Os autores agradecem Neil Whitehouse (WPI, Higgins Loja Machine) por sua ajuda com usinagem CNC. Além disso, gostaríamos de agradecer a Adriana Hera (WPI Computing e Communications Center) por sua ajuda com MATLAB programação, bem como bebidas e Kate Cotnoir Joseph (WPI Acadêmica Centro de Tecnologia) para obter assistência com Camtasia. Sophie Burke e Jacleen Becker (WPI Acadêmica Centro de Tecnologia), desde imagens de vídeo suplementar. Este trabalho foi financiado pelo National Institutes of Health (R15 HL097332), o UMass Medical School-WPI Pilot Research Initiative, a American Heart Association (bolsa de pesquisa de graduação para JZH), e Instituto Politécnico de Worcester (Verão Bolsa de Investigação para JZH Graduação e institucionais start-up fundos para MWR).

1. Célula de fabricação de moldes semeadura Começar por uma moagem 02/01 pedaço &quot;de espessura de policarbonato para criar 15, de fundo redondo, poços anular com um diâmetro poste central de 2 mm. Os canais moídos são de 6 mm de profundidade e 3,75 milímetros de largura. Limpe e seque o molde de policarbonato para remover todos os detritos de plástico a partir do processo de moagem. Mix polidimetilsiloxano (PDMS) na proporção 10:1 (w / w) de base para agente de cura, Degas para remover todas as bolhas de ar, e despeje no molde de policarbonato. Degas novamente para remover as bolhas restantes, e cura em estufa a 60 ° C por 4 horas. Uma vez curado, remova cuidadosamente o modelo PDMS por lentamente descascando-lo longe do policarbonato, lave com água e sabão, e autoclave. Autoclave também uma solução de dois por cento agarose (w / v) dissolvido em Dulbecco modificado Eagle (DMEM). Coloque o modelo PDMS sobre uma superfície plana e encher com aga fundido<html> subiu pela primeira pipetagem agarose em cada um dos moldes pós-center, e depois de pipetagem para o espaço em torno dela. Permitir que a agarose para solidificar (cerca de 15 minutos), depois inverta o molde para liberar a agarose do PDMS. Cortar o excesso de agarose em torno de cada um dos poços, e colocar os poços de agarose em 12-lamelas. 2. Cultura de células e semeadura anel Adicionar cultura de células de mídia (DMEM com 10% de SFB e 1% de penicilina-estreptomicina) em torno do exterior do molde agarose, sem cobrir a parte superior dos moldes. Coloque as placas na incubadora e permitir-lhes estabilizar durante cerca de 15 minutos enquanto você prepara as células. Na confluência de 90%, trypsinize rat aorta células musculares lisas (rSMCs) e ressuspender em uma concentração de 5x10 6 células / ml. Pipetar 100 mL da suspensão de células em cada um dos poços de anular agarose, usando um movimento circular para aplicar as células em cada poço. content &quot;&gt; Make prateleiras certeza incubadora são de nível, em seguida, coloque as placas na incubadora e permitir-lhes a sentar-se em repouso por 24 horas. meio de troca a cada dois dias depois, aspirando a mídia de todo o bem, e re-encher bem cada até o agarose bem é completamente submerso. 3. Colheita de tecidos de toque e medidas de espessura Na conclusão da cultura, remover o anel do molde agarose, deslizando-a sobre a parte superior do poste central. Coloque o anel em uma pequena placa de Petri com tampão fosfato (PBS), o centro da amostra, e adquirir uma imagem usando um sistema de imagem digital. Use um programa de análise de imagem para detecção de bordas e medir a espessura do anel em 4 posições (superior, inferior, direita e esquerda) em torno de sua circunferência. Calcular o valor de espessura média (t) para cada anel individual, e usar esse valor para calcular a área da seção transversal(2π (t / 2) 2). 4. Ensaios mecânicos de anéis Configurar a máquina de ensaio de tração (Instron EPS 1000) na posição horizontal. Anexar uma célula de carga 1N ± 1mn e personalizados apertos fio fino (criado por flexão de arame de aço inoxidável). Monte a amostra anel sobre os dois apertos de arame fino. Estender as garras até a 5 mN de carga de tara é aplicada à amostra. Entrar na área transversal (calculado a partir de medições de espessura) e gravar o tamanho calibre. Pré-ciclo dos anéis 8 vezes entre a carga de tara e 50 kPa estresse a uma taxa de 10 mm / min. Após o oitavo pré-ciclo, retire a falha em 10 mm / min. Falha é observada como uma diminuição em vigor em 40% a partir de uma medição para outra. Para cada amostra de anel, medir a tensão de rotura (UTS) e tensão de falha e calcular o máximo tangente módulo (MTM) a partir dos dados adquiridos. 5. Anel de tecido comoassembléia para fabricar tubos de tecido Suportes para tubos personalizadas são usinadas a partir de discos round 5 centímetros de policarbonato com recortes retangulares (2 cm x 3 cm). Faça furos com rosca através destes discos para permitir que dois titulares para ser aparafusada. Autoclave titulares tubo de costume, e depois transferir para o gabinete de biossegurança e colocar em uma placa de Petri vazia. Use uma tesoura cirúrgica para cortar as extremidades dos tubos de silicone 1,9 milímetros de diâmetro em um ângulo para criar bordas chanfradas, e depois autoclave os tubos de silicone. Enquanto os tubos estão sendo autoclavado, preencher uma placa de Petri com meios de comunicação. Remova os anéis dos poços agarose e colocá-los na placa de Petri com meios de comunicação. Coloque autoclavado tubos de silicone na mesma placa de Petri para molhar-los antes de usar. Use uma pinça para colocar um fim chanfrado de um tubo de silicone para o centro de um anel e deslize o anel para o tubo de silicone. Repita com o número desejado de rings. Deslize os anéis em contato um com o outro delicadamente empurrá-los sucessivamente em ambas as direções ao longo do tubo. Este método pode ser usado para anéis colhidos depois de apenas um dia de cultura. Uma vez que os anéis são montados, alinhar tubos de silicone dentro do parafuso titulares policarbonato e as duas partes do titular juntos. Coloque o suporte em 100 milímetros prato Petri e adicionar 55 mL de mídia. Troca de mídia a cada 3 dias para a duração da cultura. Para remover os tubos de tecido, solte os tubos de silicone do detentor de policarbonato e utilize uma pinça para deslizar os tubos de tecido fora do tubo de silicone e em uma placa de Petri cheia de PBS. 6. Resultados representativos: Quando o protocolo é realizado corretamente, as células agregadas para formar anéis de tecido com um diâmetro interno igual ao diâmetro do pino molde correspondente no prazo de 24 horas. As bordas do anel são geralmente liso na aparência e (se cultured em uma superfície plana), são uniformes em espessura em torno de toda a circunferência. Os anéis de tecido são de fácil manuseio e podem ser removidos de seus poços para análise mecânica e histológica subseqüente (ver na Gwyther et al., 2011 8). Morfologia do tecido de toque, composição da matriz, e propriedades mecânicas variam de acordo com o número eo tipo de células inoculadas. Resultados representativos dos anéis de tecido feito de diferentes tipos de células, semeando as condições e duração da cultura são apresentados na Tabela 1. Dois anéis mm de diâmetro criados a partir de 5x10 5 rSMCs exibiu uma UTS superior anéis criados a partir de nosso previamente publicados dois anéis mm (feita a partir de 6.6x10 5 rSMCs) 8. Similares às células de rato, células do músculo liso (hSMC) prontamente agregadas para formar tecidos anéis, que continham uma grande quantidade de colágeno após apenas 14 dias em cultura (dados não mostrados). Células-tronco mesenquimais (hMSC) também agregados e anéis formados coesa, mas laforça cked mecânica e quebrou durante as fases iniciais de precycling testes de tração uniaxial. <table border="1"><tbody><tr><td></td><td> n </td><td> Número de células </td><td> Espessura (mm) </td><td> Comprimento da cultura (dias) </td><td> UTS (kPa) </td><td> MTM (kPa) </td><td> Tensão de falha (mm / mm) </td></tr><tr><td> RSMC anéis </td><td> 6 </td><td> 660000 </td><td> 0,94 ± 0,12 </td><td> 14 </td><td> 97 ± 30 </td><td> 497 ± 91 </td><td> 0,50 ± 0,08 </td></tr><tr><td> RSMC anéis </td><td> 4 </td><td> 500000 </td><td> 0,53 ± 0,02 </td><td> 7 </td><td> 113 ± 8 </td><td> 189 ± 15 </td><td> 0,88 ± 0,05 </td></tr><tr><td> hSMC anéis </td><td> 3 </td><td> 750000 </td><td> 0,51 ± 0,05 </td><td> 14 </td><td> 160 ± 30 </td><td> 270 ± 20 </td><td> 0,92 ± 0,08 </td></tr><tr><td> hMSC anéis </td><td> 3 </td><td> 750000 </td><td> 0,40 ± 0,07 </td><td> 14 </td><td> N / A </td><td> N / A </td><td> N / A </td></tr></tbody></table> Tabela 1. Tabela mostrando os parâmetros da cultura e propriedades mecânicas de dois anéis milímetros gerados a partir de diferentes tipos de células, as concentrações de semeadura, e durações cultura. <img alt="Figura 1" src="/files/ftp_upload/3366/3366fig1.jpg" /> Figura 1. (A) Esquema do processo de geração de tecido do anel. (B) Custom molde de policarbonato com fresado poços anular. Diâmetros pós Central são 2 mm. (C) modelo PDMS depois de ter sido desfeito, a partir do molde de policarbonato. (D) anel de tecido agregados cultivadas em um molde de agarose com um posto de 2 mm. (E) anel de tecido Dois mm de diâmetro na PBS. Barra de escala = mm 6 mm (B, C) e 2 (D, E). <img alt="Figura 2" src="/files/ftp_upload/3366/3366fig2.jpg" /> Figura 2. Enredo Representante da curva de tensão-produced do ensaio de tração uniaxial.

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Recentemente, tem havido um crescente interesse na célula-base ou &quot;scaffold-less&quot; métodos de engenharia de tecidos para tratar de algumas das limitações do andaime abordagens baseadas em engenharia de tecidos. Uma vez que células derivadas de tecidos são criados a partir de células e da matriz que produzem, eles inerentemente contêm densidades celulares muito maior, não contêm materiais exógenos, e pode ser feita inteiramente a partir de células humanas e proteínas. Enxertos vasculares feita a partir de células humanas podem atingir resistência mecânica substanciais na ausência de andaimes exógenas (por exemplo, pressões de ruptura 3400 mmHg em comparação a 1600 mmHg para veias safenas humanas) 12. Embora baseada em células de tecido vascular apresentam densidade celular melhorada e resistência mecânica, métodos de fabricação mais atuais (como &quot;folha-base engenharia&quot; 3,4,12 ou &quot;bioprinting&quot; 5,13) requerem períodos cultura extensa (&gt; 3 meses) ou equipamentos especializados para a construção do tecido 3D. O anel de tecido derivadas de células-method descrito aqui permite rápida celulares auto-montagem para formar robusto constrói tecido 3D dentro de um curto período de tempo e sem o uso de equipamentos especializados. Este protocolo detalha o procedimento foi desenvolvido para criar dois milímetros de diâmetro interior de ratos células derivadas do tecido do músculo liso anéis. No presente exemplo, os anéis de tecido foram cultivados por 7 dias (então cultivadas por mais sete dias para a fusão de anel e formação do tubo). No entanto, dois ratos mm (e humanos) anéis de células musculares lisas podem ser removidas dos poços e coesa o suficiente para o tratamento (por exemplo, transferir para tubos de silicone) tão cedo como um dia após a semeadura de células. Além disso, os anéis de tecido robusto com diferentes diâmetros internos (2, 4 e 6 mm) podem ser criadas com este método, simplesmente alterando o diâmetro post do molde de policarbonato original. 8 Nós também recentemente modificado o projeto de moldes de policarbonato para habilitar cinco dois milímetros poços semeadura para ser lançado em um único multi-câmara de agarose bem, o queusa menos PDMS e agarose, e se encaixa em um poço de uma placa de 6 poços (dados não mostrados). Alterações no diâmetro post, a largura da semeadura bem, o raio de curvatura da parte inferior arredondada, o número de poços de semeadura, ou a profundidade dos poços semeadura podem ser modificadas simplesmente mudando as especificações no arquivo CAD para CNC usinagem do molde de policarbonato. Finalmente, um molde de policarbonato único pode ser usado para fabricar um número ilimitado de modelos PDMS, e cada modelo PDMS podem ser limpos, autoclavados e reutilizados dezenas de vezes. Além de alterar o tamanho dos anéis de tecido, fizemos anéis de muitos tipos de células diferentes, incluindo: SMCs rato primária (Applications Cell, R354-05), humanos primários SMCs arterial coronariana (Lonza, CC-2583), humanas primárias fibroblastos dérmicos 11 (dom generoso de Pinos Dr. George, WPI Departamento de Engenharia Biomédica), fibroblastos de pulmão de rato (RFL-6, ATCC CCL-192), e as células-tronco mesenquimais (Lonza, PT-2501). Cada um dos tipos de célula destes agregados e contratos em todo o posts centro para formar anéis de tecido, embora a organização celular, composição ECM, e propriedades mecânicas das construções variam para cada tipo de célula. Os parâmetros de semeadura para cada tipo de célula deve ser empiricamente determinada com base no tamanho das células e sua capacidade de agregar. Portanto, enquanto este sistema de criação de anéis de células derivadas de tecido é extremamente versátil, o protocolo pode precisar de pequenos ajustes para a formação de tecido ideal com diferentes tipos de células. Geometria do anel de tecido facilita o carregamento fácil e avaliação das propriedades do material tecido por testes de tração uniaxial, conforme descrito. Há também precedentes substancial para o uso segmentos sangue anel navio para medir a contração vascular e função fisiológica. Estudos preliminares indicam que os anéis de células derivadas de tecido pode ser montado em um dispositivo myograph fio para a medição da capacidade de resposta farmacológica egeração de força contrátil (dados não mostrados). Ao todo, a capacidade de rapidamente fabricar anéis de auto-montagem de células para análise histológica, mecânicos, fisiológicos, bioquímicos e sugere uma nova e poderosa ferramenta que pode ser útil para a modelagem de estrutura do tecido vascular e função na saúde e na doença.

<table border="1"><tbody><tr><th> Nome do reagente / equipamentos </th><th> Companhia </th><th> Catalogo </th></tr><tr><td> Polidimetilsiloxano (PDMS) </td><td> Dow Corning </td><td> Sylgard 184 </td></tr><tr><td> Agarose </td><td> Lonza </td><td> 50000 </td></tr><tr><td> DMEM </td><td> Mediatech </td><td> 15-017-CV </td></tr><tr><td> De soro fetal bovino (FBS) </td><td> PAA </td><td> A05-201 </td></tr><tr><td> Penicilina / estreptomicina </td><td> Mediatech </td><td> 30-002-CI </td></tr><tr><td> Sistema de imagem digital </td><td> TVP Corporação </td><td> Modelo 630 </td></tr><tr><td> Máquina de ensaio uniaxial </td><td> Instron </td><td> ElectroPuls E1000 </td></tr><tr><td> Software de detecção de borda </td><td> TVP Corporação </td><td> Quadro 2.4.6 </td></tr></tbody></table>

directed cellular self-assembly to fabricate cell-derived tissue rings for biomechanical analysis and tissue engineering

Cada ano, centenas de milhares de pacientes submetidos a cirurgia de revascularização do miocárdio nos Estados Unidos 1. Aproximadamente um terço destes pacientes não têm adequado navios dadores autólogo devido à progressão da doença ou safra anterior. O objetivo da engenharia de tecidos vasculares é desenvolver uma fonte alternativa adequada para estas pontes. Além disso, o tecido vascular engenharia pode revelar-se útil como modelos vivos vascular para estudar doenças cardiovasculares. Várias abordagens promissoras para os vasos sanguíneos de engenharia têm sido exploradas, com muitos estudos recentes com foco no desenvolvimento e análise de métodos baseados em células. 2-5 Aqui, apresentamos um método para rapidamente auto-montar células em anéis de tecido 3D que pode ser usado em vitro para modelar os tecidos vasculares. Para fazer isso, suspensões de células musculares lisas são semeados no fundo redondo de anular agarose poços. As propriedades não-adesivo do agarose permitem ªcélulas e para resolver, agregar e contrato em torno de um posto no centro do poço para formar um anel de tecido coeso. 6,7 Estes anéis podem ser cultivadas por vários dias antes da colheita para a mecânica, análise fisiológica, bioquímica ou histológica. Nós mostramos que estes anéis de células derivadas de tecidos de rendimentos na resistência à tração 100-500 kPa final 8, que excede o valor reportado para outras construções de tecido vascular engenharia cultivadas por períodos semelhantes (&lt;kPa 30). 9,10 Nossos resultados demonstram que a célula robusta derivados de geração de anel vascular do tecido pode ser alcançada dentro de um curto período de tempo, e oferece a oportunidade para avaliação direta e quantitativa das contribuições de células e de células derivadas de matriz (MDL) para a estrutura do tecido vascular e função.

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Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

Este artigo descreve um método versátil para criar anéis de células derivadas do tecido celular por auto-montagem. Células musculares lisas semeadas em forma de anel-agarose agregado poços e contrato de forma robusta em três dimensões (3D) tecidos dentro de 7 dias. Anéis milímetro escala de tecido são conducentes a testes mecânicos e servem como blocos de construção para a montagem dos tecidos.

Dirigido Cellular Automontagem para Fabricar Anéis Cell-Derived Tecido para Análise Biomecânica e Engenharia de Tecidos

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do ...

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18.9K visualizações.  Worcester Polytechnic Institute. Este artigo descreve um método versátil para criar anéis de células derivadas do tecido celular por auto-montagem. Células musculares lisas semeadas em forma de anel-agarose agregado poços e contrato de forma robusta em três dimensões (3D) tecidos dentro de 7 dias. Anéis milímetro escala de tecido são conducentes a testes mecânicos e servem como blocos de construção para a montagem dos tecidos.

Vídeo: Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

Campo elétrico controlado de migração dirigida de células progenitoras neurais em ambientes 2D e 3D

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

Bioengenharia

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

Sem contato, Monitoramento Label livre de células e matriz extracelular utilizando Espectroscopia Raman

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

Capilar Força Litografia de Engenharia tecido cardíaco

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

Avaliação de Entrega Gene Polimérico Nanopartículas por Análise Nanoparticle Rastreamento e alta capacidade de Citometria de Fluxo

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Engenharia de Tecidos de um 3D Humano<em&gt; In vitro</em&gt; Sistema de Teste de Tumor

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Andaimes auto-relato para cultura de células 3-Dimensional

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Engenharia 3D celularizados Colágeno Gel para Vascular Regeneração Tecidual

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Microambientes sanduíche-como aproveitarar Telemóveis / interações materiais

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

Preparação de Thermoresponsive nanoestruturados Surfaces para Engenharia de Tecidos

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

Engenharia de Tecidos por Intrínseca Vascularização numa<em&gt; In Vivo</em&gt; Câmara Engenharia de Tecidos

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...