Nós apreciamos profundamente Dr. Takeshi Ikeya e pessoal técnico em Toyo Gosei, Tokyo, Japão por fornecer placas de cultura de micropadronadas termossensíveis, bem como os pareceres científicos. Agradecemos também Ms. Satomi Ogura, Ms. Sae Suzuki, Ms. Asuka Miyoshi e Ms. Katsue Morii de assistência técnica com experiências em animais. Este trabalho foi apoiado financeiramente em parte pela JSPS KAKENHI Grant-in-Aid para a Investigação Científica, o Centro de Inovação Programa (COI) eo programa de inovação S- da Agência de Ciência e Tecnologia do Japão (JST), ea JSPS Core- para-Core Programa, A. avançadas redes de pesquisa.

Todos os estudos com animais foram realizados com a aprovação do Comitê de Cuidado e Uso Animal da Universidade de Tokyo, Tokyo, Japão. 1. Preparação celular <ol><li> Para hepatócitos primários, seguir o protocolo para o isolamento de hepatócitos de rato por uma de duas etapas processo de digestão de colagenase modificado 13,14. <ol><li> Anestesiar Sprague Dawley (SD) ratos (machos, 5 semanas de idade), sob anestesia inalatória com isoflurano. Colocar um rato numa câmara ligada a uma máquina de anestesia com isoflurano para proporcionar a câmara. Retire o rato depois de adormecer, e configurá-lo na mesa de operação com ventilação usando uma máscara. Controlar o fluxo de isoflurano a cerca de 0,4-0,7 L / min, verificando as condições do rato. Perfundir os fígados de ratos Sprague Dawley (SD) ratos (machos, 5 semanas de idade) a partir da veia da porta hepática, com uma solução especial composto de cloreto de sódio 8 g / L (NaCl), 400 mg / L de cloreto de potássio (KCl), 78 mg /EUdi-hidrato de di-hidrogenofosfato de sódio (NaH 2 PO 4 · 2H 2 O), 151 mg / L de fosfato dissódico dodeca-hidrogénio (Na 2 HPO 4 · 12H 2 O), 2,38 g / L de 2- [4- (2-hidroxietil) -1 piperazinil] etanossulfónico (HEPES), 190 mg / L de etileno glicol de ácido tetraacético (EGTA), 350 mg / L de carbonato de hidrogénio de sódio (NaHCO3), e 900 mg / L de glucose. </li><li> Circular solução de colagenase através do fígado. NOTA: A solução é composta de 500 mg / L de colagenase, 9,8 g / salina tamponada de L Hank, 2,38 g / ml de HEPES, 556 mg / hidrato de cloreto de cálcio ml (CaCl 2 H 2 O), 350 mg / L NaHCO3, e 50 mg / L de inibidor de tripsina de, com o pH ajustado para 7,2. </li><li> Remover cuidadosamente o fígado, e mediu-o suavemente em um prato usando uma lâmina de bisturi, adicionar Meio de Eagle Modificado por Dulbecco (DMEM) suplementado com 10% de soro fetal de bovino (FBS), e filtra-se a suspensão de células através de um 100 pmmalha de nylon. Para remover os detritos adicional, centrifugar a suspensão de células a 20 xg durante 1 min. Neste passo, os hepatócitos são no sobrenadante. </li><li> Repetir o passo de centrifugação, duas vezes (de um total de três centrifugações). Finalmente, centrifugar os hepatócitos a 50 x g durante 3 minutos para recuperá-los sob a forma de um pelete. </li><li> Re-suspender os hepatócitos a uma concentração de 4 x 10 5 culas / ml num meio de cultura especial, composto por DMEM suplementado com 10% FBS, 1% de Pen-Strep-Glut (QSP), 1% de dimetilsulfóxido (DMSO), 0,1 nmol / L dexametasona, 0,5 ug / ml de insulina, 10 mmol / L de nicotinamida, 0,2 mmol / L fosforilada ascorbato (Asc-2P), e 10 ng / ml de factor de crescimento epidérmico humano (hEGF) 15. Este meio especial é obrigatório para a preservação da função hepática em condições in vitro. </li></ol></li><li> Para a obtenção de MSCs de rato, eutanásia Sprague Dawley (SD) ratos (machos, 5 semanas de idade) por administração excessiva de isoflurane. Ressecar os fémures e as tíbias, e recolher as medulas ósseas através da inserção de uma agulha G 22, no eixo do osso para a nivelar-se com 10 ml de DMEM suplementado com FBS a 10%. Recolher as células por filtração através de uma malha de nylon de 100 um. </li><li> Semente as células sobre 10 cm pratos de cultura utilizando DMEM contendo 10% de FBS e 1% de penicilina / estreptomicina. Para as experiências de esferóides, utilizar as MSCs a 5 passagens. </li></ol> 2. Preparação de esferóides de células 3D <ol><li> Comercialmente obter placas de cultura de micropatterned, em que as zonas adesivas células são regularmente dispostas a 100 um de diâmetro, de forma bidimensional, rodeado por zonas não adesivas revestidas pela matriz de PEG. NOTA: Para o transplante de células, um revestimento adicional com o polímero termossensível PIPAAm é necessário para permitir a separação celular por as placas de arrefecimento (ver passo 5.1). As flutuações de temperatura pode induzir alterações na química deste polímero8-10. A 37 ° C, PIPAAm é ligeiramente hidrofóbica, permitindo que as células a ser cultivadas sob condições normais. Uma diminuição na temperatura abaixo de 32 ° C resulta em rápida hidratação do polímero, levando ao desprendimento espontâneo das células. </li><li> Semente os hepatócitos ou MSC em placas de 12 poços micropatterned a uma densidade de 4 x 10 5 células / poço andincubate-los a 37 ° C numa atmosfera humidificada contendo 5% de CO 2. As células irá acumular-se nas áreas de adesão micropatterned e gradualmente formar esferóides redondas em 2 dias. NOTA: Para a preparação de células de controlo numa cultura em monocamada, utilizar placas de 12 poços normais e semear as células a uma densidade idêntica, seguindo um procedimento semelhante ao descrito acima. </li></ol> 3. Preparação de Polyplex Nanomicelles <ol><li> Sintetizar um copolímero de bloco de PEG-PSAP (DET) (poli [N &#39;- [N - (2-aminoetil) -2-aminoetil] aspartamida]) (PEG Mw = 12,000, grau de polimerização (DP) do segmento PSAP (DET) = 59), e um homopolímero que é composta de apenas o segmento catiónico [PSAP (DET)] (DP = 55), seguindo os procedimentos anteriormente descritos pelos autores 11, 16, 17. </li><li> Prepara-se uma solução mista do (DET) copolímero em bloco PEG-PSAP e o PSAP (DET) homopolímero em uma proporção molar igual de grupos amino residuais, em HEPES 10 mM (pH 7.3) por ajuste da concentração de polímero de 33,3 ^ g / ml e 19,1 ug / ml, respectivamente. NOTA: As concentrações de polímero descritos acima são os valores representativos para a preparação de nanomicelles com uma razão molar residual do total de grupos amino nos dois polímeros para os grupos fosfato no (relação N / P) de ADNp de 10. A relação N / P pode variar dependendo do tipo de célula e da finalidade (para mais detalhes, ver discussão). A utilização combinada dos dois polímeros pode atingir tanto PEG blindagem eficaz e funcionamento do PSAP (DET) para melhorar endossomalescapar (para detalhes, ver Discussão) 18. </li><li> Prepara-se o ADNp que codifica a luciferase Gaussia, GL4 de luciferase, ou eritropoietina por clonagem do segmento de expressar os respectivos genes no plasmídeo pCAG-GS (http://www.cdb.riken.jp/pcs/protocol/vector/map/m36.html ) para se obter a expressão sob o CAG promotor / potenciador usando um kit comercial de acordo com o protocolo do fabricante. Amplificar o pDNA em uma cepa Escherichia coli competente e purificá-la usando um plasmídeo sistema de purificação de ADN livre de endotoxina. Determinar a concentração de ADNp a uma absorvância de 260 nm para se obter uma solução / ml 150 ug em tampão HEPES 10 mM (pH 7,3). </li><li> Para preparar as nanomicelles poliplex, homogeneiza-se a solução de ADNp (150 ug / ml em tampão HEPES 10 mM) e a solução pré-misturada dos dois polímeros numa proporção de 2: 1 (em volume). </li></ol> 4. Gene transfecção em esferóides <ol><li> Incubar as células (hepatócitosou MSC) durante 72 horas após a sementeira em placas micropatterned para permitir a formação de esferóides maduros. Para a transfecção de genes, adicionar 100 uL da solução Polyplex nanomicelle (contendo 10 ug de ADNp) a cada poço após a substituição do meio de cultura com 1 ml de meio fresco. Continuar a incubação com a solução nanomicelle durante 24 horas. </li><li> Para um controlo usando um reagente de transfecção à base de lípidos, misturar soluções pADN com o reagente em uma proporção em peso de reagente / pDNA de 3. Ajustar a dose final de pADN para ser igual, tanto para o reagente à base de lípidos e métodos nanomicelle. </li></ol> 5. Recuperação e Transplante de esferóides celulares <ol><li> Substituir o meio de cultura com 200 ul de PBS arrefecido e colocar as placas em gelo. NOTA: De um modo geral, os esferóides podem separar em cerca de 15 min e pode ser recuperada na forma de uma suspensão para o transplante. </li><li> Suavemente aspirar as células em 200 ul dosuspensão utilizando uma seringa com uma 23 G ou 27 G agulha para injeções in vivo. </li></ol> 6. Avaliação da Transgene Expression <ol><li> Para a avaliação in vitro da expressão de luciferase Gaussia segregada no meio de cultura, recolher 50 - 100 ul do meio de precisamente 24 horas após a substituição com meio fresco. Estimar a expressão da luciferase utilizando um sistema de ensaio de luciferase de Renilla comercial e um luminómetro de acordo com o protocolo do fabricante. NOTA: A luciferase Gaussia permanece estável no meio de cultura durante mais de uma semana. Assim, para avaliar a eficiência em tempo real da expressão do transgene, substituir o meio com um meio fresco antes da recolha do meio da amostra. O momento da mudança de meio pode ser flexível. </li><li> Para a avaliação in vivo de expressão do transgene em animais hospedeiros, após o transplante de células, anestesiar ratos BALB / c nu (fêmea; 7 semanasidade), sob anestesia inalatória com isoflurano. <ol><li> Colocar um rato numa câmara ligada a uma máquina de anestesia com isoflurano para proporcionar a câmara. Retire o mouse depois de adormecer, e configurá-lo na mesa de operação com ventilação usando uma máscara. Controlar o fluxo de isoflurano a cerca de 0,2-0,5 L / min, verificando as condições do mouse. </li><li> Injectar 200 ul da suspensão de células contendo esferóides transfectadas com os ADNp que expressam luciferase GL4 (como descrito em 4.1) para dentro do tecido subcutâneo da região abdominal. </li><li> Imediatamente após a injecção de D-luciferina (150 mg / kg; via intravenosa), medir a expressão de luciferase utilizando o sistema de imagiologia IVIS de acordo com o protocolo do fabricante. </li></ol></li><li> Para avaliar os efeitos terapêuticos do transplante de células, injectar 200 ul da suspensão de células contendo esferóides transfectadas com os ADNp que codifica para a eritropoietinatecido subcutâneo da região abdominal. <ol><li> Coletar amostras de sangue por sangramento submandibular obter cerca de 200 l de sangue 19. Medir a hemoglobina e hematócrito usando um analisador de amostra de sangue. NOTA: O número de células para transplante é regulada pelo número semeadas nas placas. Infelizmente, é difícil determinar o número de células exactos, porque o número dentro de esferóides não pode ser medido. </li></ol></li><li> Depois de experiências, os ratinhos colocar sobre uma almofada de aquecimento ligada a um controlador de temperatura até que o despertar da anestesia. </li></ol>

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The authors declare that they have no competing financial interests.

Neste protocolo, é fundamental a manutenção da estrutura 3D de esferóides durante os passos de introdução de gene e recuperação esferóide. É essencial manter uma microambientes favoráveis ​​para as células para evitar a morte ou perda de actividade de célula a célula. Por exemplo, a secreção de albumina, uma função representativa inato de hepatócitos, é bem preservada nos esferóides de hepatócitos, enquanto que os hepatócitos em cultura em monocamada convencionais perdem rapidamente a sua capa...

Terapia de transplante de células atraiu atenção generalizada para o tratamento de várias doenças incuráveis. A actividade e semi-vida de factores bioactivos, que são secretadas pelas células transplantadas são essenciais para a melhoria da eficácia terapêutica de um sistema de transplante de células. A modificação genética das células antes de transplante é uma técnica benéfica para regular e manipular as funções celulares, incluindo a secreção dos factores bioactivos. É também importante para manter um microambiente favorável para as células para evitar a morte ou perda de actividade de célula a célula. Cultura tridimensional (3D) esferóide célula, em que as interacções célula-para-célula são bem preservada, é promissor para este fim, por exemplo, para melhorar a secreção de albumina a partir de hepatócitos primários e promover a diferenciação de várias linhagens de células estaminais mesenquimais (MSCs ) 1-7. Neste estudo, um novo sistema de combinação de spheroid cultura e transfecção do gene é usado para servir como uma plataforma para o transplante de células modificadas geneticamente. Para a criação de células de esferóides, um sistema de cultura em placas de cultura esferóide micropatterned é usado. Nessas placas, áreas de adesão celular de 100 um de diâmetro são regularmente dispostas de forma bidimensional e estão rodeados por áreas não adesivas revestidas por uma matriz de PEG 3. Semeando um número adequado de células, esferóides de matrizes 3D de 100 um de diâmetro são formadas correspondente ao leito de cultura micropadronadas. Os esferóides são recuperados sem perturbar a sua estrutura 3D, utilizando placas de cultura celular termossensíveis, que foram revestidos com um polímero termossensível, poli (iso-propilacrilamida) (PIPAAm) 8-10. A arquitetura micropadronadas é construído sobre as placas termossensíveis (custom-built). Por simplesmente baixando a temperatura das placas, os esferóides são separadas do leito de cultura e dispersard em tampão fosfato salino (PBS). Assim, um grande número de esferóides com um tamanho uniforme de 100 mm pode ser obtido sob a forma de uma suspensão injectável. <img alt="Figura 1" p="" src="/files/ftp_upload/52384/52384fig1.jpg" /> Figura 1. Representação esquemática do sistema de cultura de esferóide sobre uma placa micropadronadas. A modificação genética é alcançado através de transfecção de genes utilizando o transportador do gene não virai original, nanomicelle Polyplex. É composto de plasmídeo de ADN (pADN) e polietileno glicol (PEG), copolímeros de bloco -polycation 11. Estes têm uma estrutura de núcleo-shell característica que consiste em um shell de PEG e um núcleo interno de pADN condensado, permitindo introdução gene segura e eficaz em células para fins terapêuticos 11. <a href="https://www.jove.com/files/ftp_upload/52384/52384fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior do thi</a>s figura. <img alt="Figura 2" src="/files/ftp_upload/52384/52384fig2.jpg" /> Figura 2. Estrutura do nanomicelle Polyplex formado pelo complexo de ácidos nucleicos e copolímeros de bloco de PEG-bloco-policatião. Neste estudo, a principal vantagem desta técnica é que a estrutura de esferóides não é interrompido durante a transfecção de genes pelos nanomicelles. Após as transfecções mediada por nanomicelle de esferóides de hepatócitos de rato primária, a expressão do transgene prolongada é obtida por mais de um mês, com a secreção de albumina a partir dos hepatócitos contínua a um nível comparável ao de esferóides não transfectadas 12. A expressão do transgene e a secreção de albumina a partir dos esferóides também são mantidas após recuperação a partir das placas termossensíveis. É evidente que nanomicelles pode facilitar a introdução do gene de forma segura, sem prejudicar as funções inatas do hepatocytes. Assim, a combinação de células cultivadas em placas de esferóides micropatterned termossensíveis com introdução de genes utilizando nanomicelles é uma plataforma promissora para o transplante de células geneticamente modificadas. <a href="https://www.jove.com/files/ftp_upload/52384/52384fig2large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a>

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Pen-Strep-Glut</td>
			<td>GIBCO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Wako Pure Chemical Industries</td>
			<td>041-18861</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Wako Pure Chemical Industries</td>
			<td>141-01202</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#8217;s buffered salt and L-ascorbic acid 2-phosphate (Asc-2P)</td>
			<td>Sigma-Aldrich</td>
			<td>A8960</td>
			<td></td>
		</tr>
		<tr>
			<td>Human epidermal growth factor (hEGF)</td>
			<td>Toyobo</td>
			<td>PT10015</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell-able multi-well plates</td>
			<td>Toyo Gosei</td>
			<td>PP-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermosensitive cell culture plates (Upcell)</td>
			<td>CellSeed Inc</td>
			<td></td>
			<td>The micropatterned architecture is constructed on the thermosensitive plates (custom-built by Toyo Gosei)</td>
		</tr>
		<tr>
			<td>Lipid-based transfection reagent (FuGENE HD)</td>
			<td>Promega</td>
			<td>E2311</td>
			<td></td>
		</tr>
		<tr>
			<td>Renilla Luciferase Assay System</td>
			<td>Promega</td>
			<td>E2810</td>
			<td></td>
		</tr>
		<tr>
			<td>pGL4 Luciferase Reporter Vector</td>
			<td>Promega</td>
			<td>E6651</td>
			<td></td>
		</tr>
		<tr>
			<td>pDNA expressing Gaussia luciferase</td>
			<td>New England BioLabs</td>
			<td>N8082S</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse erhthropoietin-expressing vector</td>
			<td>Origene</td>
			<td>MC208445</td>
			<td></td>
		</tr>
		<tr>
			<td>pCAG-GS</td>
			<td></td>
			<td></td>
			<td>Kindly provided by Laboratory for Pluripotent Cell Studies, Center for Developmental Biology, RIKEN</td>
		</tr>
		<tr>
			<td>Escherichia coli DH5&#945; competent cells</td>
			<td>Takara</td>
			<td>9057</td>
			<td></td>
		</tr>
		<tr>
			<td>Endotoxin-free plasmid DNA purification system</td>
			<td>Nippon Genetics</td>
			<td>NucleoBond Xtra EF</td>
			<td></td>
		</tr>
		<tr>
			<td>collagenase</td>
			<td>Wako Pure Chemical Industries</td>
			<td>639-00951</td>
			<td></td>
		</tr>
		<tr>
			<td>trypsin inhibitor</td>
			<td>GIBCO</td>
			<td>R-007-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminometer</td>
			<td>Promega</td>
			<td>GloMax&#8482; 96 Microplate Luminometer</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Imaging System</td>
			<td>Xenogen Corp.</td>
			<td>Xenogen IVIS Spectrum in vivo imaging system</td>
			<td></td>
		</tr>
		<tr>
			<td>blood sample analyzer</td>
			<td>Sysmex</td>
			<td>pocH-100i Automated Hematology Analyzer</td>
			<td></td>
		</tr>
	</tbody>
</table>

gene transfection toward spheroid cells on micropatterned culture plates for genetically-modified cell transplantation

Para melhorar a eficiência terapêutica do transplante de células, um sistema de transplante de geneticamente modificados, esferóides injectáveis ​​foi desenvolvido. Os esferóides de células são preparadas num sistema de cultura em placas de micropatterned revestidos com um polímero termossensível. Uma série de esferóides são formados nas placas, correspondentes às áreas de adesão celular de 100 um de diâmetro que são regularmente dispostas de uma forma bi-dimensional, rodeado por zonas não adesivas que são revestidos por um polietileno-glicol (PEG) de matriz. Os esferóides podem ser facilmente recuperado sob a forma de uma suspensão líquida, baixando a temperatura das placas, e a sua estrutura é bem conservado, passando-os através de agulhas de injecção com um calibre suficientemente grande (mais de 27 L). A modificação genética é alcançado através de transfecção de genes utilizando o suporte original não virai de genes, nanomicelle Polyplex, que é capaz de introduzir genes em células, sem perturbar a estrutura esferóide. Para primesferóides de hepatócitos ary transfectadas com um gene que expressa luciferase, a luciferase é sustentada obtido em animais transplantados, juntamente com a função de hepatócitos preservada, tal como indicado pela expressão de albumina. Este sistema pode ser aplicado a uma variedade de tipos de células, incluindo células estaminais mesenquimais.

Gene transfecção do Gaussia que expressam a luciferase de ADNp foi realizada nos esferóides formadas pelos hepatócitos ou MSC, utilizando nanomicelles poliplex ou o reagente de transfecção baseada em lípidos de controlo 12. Os nanomicelles induzida quase nenhuma alteração na estrutura esferóide comparação com esferóides não transfectadas nas placas micropatterned, ao passo que o reagente de controlo perturbado significativamente a estrutura de um dia após a transfecção (Fig...

Watch this Scientific Journal Video about Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation at JoVE.com

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

A transfecção Gene direção Células Spheroid em placas micropadronadas Cultura para Transplante de Células geneticamente modificados

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

gene-transfection-toward-spheroid-cells-on-micropatterned-culture

Research

JoVE Journal

Bioengineering

8.7K visualizações.  The University of Tokyo. This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

Vídeo: Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

Superfícies micropatterned para estudar as interações ácido hialurônico com células de câncer

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

Bioengenharia

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck 1-2.
To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.

Visualização melhorada e Análise Quantitativa dos Efeitos de Drogas Usando Células micropatterned

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

Fabricação de hidrogéis micropatterned para Sistemas de Cultura Neural usando fotolitografia Projeção Dinâmica Máscara

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

Sistemas Célula de rotação de cultura para cultura de células humanas: células trofoblásticas Humanos como um modelo

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

Citometria de massa: Protocolo para Ajuste Diário e amostras de células em execução em um citômetro de Massa CyTOF

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Transplante de Progenitores Miogênicos derivados de células-tronco pluripotentes induzidos em modelos de regeneração muscular

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Stretching Micropatterned Cells on a PDMS Membrane

Mechanical forces exerted on cells and/or tissues play a major role in numerous processes. We have developed a device to stretch cells plated on a PolyDiMethylSiloxane (PDMS) membrane, compatible with imaging. This technique is reproducible and versatile. The PDMS membrane can be micropatterned in order to confine cells or tissues to a specific geometry. The first step is to print micropatterns onto the PDMS membrane with a deep UV technique. The PDMS membrane is then mounted on a mechanical stretcher. A chamber is bound on top of the membrane with biocompatible grease to allow gliding during the stretch. The cells are seeded and allowed to spread for several hours on the micropatterns. The sample can be stretched and unstretched multiple times with the use of a micrometric screw. It takes less than a minute to apply the stretch to its full extent (around 30%). The technique presented here does not include a motorized device, which is necessary for applying repeated stretch cycles quickly and/or computer controlled stretching, but this can be implemented. Stretching of cells or tissue can be of interest for questions related to cell forces, cell response to mechanical stress or tissue morphogenesis. This video presentation will show how to avoid typical problems that might arise when doing this type of seemingly simple experiment.

This manuscript presents a technique to apply or release forces on adherent cells or tissues using unidirectional stretching.

Alongamento Células micropatterned num PDMS Membrana

Mechanical forces exerted on cells and/or tissues  play a major role in numerous processes. We have developed a device to stretch  cells plated on a ...

Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular behaviors on disease-relevant time scales. Previously, microcontact printing has been used to fabricate two-dimensional functional arterial mimics through patterning of extracellular matrix protein as guidance cues for tissue organization. Vascular muscular thin films utilized these mimics to assess functional contractility. However, the microcontact printing fabrication technique used typically incorporates hydrophobic PDMS substrates. As the tissue turns over the underlying extracellular matrix, new proteins must undergo a conformational change or denaturing in order to expose hydrophobic amino acid residues to the hydrophobic PDMS surfaces for attachment, resulting in altered matrix protein bioactivity, delamination, and death of the tissues.
Here, we present a microfluidic deposition technique for patterning of the crosslinker compound genipin. Genipin serves as an intermediary between patterned tissues and PDMS substrates, allowing cells to deposit newly-synthesized extracellular matrix protein onto a more hydrophilic surface and remain attached to the PDMS substrates. We also show that extracellular matrix proteins can be patterned directly onto deposited genipin, allowing dictation of engineered tissue structure. Tissues fabricated with this technique show high fidelity in both structural alignment and contractile function of vascular smooth muscle tissue in a vascular muscular thin film model. This technique can be extended using other cell types and provides the framework for future study of chronic tissue- and organ-level functionality.

We present a method for microfluidic deposition of patterned genipin and fibronectin on PDMS substrates, allowing extended viability of vascular smooth muscle cell-dense tissues. This tissue fabrication method is combined with previous vascular muscular thin film technology to measure vascular contractility over disease-relevant time courses.

Técnica microfluídico genipina Deposição de Cultura Extensão da micropatterned vasculares musculares Filmes Finos

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular ...

Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and multidirectional shear stresses have all been postulated to stimulate a pro-atherosclerotic phenotype in endothelial cells, whereas high magnitude and unidirectional or uniaxial shear are thought to promote endothelial homeostasis. These hypotheses require further investigation, but traditional in vitro techniques have limitations, and are particularly poor at imposing multidirectional shear stresses on cells. 
One method that is gaining increasing use is to culture endothelial cells in standard multi-well plates on the platform of an orbital shaker; in this simple, low-cost, high-throughput and chronic method, the swirling medium produces different patterns and magnitudes of shear, including multidirectional shear, in different parts of the well. However, it has a significant limitation: cells in one region, exposed to one type of flow, may release mediators into the medium that affect cells in other parts of the well, exposed to different flows, hence distorting the apparent relation between flow and phenotype. 
Here we present an easy and affordable modification of the method that allows cells to be exposed only to specific shear stress characteristics. Cell seeding is restricted to a defined region of the well by coating the region of interest with fibronectin, followed by passivation using passivating solution. Subsequently, the plates can be swirled on the shaker, resulting in exposure of cells to well-defined shear profiles such as low magnitude multidirectional shear or high magnitude uniaxial shear, depending on their location. As before, the use of standard cell-culture plasticware allows straightforward further analysis of the cells. The modification has already allowed the demonstration of soluble mediators, released from endothelium under defined shear stress characteristics, that affect cells located elsewhere in the well.

This protocol describes a coating method to restrict endothelial cell growth to a specific region of a 6-well plate for shear stress application using the orbital shaker model.

Segmentando o crescimento de células endoteliais em placas de 6-bem em um agitador orbital para estudos mecanobiológicos

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

Um método cultural para manter células-tronco hematopoiéticas humanas quiescentes

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...