Os autores gostariam de agradecer o financiamento parcial do Projeto de Pesquisa FP7 NAMDIATREAM Europeia (NMP4-LA-2010-246479, http://www.namdiatream.eu) eo projecto INTERREG IV França-Suíça NAOMI.

1. Cultura e Expansão da ESC Humano <ol><li> Preparar o meio de propagação da célula (chamado PM) contendo Knockout DMEM suplementado com 20% de Soro KO, 1% MEM não-aminoácidos essenciais, 1% de L-glutamina 200 mM, 1% de penicilina-estreptomicina, e 3,5 ul β-mercaptoetanol. </li><li> Descongele ESC humana (hESC) em 8 ml de meio PM e centrifugar para 5 min a 115 xg para descartar suplementados com DMSO meio de congelamento. </li><li> Adicione 1 ml de meio PM contendo 10 inibidor Rocha mM para evitar a apoptose e melhorar a sobrevivência das células após a descongelação (apenas 24 incubação h). </li><li> Adicionar o factor de crescimento de fibroblastos básico (bFGF), para a AM para a concentração desejada (4-10 ng / ml) para a manutenção da pluripotência. </li><li> Semear a células estaminais embrionárias humanas no prepúcio humano irradiado células alimentadoras (γHFF) (Figura 1A), anteriormente colocadas em placas a uma concentração de 2 x 10 4 células / cm 2. </li><li> Mude média diária e, quando necessário, remover partes colonies começando a diferenciar por aspiração com uma pipeta de Pasteur acentuadamente alongada cortada. </li><li> Uma vez por semana, colônias passagem enzimaticamente: <ol><li> Remover o meio e lava-se as colónias, com 2 ml de PBS. </li><li> Remover PBS e incubar com 0,5 ml por poço de quente colagenase IV a 1 mg / ml durante 10 min a 37 ° C, 5% de CO 2. </li><li> Coletar fragmentos de colônias em 2 ml novo PM e centrifugar a 115 xg por 3 min. </li></ol></li><li> Alternativamente, as colónias podem ser passadas através de raspagem mecânica: <ol><li> Remover o meio e lava-se as colónias, com 2 ml de PBS. </li><li> Raspar manualmente colónias usando a ferramenta rolo raspador StemPro EZPassage (Figura 1B). </li><li> Recolher fragmentos de colónias e depois centrifugar durante 3 minutos a 115 xg para remover o sobrenadante. </li><li> Fragmentos de placa em γHFF ou processá-los para a diferenciação em corpos embrióides (EBS). </li></ol></li></ol> 2. Diferenciação ESC HumanoProtocolo <ol><li> Preparar o meio de diferenciação (MS) usando Knockout DMEM suplementado com 2% de soro de Hyclone, 1,0% MEM não-aminoácidos essenciais, 1,0% de L-glutamina 200 mM, 1% de penicilina-estreptomicina, 3,5 ul β-mercaptoetanol. </li><li> Remover o meio e lava-se as colónias, com 2 ml de PBS. </li><li> EB formação de fragmentos de colônias em suspensão: <ol><li> Remover PBS e incubar com 0,5 ml por poço de quente colagenase IV a 1 mg / ml durante 10 min a 37 ° C, 5% de CO 2. </li><li> Coletar fragmentos de colônias em 2 ml novo DM e centrifugar a 115 xg por 3 min. </li><li> Cultura los em suspensão em ultra-baixo de fixação placas de 6 poços (2 ml por poço) durante 4 dias (Figura 1C). </li><li> Recolha e placa os CEs recém formados em 0,1% revestidas com gelatina de 24 poços, placas (cerca de 3 EB / poço) (Figura 1D). </li></ol></li><li> Alternativamente, a agregação de células individuais forçado a EBS usando a placa Aggrewell: <ol><li> Remover PBS edissociar colónias em células individuais utilizando Accutase (0,5 ml / poço). </li><li> Centrifugar células e ressuspender em DM em 1.2 x 10 6 / ml. </li><li> Adicionar 2 ml por Aggrewell câmara durante 1 dia (Figura 1B). </li><li> Recolhe CEs recém-formados (Fig. 1C &#39;) e placa-los em 0,1% revestidos com gelatina placas de 24 poços (cerca de 3 EB / poço). </li><li> Mude DM a cada 2-3 dias até o aparecimento de bater aglomerados de cardiomiócitos (15-30 dias). </li></ol></li></ol> 3. Ar líquidos culturas 3D de batendo Clusters e Rotulagem com HNPs <ol><li> Identificar e manualmente dissecar aglomerados de batendo cardiomiócitos (Figura 1D), normalmente aparecem após 2-4 semanas de cultura, utilizando uma pipeta Pasteur alongada cortou drasticamente. </li><li> Depósito 4 filtros de PTFE por pastilha, que serão colocados em uma placa de 6 poços (Figura 1F). </li><li> Adicione 1 ml de meio de DM em cima de each bem. </li><li> Adicionar um grupo dissecado batendo em cada filtro de PTFE (Figura 1E), máxima 4/well, ou seja, 24 agregados por placa (Figura 1F). </li><li> Mude médio DM cada 2-3 dias. </li><li> Para identificar os clusters, retire o filtro PTFE contendo o cluster surra e colocá-lo em 3,5 centímetros prato de vidro de fundo (170 mm de espessura). </li><li> Adicionar 1 ml de PNH a uma concentração de 50 ug / ml durante 30 min. </li></ol> 4. Imagem óptico não-linear <ol><li> A preparação das amostras. Após a marcação PNH (ver passo 3.6), transferir ou inteiro diferenciadores HeBS início ou clusters batimento cardíaco (dissecados na aparência contração) em filtros de PTFE sobre um prato de 170 mm de espessura com fundo de vidro compatível com a distância de trabalho de altos objetivos abertura numérica. Como alternativa, aplique uma camada de bater direto clusters para o prato fundo de vidro pré-revestido com 0,1% de gelatina. </li><li> Amostras de transferência para umatudo-em-um microscópio equipado com câmara de incubação garantindo temperatura estável, umidade e CO 2 controle ao longo de períodos de tempo prolongados. </li><li> Após a inspeção inicial, a imagem da amostra através de imagens de campo amplo com iluminação de luz branca para identificar as estruturas de interesse dentro EBs diferenciados ou clusters batendo ativos que serão selecionados para posteriores investigações. </li><li> Se a autofluorescência das células a partir de NADH e SH de HNPs tem que ser adquirido simultaneamente, defina um menor comprimento de onda de excitação de laser (isto é, 720 nm) para coincidir com a absorção molecular. Use um filtro passa-banda espectral estreita centrada na metade do comprimento de onda de excitação (ou seja, λ = 360 ± 10 nm) para detectar SH, e outro para detectar autofluorescência. </li><li> Em primeiro lugar fazer uma, de baixa resolução rápida, digitalização tridimensional para reconstruir a morfologia geral do cluster cardíaca e selecionar um plano de imagem dentro do volume de cluster com vários PNH visívels. </li><li> Se necessário, altere o comprimento de onda de excitação livremente para melhor corresponder propriedades ópticas da amostra ou para evitar a fotodegradação, danos foto e imagem mais profunda nos tecidos, com um comprimento de onda IR (ou seja, 790 nm) e substituir o segundo filtro de harmônicas em conformidade (ou seja, 395 nm ). Otimizar as configurações do pulso de laser pré-compressor no comprimento de onda de trabalho de escolha, maximizando o segundo sinal harmônico de HNPs depositados na amostra. </li><li> Para acompanhar em tempo real as contrações cardíacas de cluster, configure o scanner óptico do microscópio no modo mais rápido, como o modo de ressonância, permitindo a aquisição de alta velocidade de imagens bidimensionais. </li><li> Escolha as configurações como melhor compromisso entre o contraste de imagem e velocidade de aquisição, tais como: <ul type="disc"><li> Média de digitalização: 4 </li><li> Pixel tempo de permanência: 0,1 ms </li><li> Taxa Image: 3,75 frames por segundo (fps) </li></ul></li><li> Poder do laser é ajustada de acordo com focal local size e velocidade de varredura. 50 mW (medido na entrada da objectiva) é um valor típico de 0,1 ms de pixel do tempo de permanência e Plano APO 20X NA 0,75 objectivo preservando a viabilidade celular. </li></ol> 5. Image Analysis <ol><li> Procedimento de calibração Axial. Selecione uma única PNH depositados sobre um substrato nu. Ajustar a focagem (isto é, a posição axial da nanopartícula) para maximizar a intensidade SH. Variar de uma forma controlada a posição axial do estágio de microscópio para obter a curva de calibração relativa intensidade SH como uma função da PNH deslocada do plano focal. O resultado deste procedimento de calibração para o objectivo 0,75 Plano APO 20X NA está relatado na Figura 4C. </li><li> Selecione HNPs presentes em todos os quadros de vídeo e determinar seus movimentos periódicos. Este procedimento pode ser facilmente automatizado, por exemplo, um código personalizado em busca de pontos de sinal máxima intensidade em uma região definida de interesse econectá-los entre quadros diferentes (um código de exemplo escrito em Matlab R2009 b usando a função &#39;adereços região da caixa de ferramentas de processamento de imagem é apresentada). </li><li> Avaliar a quantificação dos deslocamentos fora do plano, associadas HNPs não rastreáveis ​​continuamente em movimento ao longo do eixo óptico, através da conversão de suas modulações de intensidade ao longo de quadros diferentes para o deslocamento axial utilizando a curva de calibração estabelecida no Passo 5.1. Note-se que este procedimento dá acesso aos movimentos axiais, mas não pode ser utilizada para determinar a orientação absoluta (para cima ou para baixo). </li></ol>

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A aplicação da nanotecnologia à investigação em células estaminais é um relativamente novo, mas em rápida expansão campo. Como foi assinalado por vários artigos de revisão sobre o assunto, o uso de nanopartículas pode ser aplicada para realizar tarefas diferentes de pesquisa, variando a partir de células de monitoramento (tanto in vitro como in vivo), para a distribuição intracelular de proteínas e genes, não menos importante, a criação de ambientes celulares biomiméticos para prefe...

Sistemas de microscopia não-lineares, graças às suas inerentes três capacidades de seccionamento dimensionais, têm cada vez desencadeada a demanda por fluoróforos foto-estável, com bandas de absorção de dois fótons do infravermelho próximo. Só no último par de anos, para complementar o desenvolvimento de rótulos baseados em fluorescência (corantes, pontos quânticos, a converter nanopartículas), uma metodologia diferente de imagem vem explorando o uso de uma nova família de nanopartículas inerentemente não-lineares como rótulos, ou seja, nanopartículas de harmônicas (HNPs) que foram desenvolvidos especificamente para microscopia multi-fotão. Esses rótulos, baseadas em cristais inorgânicos, noncentrosymmetric exercer contraste óptico gerando o SH da freqüência de excitação: por exemplo, através da conversão de uma fração de perto a luz de excitação pulsada infravermelho (λ = 800 nm) para a luz azul visível (λ / 2 = 400 nm) . Vários autores no passado recente têm testado diferentes materiais, incluindo iodato de ferro Fe (IO <sub&gt; 3) 3 1, niobato de potássio (KNbO 3) 2, de niobato de lítio (LiNbO 3) 3, o titanato de bário (BaTiO3) 4,5, fosfato de potássio de titanilo (KTiOPO4, KTP) 6-8, e o óxido de zinco (ZnO ) 5,9,10. Em comparação com sondas fluorescentes, HNPs possuem uma série de propriedades interessantes, como ausência completa de branqueamento e piscando, bandas de emissão estreitas, tunability excitação-comprimento de onda (do ultravioleta ao infravermelho), a capacidade de recuperação de orientação e resposta óptica coerente. Estas propriedades únicas foram recentemente explicou em dois artigos de revisão abrangente 11,12. A possibilidade de trabalhar na região espectral do infravermelho, o que aumenta a profundidade da imagem, minimizando a dispersão e absorção, também limita drasticamente a degradação foto da amostra 13,14. Além disso, o sinal de foto-estável infinitamente garantida pelo HNPs torna sondas ideais para rastreamento de células de longo prazo, que ié particularmente atraente para aplicações de medicina regenerativa 15. Neste experimento visualizados, detalhes do protocolo são fornecidos para marcação in vitro de células-tronco embrionárias humanas (hESC) com HNPs não funcionalizados. A síntese e preparação de suspensões coloidais é detalhado em publicação anterior e em referências citadas 16 e está fora do escopo deste trabalho. Metodologias de investigação hESC por microscopia multi-fotão e sua diferenciação em clusters cardíacos (mantidos como culturas de ar líquido longo prazo) são apresentados. CES humana pode ser deixado para diferenciar dentro chamados corpos embrióides (EBS) de duas maneiras diferentes, ou por formação de EB fragmentos de colónias em suspensão ou, em alternativa, a agregação de células individuais em EB utilizando a placa Aggrewell forçados, como ilustrado na Figura 1A . Cultivar batendo aglomerados de células cardíacas com politetrafluoretileno (PTFE) filtros porosos facilitates sua manutenção a longo prazo para estudos posteriores (por exemplo medidas eletrofisiológicas de potenciais de ação). A fonte de excitação do microscópio de varrimento deve ser capaz de fornecer impulsos de ultrashort (com uma duração de impulso mais pequena do que 300 FSEC na amostra), a fim de atingir a potência de pico necessária para realizar segunda imagem harmónica de HNPs. Por exemplo, o FSEC-fonte mais comum usado para geração de imagens são ajustáveis ​​Ti: Safira lasers. Alternativamente, outros lasers ultra-rápidos podem ser empregados, por exemplo, de íons de érbio 17, cromo forsterite 18 ou Ti: Safira bombeado infravermelhos osciladores paramétricos ópticos. O microscópio pode ser equipado com uma objectiva com uma preferência relativamente elevada abertura numérica. Muito importante, antes de medições, e cada vez que o objetivo é substituído, é obrigatório para minimizar a dispersão presente no set-up (lentes), otimizando as configurações do pulso de laser pré-compressor no trabalhocomprimento de onda de escolha. Este procedimento, detalhado no protocolo, garante que o impulso de laser, é o mais próximo possível para o transformar duração limitada (ou seja, mais curto quanto possível) no plano focal e maximiza a resposta da amostra não-linear. O objectivo da análise de imagem descrito no final do protocolo é a identificar e controlar os movimentos em 3D HNPs associados com as contracções rítmicas do bater aglomerados cardíacas. Nanopartículas de rastreamento no plano de imagem é simplesmente realizado através da identificação de suas posições em quadros do filme sucessivos. Para extrair a informação sobre o movimento axial, uma calibração prévia da resposta não linear da intensidade como uma função do deslocamento axial é obrigatório. Note-se que para a medição a longo prazo, um controlo activo interferométrico da posição axial da amostra é requerida para manter a validade da curva de calibração, na presença de desvios térmicos e / ou mecânicos. Os HNPs utilizados aqui para traccélulas e batendo nos agregados são baseados em óxido de potássio niobato (KNbO 3), mas outros nanomateriais não lineares disponíveis são analisados ​​em detalhe no trabalho de Staedler et al 16. As eficiências ópticos não-lineares da maioria dos nanomateriais investigados até agora são muito comparáveis. A escolha para KNbO 3 foi motivado essencialmente pela boa estabilidade da solução coloidal e sua boa biocompatibilidade, testado em várias linhas de células humanas, mesmo em concentração razoavelmente elevada e longos tempos de exposição 16. Dada a novidade do nanomaterial empregada para este trabalho, as principais características do HNPs em comparação com / luminescentes bio-marcadores fluorescentes são mostrados em uma animação de vídeo original do computador curta realizado pelos autores.

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harmonic nanoparticles for regenerative research

Neste experimento visualizados, detalhes do protocolo são fornecidos para marcação in vitro de células-tronco embrionárias humanas (hESC) com nanopartículas de geração harmônica segundo (HNPs). Estes últimos são uma nova família de sondas introduzidas recentemente para a rotulagem de amostras biológicas para imagens multi-fotão. HNPs são capazes de dobrar a freqüência de luz de excitação pelo processo de óptica não-linear de geração de segundo harmônico, sem restrição do comprimento de onda de excitação. Multi-fóton metodologias para hESC diferenciação em clusters cardíacos (mantidos como culturas de ar líquido longo prazo) com base são apresentados em detalhe. Em particular, as evidências sobre como maximizar a intensa segundo harmônico (SH) emissão de HNPs isoladas durante o monitoramento 3D de bater o tecido cardíaco em 3D é exibido. A análise das imagens resultantes para obter padrões de deslocamento 3D também é detalhado.

Antes de avaliar a actividade de batimento de imagiologia confocal, uma caracterização cuidadosa da resposta óptica não-linear dos filtros de PTFE foi realizada, quer isoladamente ou na presença de HNPs em concentração elevada (1 mg / ml). Assegurou-se que: i) o substrato utilizando dois fotões fluorescência animado é muito fraca e não pode impedir a medição das amostras biológicas relevantes, e ii) a emissão SH de HNPs isolados podem ser facilmente adquiridos por imagiologia através do substrato no modo...

Watch this Scientific Journal Video about Harmonic Nanoparticles for Regenerative Research at JoVE.com

Harmonic Nanoparticles for Regenerative Research

Detalhes do protocolo estão previstas in vitro de células-tronco embrionárias humanas em matéria de rotulagem com a segunda geração de nanopartículas harmônicas. Também são apresentadas as metodologias de investigação hESC por microscopia multi-fotão e sua diferenciação em aglomerados cardíacas.

Harmonic Nanopartículas Regenerativa Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

harmonic-nanoparticles-for-regenerative-research

Research

JoVE Journal

Bioengineering

11.2K visualizações.  University of Geneva.  Detalhes do protocolo estão previstas in vitro de células-tronco embrionárias humanas em matéria de rotulagem com a segunda geração de nanopartículas harmônicas. Também são apresentadas as metodologias de investigação hESC por microscopia multi-fotão e sua diferenciação em aglomerados cardíacas. 

Vídeo: Harmonic Nanoparticles for Regenerative Research

Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively for tissue regeneration, a number of variables must be taken into account. These variables include: the total volume and surface area of the implantation site, the mechanical properties of the tissue and the tissue microenvironment, which includes the amount of vascularization and the components of the extracellular matrix. Therefore, the materials being used to deliver these cells must be biocompatible with a defined chemical composition while maintaining a mechanical strength that mimics the host tissue. These materials must also be permeable to oxygen and nutrients to provide a favorable microenvironment for cells to attach and proliferate. Chitosan, a cationic polysaccharide with excellent biocompatibility, can be easily chemically modified and has a high affinity to bind with in vivo macromolecules4-5. Chitosan mimics the glycosaminoglycan portion of the extracellular matrix, enabling it to function as a substrate for cell adhesion, migration and proliferation. In this study we utilize chitosan in the form of microspheres to deliver adipose-derived stem cells (ASC) into a collagen based three-dimensional scaffold6. An ideal cell-to-microsphere ratio was determined with respect to incubation time and cell density to achieve maximum number of cells that could be loaded. Once ASC are seeded onto the chitosan microspheres (CSM), they are embedded in a collagen scaffold and can be maintained in culture for extended periods. In summary, this study provides a method to precisely deliver stem cells within a three dimensional biomaterial scaffold.

A major hurdle in current stem cell therapies is determining the most effective method to deliver these cells to host tissues. Here, we describe a chitosan-based delivery method that is efficient and simple in approach, while allowing adipose-derived stem cells to maintain their multipotency.

Construindo um hidrogel de colágeno para a entrega de células-tronco carregado microesferas

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively ...

Bioengenharia

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

Nanopartículas virais para<em&gt; Em vivo</em&gt; Imaging Tumor

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, complementary imaging techniques. These imaging agents facilitate the real-time assessment of pathways and mechanisms in vivo, which enhance both diagnostic and therapeutic efficacy. This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) - a novel class of agents for use in multimodal, molecular imaging applications. The imaging modalities incorporated in the nanoparticles, fluorescence imaging and magnetic resonance imaging (MRI), have complementary features. The PB NPs possess a core-shell design where gadolinium and manganese ions incorporated within the interstitial spaces of the PB lattice generate MRI contrast, both in T1 and T2-weighted sequences. The PB NPs are coated with fluorescent avidin using electrostatic self-assembly, which enables fluorescence imaging. The avidin-coated nanoparticles are modified with biotinylated ligands that confer molecular targeting capabilities to the nanoparticles. The stability and toxicity of the nanoparticles are measured, as well as their MRI relaxivities. The multimodal, molecular imaging capabilities of these biofunctionalized PB NPs are then demonstrated by using them for fluorescence imaging and molecular MRI in vitro.

This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting.

Bioativa de Prussian Blue Nanopartículas para Multimodal Imagem Molecular Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

Nanopartículas Lipídicas Sólidas (NLS) para intracelular Segmentação Aplicações

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron microscopy.

Correlativa luz e de Microscopia Eletrônica Usando Quantum Dot Nanopartículas

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using ...

Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

The only definitive therapy for end stage heart failure is orthotopic heart transplantation. Each year, it is estimated that more than 100,000 donor hearts are needed for cardiac transplantation procedures in the United States1-2. Due to the limited numbers of donors, only approximately 2,400 transplants are performed each year in the U.S.2. Numerous approaches, from cell therapy studies to implantation of mechanical assist devices, have been undertaken, either alone or in combination, in an attempt to coax the heart to repair itself or to rest the failing heart3. In spite of these efforts, ventricular assist devices are still largely used for the purpose of bridging to transplantation and the utility of cell therapies, while they hold some curative promise, is still limited to clinical trials. Additionally, direct xenotransplantation has been attempted but success has been limited due to immune rejection. Clearly, another strategy is required to produce additional organs for transplantation and, ideally, these organs would be autologous so as to avoid the complications associated with rejection and lifetime immunosuppression. Decellularization is a process of removing resident cells from tissues to expose the native extracellular matrix (ECM) or scaffold. Perfusion decellularization offers complete preservation of the three dimensional structure of the tissue, while leaving the bulk of the mechanical properties of the tissue intact4. These scaffolds can be utilized for repopulation with healthy cells to generate research models and, possibly, much needed organs for transplantation. We have exposed the scaffolds from neonatal mice (P3), known to retain remarkable cardiac regenerative capabilities,5-8 to detergent mediated decellularization and we repopulated these scaffolds with murine cardiac cells. These studies support the feasibility of engineering a neonatal heart construct. They further allow for the investigation as to whether the ECM of early postnatal hearts may harbor cues that will result in improved recellularization strategies.

In these studies, we provide methodology for novel, neonatal, murine cardiac scaffolds for use in regenerative studies.

Neonatal Cardiac Scaffolds: Matrizes novas para Estudos Regenerativa

The only definitive therapy for end stage heart failure is orthotopic heart transplantation.  Each year, it is estimated that more than 100,000 donor ...

Production of Metal Nanoparticles by Pulsed Laser-ablation in Liquids: A Tool for Studying the Antibacterial Properties of Nanoparticles

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of medicine. In addition to efforts to identify novel small-molecule antimicrobial drugs, there has been great interest in the use of metal nanoparticles as coatings for medical devices, wound dressings, and consumer packaging, due to their antimicrobial properties. The wide variety of methods available for nanoparticle synthesis results in a broad spectrum of chemical and physical properties which can affect antibacterial efficacy. This manuscript describes the pulsed laser-ablation in liquids (PLAL) method to create nanoparticles. This approach allows for the fine tuning of nanoparticle size, composition, and stability using post-irradiation methods as well as the addition of surfactants or volume excluders. By controlling particle size and composition, a large range of physical and chemical properties of metal nanoparticles can be explored which may contribute to their antimicrobial efficacy thereby opening new avenues for antibacterial development.

The antimicrobial properties of metals such as copper and silver have been recognized for centuries. This protocol describes pulsed laser-ablation in liquids, a method of synthesizing metal nanoparticles that provides the ability to fine tune the properties of these nanoparticles to optimize their antimicrobial effects.

Produção de nanopartículas metálicas por ablação a laser pulsada em líquidos: uma ferramenta para estudar as propriedades antibacterianas das nanopartículas

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of ...

20 mJ, 1 ps Yb:YAG Thin-disk Regenerative Amplifier

This is a report on a 100 W, 20 mJ, 1 ps Yb:YAG thin-disk regenerative amplifier. A homemade Yb:YAG thin-disk, Kerr-lens mode-locked oscillator with turn-key performance and microjoule-level pulse energy is used to seed the regenerative chirped-pulse amplifier. The amplifier is placed in airtight housing. It operates at room temperature and exhibits stable operation at a 5 kHz repetition rate, with a pulse-to-pulse stability less than 1%. By employing a 1.5 mm-thick beta barium borate crystal, the frequency of the laser output is doubled to 515 nm, with an average power of 70 W, which corresponds to an optical-to-optical efficiency of 70%. This superior performance makes the system an attractive pump source for optical parametric chirped-pulse amplifiers in the near-infrared and mid-infrared spectral range. Combining the turn-key performance and the superior stability of the regenerative amplifier, the system facilitates the generation of a broadband, CEP-stable seed. Providing the seed and pump of the optical parametric chirped-pulse amplification (OPCPA) from one laser source eliminates the demand of active temporal synchronization between these pulses. This work presents a detailed guide to set up and operate a Yb:YAG thin-disk regenerative amplifier, based on chirped-pulse amplification (CPA), as a pump source for an optical parametric chirped-pulse amplifier.

A protocol for the operation of a high-energy, high-power optical parametric chirped pulse amplifier pump source based on an Yb:YAG thin-disk regenerative amplifier is presented here.

20 mJ, 1 ps Yb: amplificador regenerativo de disco fino YAG

This is a report on a 100 W, 20 mJ, 1 ps Yb:YAG thin-disk regenerative amplifier. A homemade Yb:YAG thin-disk, Kerr-lens mode-locked oscillator with ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

Microtissues 3D para terapia regenerativa injetável e triagem de drogas de alto rendimento

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.