * Nota: P1.1-7 também podem ser usados ​​para isolar FAC de carcinomas de mama obtidos por uma mastectomia 12. </li></ol> 2. Geração de GFP-etiquetadas, puromicina resistente, imortalizado humano normal fibroblastos mamárias <ol><li> Para imortalizar o principal fibroblastos humanos normais mamárias isoladas em P1.7), introduzir um pMIG retroviral (MSCV-IRES-GFP) vector 12, expressando tanto hTERT e GFP. Cultura das células para os dias 05/04 e, em seguida, classificar a GFP células positivas por citometria de fluxo. </li><li> Introduzir uma retroviral pBabe-puro construir codificação de um gene de resistência puromicina para os fibroblastos GFP-etiquetadas imortalizado. Cultura das células durante 5-7 dias na presença de puromicina (concentração final: 1 mg / ml) para isolar a GFP-positivo (GFP +), puromicina-resistente (R puro </sup&gt;) imortalizado fibroblastos humanos mamária. </li><li> Em seguida, examinar se esses fibroblastos produzem vírus ativo, o que pode levar à transferência horizontal de genes que codificam GFP e resistência puromicina, crescer 2.5x10 5 GFP + puro R imortalizado fibroblastos humanos mamária em uma placa de Petri seis centímetros por 2 dias. </li><li> Meio filtrante condicionada por estes fibroblastos utilizando um filtro de seringa (tamanho dos poros: 0,45 mm) e adicioná-lo em 10T1 / 2 células de camundongos fibroblástica por 12h na presença de sulfato de protamina (5 mg / ml), que aumenta a eficiência da infecção pelo vírus. O meio é, então, alterado para 10% FCS-DMEM por mais 2 dias. </li><li> Examine as células por uma microscopia de fluorescência. 10T1 / 2 células infectadas pelo vírus da GFP se tornará GFP-positivo. Também tratar as células com puromicina (1 mg / ml) por 5-7 dias e observar a placa se houver puro R 10T1 / 2 células são colônias presentes e forma após o tratamento puromicina. </li><li> Note que o gene horizontal transfer por vírus ativo produzido pelo GFP parental puro + R fibroblastos humanos mamárias pode tornar as células ao redor murino e / ou carcinoma de células GFP + R puro dentro xenoenxertos tumor. Isto pode dificultar os processos de isolar a GFP inicialmente injetado + puro R fibroblastos humanos mamárias da xenoenxertos tumor. </li></ol> 3-A. Coinjection de fibroblastos humanos com células mamárias carcinoma de mama em um mouse imunodeficientes Procedimentos experimentais para a geração de exp-FAC são ilustrados na figura. 1BA. <ol><li> Mix 1x10 6 MCF-7-ras células de carcinoma de mama humano e 3x10 6 GFP + R puro, imortalizado fibroblastos humanos mamárias gerada em P2.2). Prepare as células em 400 mL de meio de cultura com 50% (v / v) Matrigel por injeção. </li><li> Injete a mistura de células por via subcutânea em um mouse imunodeficientes nude. Quando FAC são extraídos de forma independente a partir de diferentestumores, implantes cada mistura em flancos direito e esquerdo de vários ratos. </li></ol> 3b. Injeção de fibroblastos humanos mamárias em um mouse imunodeficientes <ol><li> Para gerar fibroblastos controle contra exp-FAC, prepare 3x10 6 GFP + puro R imortalizado fibroblastos humanos mamárias gerada em P2.2 em 400 mL de meio de cultura com 50% (v / v) Matrigel por injeção (Fig. 1BB). NÃO misturar os fibroblastos com células de carcinoma. </li><li> Implante os fibroblastos preparado em P3b.1 em sites subcutânea de camundongos nude. </li></ol> * Note-se que os fibroblastos inoculados formarão o tecido fibroblástico tumor, mas não sob a pele. 4a. Dissecção do tumor xenoenxerto <ol><li> Eutanásia o mouse abrigar um subcutânea humana xenoenxerto de câncer de mama 42 dias pós-implantação (Fig. 1BA). * Nota que, quando células de carcinoma outros e / ou fibroblastos são coimplanted, Fibroblastos podem precisar de ser incubadas por mais de 42 dias dentro do xenoenxerto tumor para iniciar seu fenótipo miofibroblástica. </li><li> Colheita do xenoenxerto tumor do flanco do mouse por dissecção romba com a pinça e tesoura. Mergulhe o tecido tumoral em PBS. </li></ol> 4b. Dissecação de xenoenxerto fibroblastos <ol><li> Eutanásia o mouse abrigar o xenoenxerto de fibroblastos 42 dias pós-implantação (Fig. 1BB). </li><li> Dissecar o xenoenxerto de fibroblastos subcutânea usando uma pinça e tesouras. Coloque o tecido fibroblástico em PBS. * Nota que um xenoenxerto de fibroblastos, que aparece como um tecido transparente pequena, pode ser difícil encontrar sob a pele. </li></ol> 5. Preparação de células primárias cultivadas a partir de xenoenxertos <ol><li> Use um gabinete de biossegurança estéril para dissecar o tumor (~ 0,5 grama) ou tecido fibroblástico (~ grama 0,1). Transferir os tecidos extirpados para o topo de uma cultura de 15 centímetros dish com 1 ml de PBS. Picar os tecidos em fragmentos de tecido pequenos (&lt;1,5 mm 3) com lâminas de barbear estéril. * Observe que se o xenoenxerto de fibroblastos pesa menos de 0,1 grama, recolher alguns xenoenxertos fibroblastos adicionais e misturá-los juntos para aumentar o número de células para isolamento. </li><li> Transferir os fragmentos de tecido em um tubo cônico de 15 ml que contém a célula dissociação buffer (10 ml por 0,5 grama de tecido) e vortex por 1 min em velocidade máxima. </li><li> Incubar a suspensão de células por 3 horas a 37 ° C com agitação lenta e contínua. </li></ol> 6. Isolamento de puromicina resistentes células em cultura <ol><li> Centrifugar a suspensão de células dissociadas a partir de fibroblastos de tumor ou xenoenxerto por 5 min a 250 X g e ressuspender o pellet celular em PBS. Centrifugar novamente por 5 min a 250 g. X Ressuspender o pellet celular, resultando em 10% FCS-DMEM. Cultura as células em um prato de 15 centímetros de petri na presença de puromicina (1 mg / ml) a 37 ° Ce dióxido de carbono de 5%, a fim de eliminar quaisquer células de carcinoma contaminação e / ou murino células do estroma. Propagar as células até confluentes (2-4 semanas). Armazenar as células a -80 ° C, utilizando meio de congelamento. * Observe que o resultado puro células R, designado exp-Caf1 de 42 dias de idade (Fig. 1BA) ou controle de fibroblastos-1 células (Fig. 1BB), são imortais e positivas para GFP. É recomendável verificar se essas células produzem vírus ativo usando 10T1 / 2 células, como descrito em P2.3-6. </li></ol> 7a. Isolamento de exp CaF2-células em cultura Para impulsionar ainda mais o fenótipo ativado miofibroblástica da FAC, misture os 42 dias de idade exp-Caf1 células com MCF-7-ras células e injetá-las novamente por via subcutânea em um rato nu por períodos adicionais de 200 dias. Dissecar e dissociar a xenoenxerto tumor em uma suspensão de uma única célula e cultura as células em um prato de 15 centímetros de Petri com 10% FCS-DMEM, na presença de puromicina (1 mg / ml), como indicado na P6.1. Propagar as células até confluentes (8-10 dias). Armazenar as células a -80 ° C, utilizando meio de congelamento. O resultado células são nomeadas puro R 242 dias de idade exp-CaF2 células (Fig. 1BA). 7b. Extração de controle de fibroblastos-2 células em cultura Para gerar fibroblastos controle contra exp-CaF2 células, injetar 42 dias de idade controle de fibroblastos-1 células sozinho, sem células de carcinoma por via subcutânea em um rato nu adicionalmente para 200 dias. Dissecar e dissociar o tecido fibroblástico em uma suspensão de células individuais e cultura as células em um prato de 15 centímetros de Petri com 10% FCS-DMEM, na presença de puromicina (1 mg / ml), conforme indicado em P6.1. Propagar as células até confluentes (3-4 semanas). Armazenar as células a -80 ° C, utilizando meio de congelamento. O resultado células puro R são nomeados de fibroblastos-2 controle de células (Fig. 1BB). 8. Resultados representativos: Controle de fibroblastos-2 e exp-CaF2 cells, que foram extraídos de tumor de mama xenoenxertos, corados fortemente positiva para marcadores mesenquimais, incluindo humanos específicos vimentina, prolil-4-hidroxilase, 1A colagénio, fibronectina, S100A4, e proteína de superfície de fibroblastos (Fig. 2A) 11, indicando humana origem ea natureza dessas células mesenquimais. Em contraste, a citoqueratina, um marcador de células epiteliais, não foi manchado nestes + GFP fibroblastos (Fig. 2B). Esses achados, portanto, sugerem que o extraído exp CaF2 e controle de fibroblastos-2 células ter se originado a partir da dos pais humanos fibroblastos mamárias, inicialmente introduzido xenoenxerto mouse. Importante, uma maior proporção de exp-CaF2 células coradas positivas para α-SMA e da glicoproteína da matriz extracelular tenascina-C 5, sendo que ambos são marcadores de miofibroblastos em comparação com 42 dias de idade exp Caf1 e controle de fibroblastos-2 células (Fig . 2C, D) 11. Estes dados indicam que fibr humana residente mamáriasoblasts progressivamente evoluir para miofibroblastos CAF dentro xenoenxertos tumor. <img alt="Figura 1" src="/files/ftp_upload/3201/3201fig1.jpg" /> Figura 1 Representação esquemática do isolamento de fibroblastos humanos mamária. A) O tecido mamoplastia de redução foi picada utilizando lâminas de barbear estéril (P1.2) e transferido para um tubo cônico de 15 ml (P1.3). Os pequenos fragmentos de tecido foram digeridos na célula dissociação buffer (P1.4) e preparado para a cultura in vitro (P1.5-7). Para imortalizar o isolado humanos primários fibroblastos mamárias, uma pMIG retroviral (MSCV-IRES-GFP) vector, expressando tanto hTERT e GFP, foi introduzido, ea GFP-positivos resultantes células foram classificados usando citometria de fluxo (P2.1). Um vetor pBabe puro-retroviral codificação de um gene de resistência puromicina foi, então, introduzidos nessas células. Após o tratamento puromicina, GFP-etiquetadas (GFP +) puromicina-resistente (Puro R), immortalised fibroblastos humanos mamária foram isolados (P2.2). Ba) Para gerar exp-FAC, GFP + puro R imortalizado fibroblastos humanos mamária foram coinjected com MCF-7-ras células de carcinoma de mama por via subcutânea em um mouse imunodeficientes nude (P3a). O xenoenxerto tumor foi retirado em 42 dias após a implantação (P4a) e dissociada em uma suspensão de uma única célula (P5). Essas células foram então cultivadas in vitro na presença de puromicina para eliminar qualquer contaminação e células de carcinoma de células do estroma do mouse (P6). O resultado puromicina resistentes células foram denominadas experimentalmente gerados Caf1 (exp-Caf1) células. Estas células, ressecadas 42 dias pós-implantação, foram mais uma vez misturado com MCF-7-ras células e implantado por via subcutânea em um rato de acolhimento, como antes (P7a). O tumor resultante foi permitido crescer por períodos adicionais de 200 dias, em seguida, dissecado, dissociado, e cultivadas na presença de puromicina. O isoladopuromicina resistentes células foram denominadas células-exp CaF2 (242 dias de idade). Bb) Para isolar as células controle contra exp-FAC, GFP + puro R imortalizado fibroblastos humanos mamárias foram injetados em um rato nu como culturas puras sem MCF-7-ras células (P3b). O tecido fibroblástico, dissecados 42 dias pós-implantação (P4B), foi dissociada em uma única célula suspensões (P5) e puromicina resistentes células, chamado de controle de fibroblastos-1 células, foram isoladas como descrito anteriormente (P6). Esses fibroblastos foram mais uma vez implantado no subcutâneo em um rato nu sem MCF-7-ras células adicionalmente para 200 dias (p7b). O tecido fibroblástico foi dissecada, dissociado, e cultivadas na presença de puromicina. O isolado puromicina resistentes células foram denominadas de fibroblastos-2 controle de células (242 dias de idade). <img alt="Figura 2" src="/files/ftp_upload/3201/3201fig2.jpg" /> Figura 2 <strong&gt; Exp-FAC e os fibroblastos originam do controle parental humano fibroblastos mamária. (A) as análises de imunofluorescência de controle de fibroblastos-2 (controle f.) e exp-CaF2 células. Os dois tipos de células coloração positiva para marcadores mesenquimais (vermelho), incluindo humanos vimentina, prolil-4-hidroxilase, 1A colagénio, fibronectina, S100A4, e proteína de superfície de fibroblastos. (B) Em contrapartida, pan-citoqueratina, um marcador de células epiteliais , não é detectado no exp-CaF2 células expressando GFP (verde). Núcleos das células são coradas com 4&#39;-6-diamidino-2-phenylindole (DAPI) (azul). Barra de escala, 50 mm (designado de Kojima et al. 11) (C) imunofluorescência de exp-CaF2 células fibroblasto-2 e controle de células (controle f.), utilizando anticorpos contra α-SMA (vermelho) ou tenascina-C (TN-C) (vermelho). Núcleos das células são coradas com DAPI (azul). Barra de escala, 50 mm. (D) 48% de exp-CaF2 células mancha positiva para α-SMA, enquanto que um4% de 42 dias de idade exp-Caf1 e 2,5% do controle populações de fibroblastos-2 de células positivas para α-SMA. (Referido a partir de Kojima et al.11)

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A falta de marcadores específicos CAF e do nível de heterogeneidade observada entre FAC tornar a caracterização deste tipo de célula um desafio em si mesmo. FAC estudar in vitro também tem sido dificultada pela complicação adicional de que essas células senesce e parar de proliferar quando cultivadas por um longo período. Nossa tentativa anterior de imortalizar diretamente FAC primário usando uma construção hTERT retroviral cDNA foi vencida. Portanto, para investigar o tumor promover propriedades d...

<table border="1"><tr><td> Nome do material </td><td> Tipo </td><td> Companhia </td><td> Número Catálogo </td><td> Comentário </td></tr><tr><td> DMEM </td><td></td><td> Invitrogen </td><td> 61965-026 </td><td></td></tr><tr><td> Soro fetal bovino </td><td></td><td> GIBCO </td><td> 10270 </td><td></td></tr><tr><td> Penicilina-estreptomicina </td><td></td><td> Invitrogen </td><td> 15140-122 </td><td></td></tr><tr><td> Colagenase tipo I </td><td></td><td> Sigma </td><td> C0130-1G </td><td></td></tr><tr><td> hialuronidase </td><td></td><td> Sigma </td><td> H4272 </td><td></td></tr><tr><td> Vimentina anticorpos (V9) </td><td></td><td> Novocastra Laboratories </td><td> NCL-L- VIM-V9 </td><td></td></tr><tr><td> Tenascina C (BC-8) de anticorpos </td><td></td><td></td><td></td><td> um presente de Dr. <strong&gt; Luciano Zardi </td></tr><tr><td> α-SMA-Cy3 anticorpos (1A4) </td><td></td><td> Sigma </td><td> C6198 </td><td></td></tr><tr><td> Prolil-4-hidroxilase </td><td></td><td> Dako </td><td> M0877 </td><td></td></tr><tr><td> (5B5) de anticorpos </td><td></td><td></td><td></td><td></td></tr><tr><td> Colágeno type1 anticorpos 1A </td><td></td><td> Sigma </td><td> HPA011795 </td><td></td></tr><tr><td> Pan-citoqueratina de anticorpos </td><td></td><td> Sigma </td><td> C5992 </td><td></td></tr><tr><td> Anticorpos fibronectina </td><td></td><td> BD Biosciences </td><td> 610077 </td><td></td></tr><tr><td> S100A4/FSP-1 (fibroblastos proteína-1 anticorpo específico) </td><td></td><td> Dako </td><td> A5114 </td><td></td></tr><tr><td> Proteína de superfície de fibroblastos (Clone 1B10) anticorpos </td><td></td><td> Abcam </td><td> ab11333 </td><td></td> &lt;/ Tr&gt; <tr><td> MSCV-IRES-GFP construir </td><td></td><td></td><td></td><td> Pedido para os autores do </td></tr><tr><td> pBabe-puro construir </td><td></td><td></td><td></td><td> Compra de Addgene </td></tr><tr><td> Puromicina </td><td></td><td> Sigma </td><td> P8833 </td><td></td></tr><tr><td> DAPI </td><td></td><td> Sigma </td><td> D9564 </td><td></td></tr><tr><td> 15 ml tubo cônico </td><td></td><td> Corning </td><td> 430766 </td><td></td></tr><tr><td> Camundongo nu </td><td></td><td> Taconic </td><td> NCRNU-F </td><td> Feminino NCr nua </td></tr><tr><td> Celas de C3H/10T1/2 </td><td></td><td> ATCC </td><td></td><td> CCL-226 </td></tr></table>

experimental generation of carcinoma-associated fibroblasts (cafs) from human mammary fibroblasts

Os carcinomas são tecidos complexo composto de células neoplásicas e um compartimento não-cancerosas referido como o &#39;estroma&#39;. O estroma é composto de matriz extracelular (ECM) e uma variedade de células mesenquimais, incluindo fibroblastos, miofibroblastos, células endoteliais, pericitos e leucócitos 1-3. O estroma tumoral associado é sensível a sinais de substancial parácrinos liberados por células de carcinoma vizinhos. Durante o processo da doença, o estroma, muitas vezes torna-se povoado por carcinoma-associada fibroblastos (FAC), incluindo um grande número de miofibroblastos. Estas células foram previamente extraído de diversos tipos de carcinomas humanos para a cultura in vitro. A subpopulação de FAC é distinguível através da sua sobre-regulação de α-actina de músculo liso (α-SMA) expressão 4,5. Essas células são uma característica da &#39;fibroblastos ativados &quot;que compartilham propriedades semelhantes com miofibroblastos commonly observado em tecidos lesados ​​e fibrótico 6. A presença desse grupo CAF miofibroblástica é altamente relacionada com alto grau de malignidade e associado com prognósticos pobres em pacientes. Muitos laboratórios, inclusive o nosso, têm mostrado que FAC, quando injetados com células de carcinoma em ratos imunodeficientes, são capazes de promover substancialmente tumourigenesis 7-10. FAC preparados a partir de pacientes com carcinoma, no entanto, freqüentemente sofrem senescência durante a propagação da cultura limitar a extensão da sua utilização em toda a experimentação em curso. Para superar esta dificuldade, desenvolvemos uma nova técnica para gerar experimentalmente imortalizado mamárias humanas linhas celulares CAF (exp-FAC) de fibroblastos humanos mamária, utilizando um modelo de mama coimplantation xenoenxerto tumor. A fim de gerar exp-FAC, parental humano fibroblastos mamárias, obtidas a partir do tecido mamoplastia de redução, foram os primeiros immortalised com hTERT, a subunidade catalítica da telomerase holoenzima, e projetados para expressar um gene GFP e resistência puromicina. Essas células foram coimplanted com células MCF-7 de carcinoma de mama humano expressando um oncogene ras ativados (MCF-7-ras células) em um xenoenxerto mouse. Após um período de incubação in vivo, inicialmente injetados fibroblastos humanos mamária foram extraídos do xenoenxertos tumor com base em sua resistência puromicina 11. Observamos que o residente de fibroblastos humanos mamárias têm diferenciada, adotando um fenótipo miofibroblástica e adquirida tumor propriedades promotoras durante o curso da progressão do tumor. Importante, essas células, definidas como exp-FAC, imitam de perto o fenótipo do tumor de promoção miofibroblástica da FAC isolado de carcinomas de mama de pacientes dissecados. Nosso tumor xenoenxerto derivados exp-FAC, portanto, fornecer um modelo eficaz para estudar a biologia da FAC em Breas humanacarcinomas t. O protocolo descrito também pode ser estendido para a geração e caracterização de populações de vários CAF derivados de outros tipos de carcinomas humanos.

Watch this Scientific Journal Video about Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts at JoVE.com

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma associado fibroblastos (FAC) rico em miofibroblastos presentes no estroma tumoral, desempenhar um papel importante na condução de progressão do tumor. Nós desenvolvemos um modelo coimplantation xengraft tumor para gerar FAC experimentalmente a partir de fibroblastos humanos mamária. O protocolo descreve como estabelecer miofibroblastos CAF que adquirem uma capacidade de promover tumourigenesis.

Geração Experimental de Carcinoma Associado fibroblastos (FAC) de Fibroblastos Humanos mamária

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

experimental-generation-carcinoma-associated-fibroblasts-cafs-from

Research

JoVE Journal

Medicine

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

23.3K visualizações.  University of Manchester. Carcinoma associado fibroblastos (FAC) rico em miofibroblastos presentes no estroma tumoral, desempenhar um papel importante na condução de progressão do tumor. Nós desenvolvemos um modelo coimplantation xengraft tumor para gerar FAC experimentalmente a partir de fibroblastos humanos mamária. O protocolo descreve como estabelecer miofibroblastos CAF que adquirem uma capacidade de promover tumourigenesis.

Vídeo: Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

Transplante de Células-tronco em um In vitro Modelo de isquemia / reperfusão Simulated

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

Medicina

Human Internal Mammary Artery (IMA) Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis

Preclinical in vivo research models to investigate pathobiological and pathophysiological processes in the development of intimal hyperplasia after vessel stenting are crucial for translational approaches1,2.
The commonly used animal models include mice, rats, rabbits, and pigs3-5. However, the translation of these models into clinical settings remains difficult, since those biological processes are already studied in animal vessels but never performed before in human research models6,7. In this video we demonstrate a new humanized model to overcome this translational gap. The shown procedure is reproducible, easy, and fast to perform and is suitable to study the development of intimal hyperplasia and the applicability of diverse stents.
This video shows how to perform the stent technique in human vessels followed by transplantation into immunodeficient rats, and identifies the origin of proliferating cells as human.

Human Internal Mammary Artery IMA Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis

This video shows a model to study the development of intimal hyperplasia after stent deployment using a human vessel (IMA) in an immunodeficient rat model.

Humanos da artéria mamária interna (IMA) Transplante e implante de stent: um modelo humano para estudar o desenvolvimento de reestenose intra-stent

Preclinical in vivo research models to investigate pathobiological and pathophysiological processes in the development of intimal hyperplasia after vessel ...

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts in this protocol have been successfully used in the derivation of induced pluripotent stem cells to model disease1. Early passages of these fibroblasts can also be used for cell-based functional assays to study specific disease pathways, mechanisms2 and subsequent drug screening approaches. The advantage of the presented protocol over enzymatic procedures are 1) the reproducibility of the technique from small amounts of tissue derived from older patients, e.g. patients affected with Parkinson's disease, 2) the technically simple approach over more challenging methodologies using enzymatic treatments, and 3) the time consideration: this protocol takes 15-20 min and can be performed immediately after biopsy arrival. Enzymatic treatments can take up to 4 hr and have the problems of overdigestion, reduction of cell viability and subsequent attachment of cells when not handled properly. This protocol describes the dissection and preparation of a 4-mm human skin biopsy for derivation of a fibroblast culture and has a very high success rate which is important when dealing with patient-derived tissue samples. In this culture, keratinocytes migrate out of the biopsy tissue within the first week after preparation. Fibroblasts appear 7-10 days after the first outgrowth of keratinocytes. DMEM high glucose media supplemented with 20% FBS favors the growth of fibroblasts over keratinocytes and fibroblasts will overgrow the keratinocytes. After 2 passages keratinocytes have been diluted out resulting in relatively homogenous fibroblast cultures which expresses the fibroblast marker SERPINH1 (HSP-47). Using this approach, 15-20 million fibroblasts can be derived in 4-8 weeks for cell banking. The skin dissection takes about 15-20 min, cells are then monitored once a day under the microscope, and media is changed every 2-3 days after attachment and outgrowth of cells.

The fibroblast explant culture protocol from human skin punch biopsies is a technically robust and simple way to derive skin cells within 4-8 weeks for banking of about 15-20 million cells at a low passage number.

Pele da biópsia Explant Cultura para Derivação de primárias fibroblastos humanos

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts ...

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson&#039;s Disease

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

A utilização de recursos primários fibroblastos humanos de Monitoramento Fenótipos mitocondriais no Campo da Doença de Parkinson

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

Experimental Human Pneumococcal Carriage

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major source of transmission and the prerequisite of invasive disease. A model of carriage will allow accurate determination of the immunological correlates of protection, the immunizing effect of carriage and the effect of host pressure on the pathogen in the nasopharyngeal niche. Further, methods of carriage detection useful in epidemiologic studies, including vaccine studies, can be compared.
Aim
We aim to develop an EHPC platform that is a safe and useful reproducible method that could be used to down-select candidate novel pneumococcal vaccines with prevention of carriage as a surrogate of vaccine induced immunity. It will work towards testing of candidate vaccines and descriptions of the mechanisms underlying EHPC and vaccine protection from carriage1. Current conjugate vaccines against pneumococcus protect children from invasive disease although new vaccines are urgently needed as the current vaccine does not confer optimal protection against non-bacteraemic pneumonia and there has been evidence of serotype replacement with non-vaccine serotypes2-4.
Method
We inoculate with S. pneumoniae suspended in 100 &mu;l of saline. Safety is a major factor in the development of the EHPC model and is achieved through intensive volunteer screening and monitoring. A safety committee consisting of clinicians and scientists that are independent from the study provides objective feedback on a weekly basis.
The bacterial inoculum is standardized and requires that no animal products are inoculated into volunteers (vegetable-based media and saline). The doses required for colonization (104-105) are much lower than those used in animal models (107)5. Detecting pneumococcal carriage is enhanced by a high volume (ideally &gt;10 ml) nasal wash that is relatively mucus free. This protocol will deal with the most important parts of the protocol in turn. These are (a) volunteer selection, (b) pneumococcal inoculum preparation, (c) inoculation, (d) follow-up and (e) carriage detection.
Results
Our current protocol has been safe in over 100 volunteers at a range of doses using two different bacterial serotypes6. A dose ranging study using S. pneumoniae 6B and 23F is currently being conducted to determine the optimal inoculation dose for 50% carriage. A predicted 50% rate of carriage will allow the EHPC model to have high sensitivity for vaccine efficacy with small study numbers.

Experimental human pneumococcal carriage offers a natural model of carriage and a potential model for use in vaccine development. This technique is valuable yet complex and involves clinical risk by introducing a pathogen into a human. We have developed a detailed protocol.

Transporte pneumocócica experimental humana

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

Lapso de tempo de imagem de pré-neoplásicas primárias células epiteliais mamárias derivados de modelos de rato geneticamente modificado do cancro da mama

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

Isolamento e caracterização funcional de Ventricular Humano cardiomiócitos a partir de amostras cirúrgicas frescos

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

Profiling Molecular do microambiente do tumor invasivo em um modelo 3-Dimensional de células de câncer colorretal e<em&gt; Ex vivo</em&gt; Os fibroblastos

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

Avaliação de metástases pulmonares em Rato mamárias tumorais Models por Quantitative PCR em tempo real

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one step treatments for acute and chronic skin lesions. Moreover, the management of chronic but also acute wounds resulting from trauma, diabetes, infections and other causes, remains challenging. In this study we describe a new method to create autologous micro-grafts from cutaneous tissue of a single patient and their clinical application. Moreover, in vitro biological characterization of cutaneous tissue derived from skin, de-epidermized dermis (Ded) and dermis of multi-organ and/or multi-tissue donors was also performed. All tissues were disaggregated by this new protocol, allowing us to obtain viable micro-grafts. In particular, we reported that this innovative protocol is able to create bio-complexes composed by autologous micro-grafts and collagen sponges ready to be applied on skin lesions. The clinical application of autologous bio-complexes on a leg lesion was also reported, showing an improvement of both re-epitalization process and softness of the lesion. Additionally, our in vitro model showed that cell viability after mechanical disaggregation with this system is maintained over time for up to seven (7) days of culture. We also observed, by flow cytometry analysis, that the pool of cells obtained from disaggregation is composed of several cell types, including mesenchymal stem cells, that exert a key role in the processes of tissue regeneration and repair, for their high regenerative potential. Finally, we demonstrated in vitro that this procedure maintains the sterility of micro-grafts when cultured in Agar dishes. In summary, we conclude that this new regenerative approach can be a promising tool for clinicians to obtain in one step viable, sterile and ready to use micro-grafts that can be applied alone or in combination with most common biological scaffolds.

The protocol describes a new method to disaggregate human tissues and to create autologous micro-grafts that, combined with collagen sponges, give rise to human bio-complexes ready to use in the treatment of skin lesions. Further, this system preserves cell viability of micro-grafts at different times after mechanical disaggregation.

Caracterização do tecido depois de um novo método de desagregação para a pele Micro-enxertos Generation

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one ...