Os autores agradecem a Dirk Herrmann, Oliver Wendt, Siegfried Horn, Hartmut Gutzeit, e Joerg Bohn por sua ajuda substancial sobre a soldagem de vapores de solvente. Além disso, gostaríamos de agradecer Michael Hartmann, Alex Gerwald, e Daniel Leisen por sua assistência técnica.

Seqüência de processo # 1: Embossing Hot, Usinagem e Soldagem de vapor de solventes O CellChip em seu design cúbico é replicado por estampagem a quente ou micro moldagem por injeção. Para isso, usamos um molde de latão microusinados com a geometria inverso do chip. Os recipientes - dispostos em uma matriz regular de até 1.156 contêineres - têm um design cúbico com um comprimento da aresta de 300 mm. Para estampagem a quente, o processo de replicação é executada em um WUM02 convencional (Jenoptik Mikrotechnik, Alemanha). A ferramenta consiste em duas placas de metal circular. Em uma primeira etapa, uma placa fina PMMA (Lucryl, G77Q11, BASF) é colocado no centro da placa inferior da ferramenta aberta. A inserção do molde microestruturada é centralmente montada na placa superior. Em seguida, a ferramenta está fechado para a evacuação do molde e aquecido a uma temperatura acima da temperatura de transição vítrea do polímero. Pressionando as placas em conjunto, o polímero viscoso é empurrado para dentro das cavidades evacuados até que estejam completamente cheios precisamente reproduzindo a geometria do molde. Após o resfriamento da ferramenta, a parte de polímero microestruturada pode ser desmoldados. O processo requer mais massa de polímero que é realmente necessário para preenchimento das cavidades do molde. O excedente forma uma camada de polímero residual que pode ser utilizado para facilitar a desmoldagem da peça. No entanto, para criar poros de diâmetro menor que 3 mm no fundo dos recipientes, a espessa camada residual tem de ser diluído para baixo, ou até mesmo completamente removido e substituído por uma membrana porosa. Para simplificar o processo de integração dos poros, a parte de trás do CellChip replicado é completamente removido por usinagem com uma fábrica de diamantes. Para isso, as peças são fixadas em uma placa de refrigeração de montagem e, adicionalmente, as estruturas frágeis são congelados em água desionizada para protegê-los contra danos. Em uma etapa do processo passado, finalmente, um comercial de íons da membrana track-gravado (policarbonato, espessura de 10 mM, pore tamanho 3 mm, 2x10 6 poros / cm ², Pieper Filter GmbH) é ligada à parte traseira do conjunto de recipientes abertos agora tanto no superior e inferior. O processo de ligação é um processo de soldagem de vapor de solventes realizado em uma câmara, estanque aos gases aquecidos [Fig. 1], que consiste em um êmbolo superior e uma placa móvel inferior com um mandril de vácuo integrado. 4 Até quatro CellChips usinadas e-track gravado membranas são expostas em paralelo com um solvente vaporizado após a câmara foi evacuada. Então, as peças moldadas e as membranas são pressionadas juntas pelo êmbolo superior. Após um curto período de exposição (&lt;15 s), a câmara é evacuada novamente, eliminando assim o solvente. Devido ao curto tempo de contato, apenas a superfície perto material é dissolvido e uma deformação da estrutura em massa devido à carga mecânica pode ser evitado. Finalmente, a câmara é aberta eo CellChips solvente soldadas podem ser removidos e preparados para cultura de células [Fig. 2]. <a href="/files/ftp_upload/old/699_2008_5_12_7/Giselbrecht-Fig1.png"><img style="border: 1px solid black;" src="/files/ftp_upload/old/699_2008_5_12_7/Giselbrecht-Fig1.png" alt="Figura 1" width="100" /></a> Figura 1. <a href="/files/ftp_upload/old/699_2008_5_12_8/Giselbrecht-Fig2.png"><img style="border: 1px solid black;" src="/files/ftp_upload/old/699_2008_5_12_8/Giselbrecht-Fig2.png" alt="Figura 2" width="100" /></a> Figura 2. Seqüência de processo # 2: irradiação de íons pesados, microthermoforming e gravura track (processo SMART) O novo processo chamado SMART é uma tecnologia recentemente desenvolvida para a fabricação de micro-funcionalizados membrana como microestruturas. 5 A tecnologia é baseada em um processo de microtécnica de termoformagem, chamado &quot;microthermoforming&quot; 6,7. Nesta etapa do processo central, que foi adaptado a partir do macroscópica processo preso folha de termoformagem, uma aquecida fina película de polímero é formado em sua amolecida Estado de borracha elástica, por pressão de gás em uma cavidade do molde [Fig. 3]. Ao contrário de estampagem a quente ou de moldagem por injeção, esse processo não é uma formação primária e do polímero não é derretido. Devido ao fato de que o filme é formado ainda em estado sólido, com uma coesão material permanente, material de modificações com alta resolução lateral pode ser gerado no primeiro planar filmes de polímero e são preservados durante o processo de formação. Após a etapa de microthermoforming, essas modificações podem ser ainda mais seletiva processados, por exemplo, por tratamento químico úmido. <img style="border: 1px solid black;" src="/files/ftp_upload/old/699_2008_5_12_9/Giselbrecht-Fig3.png" alt="Figura 3" width="400" height="217" /> Figura 3 O processo SMART em princípio consiste de três etapas do processo: <ol><li> criação de padrões de modificação altamente resolvidas em planar filmes finos de polímero em um processo de pré- </li><li> 3D shapção de filmes por microthermoforming sem perda de (padrão) modificações </li><li> pós-processamento (opcional) para uma funcionalização final de peças de paredes finas microestruturada </li></ol> O processo INTELIGENTE atualmente estamos aplicando para a fabricação de CellChips porosa inclui os seguintes etapas de processo [Fig. 4]. Uma fina película de polímero, por exemplo, de policarbonato (Pokalon OG461Gl, 50 mm, LOFO High Tech Film GmbH, Alemanha), é irradiado com íons pesados ​​acelerados (como Xe, Au ou íons U) nas instalações do acelerador de GSI (Darmstadt, Alemanha) com energias de aprox. 1 GeV e fluências na ordem de 106 8 Após o resfriamento da ferramenta, a parte de paredes finas podem ser desmoldados. íons / cm ². Ao penetrar através do filme, cada íon produz uma trilha quase reta de material modificado, chamado de faixa latente. Os filmes pré-tratados são, então, termoformados para uma matriz de 25x25 fina microcontainers paredes, cada uma com um diâmetro e profundidade de 300 mm. O processo é realizado atualmente em um cunho modificado hot [Fig. 5], onde o filme de polímero é fixada entre duas placas de metal. A placa superior é equipada com o molde microusinados ea inferior contém a pressão e conectores de vácuo. O filme é esticado em microcavidades previamente evacuado do molde de nitrogênio com uma pressão de até 5 MPa. Os filmes são formados perto de sua temperatura de transição de vidro, evitando faixa de recozimento. <img style="border: 1px solid black;" src="/files/ftp_upload/old/699_2008_5_12_10/Giselbrecht-Fig4.png" alt="Figura 4" width="400" height="119" /> Figura 4 <a href="/files/ftp_upload/old/699_2008_5_12_11/Giselbrecht-Fig5.png"><img style="border: 1px solid black;" src="/files/ftp_upload/old/699_2008_5_12_11/Giselbrecht-Fig5.png" alt="Figura 5" width="100" /></a> Figura 5 Em um pós-processamento, as faixas de íons são seletivamente gravado para pores por imersão a microestrutura inteiro em um meio de gravação apropriado (por exemplo, 5 Mol / L NaOH, 10% w / v MeOH). Ao controlar os tempos de gravura e gravura condições, tais como temperatura, concentração e aditivos especiais (por exemplo, os promotores etch), o tamanho ea forma dos poros resultante pode ser ajustado [fig. 6]. <a href="/files/ftp_upload/old/699_2008_5_12_12/Giselbrecht-Fig6.png"><img style="border: 1px solid black;" src="/files/ftp_upload/old/699_2008_5_12_12/Giselbrecht-Fig6.png" alt="Figura 6" width="100" /></a> Figura 6

<ol>
	<li>Knedlitschek, G., Schneider, F., Gottwald, E., Schaller, T., Eschbach, E., Weibezahn, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tissue-like+culture+system+using+microstructures:+influence+of+extracellular+matrix+material+on+cell+adhesion+and+aggregation.">A tissue-like culture system using microstructures: influence of extracellular matrix material on cell adhesion and aggregation.</a> J Biomech Eng. 121, 35-39 (1999).</li><li>Gottwald, E., Giselbrecht, S., Augspurger, C., Lahni, B., Dambrowsky, N., Truckenm&#252;ller, R., Piotter, V., Gietzelt, T., Wendt, O., Pfleging, W., Welle, A., Rolletschek, A., Wobus, A. M., Weibezahn, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chip-based+platform+for+the+in+vitro+generation+of+tissues+in+three-dimensional+organization.">A chip-based platform for the in vitro generation of tissues in three-dimensional organization.</a> Lab Chip. 7, 777-785 (2007).</li><li>Heckele, M., Schomburg, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+on+micro+molding+of+thermoplastic+polymers.">Review on micro molding of thermoplastic polymers.</a> Journal of Micromechanics And Microengineering. 14, (2004).</li><li>Giselbrecht, S., Gietzelt, T., Guber, A. E., Gottwald, E., Trautmann, C., Truckenm&#252;ller, R., Weibezahn, K. -. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microthermoforming+as+a+novel+technique+for+manufacturing+scaffolds+in+tissue+engineering+(CellChips.">Microthermoforming as a novel technique for manufacturing scaffolds in tissue engineering (CellChips.</a> IEE Proc.-Nanobiotechnol. 151, 151-157 (2004).</li><li>Giselbrecht, S., Gietzelt, T., Gottwald, E., Trautmann, C., Truckenm&#252;ller, R., Weibezahn, K. -. F., Welle, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+tissue+culture+substrates+produced+by+microthermoforming+of+pre-processed+polymer+films.">3D tissue culture substrates produced by microthermoforming of pre-processed polymer films.</a> Biomed Microdevices. 8, 191-199 .</li><li>Truckenm&#252;ller, R., Rummler, Z., Schaller, T., Schomburg, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-cost+thermoforming+of+micro+fluidic+analysis+chips.">Low-cost thermoforming of micro fluidic analysis chips.</a> Journal of Micromechanics and Microengineering. 12, 375-379 (2002).</li><li>Truckenm&#252;ller, R., Giselbrecht, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microthermoforming+of+flexible,+not+buried+hollow+microstructures+for+chip-based+life+sciences+applications.">Microthermoforming of flexible, not buried hollow microstructures for chip-based life sciences applications.</a> IEE Proc.-Nanobiotechnol. 151, 163-166 (2004).</li><li>Fleischer, R. L., Price, P. B., Walker, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+tracks+in+solids.">Nuclear tracks in solids.</a> , .</li></ol>

Embora os métodos estabelecidos de polímero microrreplicação, como micro moldagem por injeção ou estampagem a quente, são adequados para a produção de microestruturas, eles não são realmente eficazes na produção de microestruturas com uma porosidade integrada e altamente controlada, como é necessário para o CellChip. Estruturas volumosas por exemplo, requerem usinagem caros para reduzir a espessura da parede de uma perfuração a laser subseqüente ou paredes têm de ser completamente substituídos por faixas gravadas membran...

microfabrication of chip-sized scaffolds for three-dimensional cell cultivation

Uso de tecnologias de microfabricação é um pré-requisito para criar andaimes da geometria reprodutíveis e de qualidade constante para tridimensional de cultivo celular. Essas tecnologias oferecem um amplo espectro de vantagens não só para a fabricação, mas também para diferentes aplicações. O tamanho ea forma dos ninhos de células formado pode ser influenciado pela arquitetura exata e reprodutível do andaime microfabricated e, portanto, o comprimento do caminho de difusão de nutrientes e gases podem ser controlled.1 Este é sem dúvida uma ferramenta útil para prevenir a apoptose e necrose de células devido a uma insuficiente de nutrientes e fornecimento de gás ou remoção de metabólitos celulares. Nosso chip de polímero, chamado CellChip, tem as dimensões exteriores de 2 x 2 cm, com uma área central microestruturada. Esta área é subdividida em uma matriz de até 1156 microcontainers com uma dimensão típica de 300 m comprimento da aresta para o projeto cúbicos (cp ou cf-chip) ou de 300 m de diâmetro e profundidade para o projeto round (r-chip). 2 Até agora, estampagem a quente ou micro moldagem por injeção (em combinação com usinagem trabalhoso subseqüentes das partes) foi usado para a fabricação dos chips microestruturada. Basicamente, micro moldagem por injeção é uma das únicas técnicas de replicação de polímero baseado em que, até agora, é capaz de produção em massa de polímero microstructures.3 No entanto, ambas as técnicas têm certas limitações indesejadas devido ao processamento de um polímero viscoso derreter com a geração de paredes muito finas ou integrados através de furos. No caso do CellChip, camadas inferiores finos são necessários para perfurar o polímero e fornecem pequenos poros de tamanho definido para abastecer as células com meio de cultura por exemplo, por perfusão microfluídicos dos recipientes. Para superar essas limitações e reduzir os custos de fabricação, desenvolvemos uma nova abordagem microtécnicos com base em um processo de baixo escala termoformagem. Para a fabricação de altamente porosa e fina fichas murada polímero, usamos uma combinação de irradiação de íons pesados, microthermoforming e gravura pista. Neste processo chamado &quot;SMART&quot; (Modificação substrato e replicação por Termoformagem) filmes finos de polímeros são irradiados com energia projéteis pesados ​​de várias centenas de MeV introdução dos chamados &quot;faixas latente&quot; Posteriormente, o filme em um estado elástico de borracha é formado em três partes dimensional sem modificar ou recozimento as faixas. Após o processo de formação, ataque químico seletivo, finalmente, converte as faixas nos poros cilíndricos de diâmetro ajustável.

Watch this Scientific Journal Video about Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation at JoVE.com

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Nós apresentamos dois processos para a microfabricação dos chips porosa de polímero para tridimensional cultivo celular. O primeiro é de estampagem a quente combinado com um processo de soldagem solvente vapor. A segunda usa um processo desenvolvido recentemente microthermoforming combinado com tecnologia de iões pista levando a uma significativa simplificação de fabricação.

Microfabricação de Chip porte Andaimes para tridimensional de cultivo celular

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

microfabrication-chip-sized-scaffolds-for-three-dimensional-cell

Research

JoVE Journal

Biology

11.7K visualizações.  Karlsruhe Research Centre. Nós apresentamos dois processos para a microfabricação dos chips porosa de polímero para tridimensional cultivo celular. O primeiro é de estampagem a quente combinado com um processo de soldagem solvente vapor. A segunda usa um processo desenvolvido recentemente microthermoforming combinado com tecnologia de iões pista levando a uma significativa simplificação de fabricação.

Vídeo: Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from non-biodegradable polymers, e.g., polycarbonate or polymethyl methacrylate by micro injection molding, micro hot embossing or micro thermoforming. But, it can also be manufactured from bio-degradable polymers. Its overall dimensions are 0.7 1 x 20 x 20 x 0.7 1 mm (h x w x l). The main features of the chips used are either a grid of up to 1156 cubic micro-containers (cf-chip) each the size of 120-300 x 300 x 300 &mu; (h x w x l) or round recesses with diameters of 300 &mu; and a depth of 300 &mu; (r-chip). The scaffold can house 10 Mio. cells in a three-dimensional configuration. For an optimal nutrient and gas supply, the chip is inserted in a bioreactor housing. The bioreactor is part of a closed steril circulation loop that, in the simplest configuration, is additionaly comprised of a roller pump and a medium reservoir with a gas supply. The bioreactor can be run in perfusion, superfusion, or even a mixed operation mode. We have successfully cultivated cell lines as well as primary cells over periods of several weeks. For rat primary liver cells we could show a preservation of organotypic functions for more than 2 weeks. For hepatocellular carcinoma cell lines we could show the induction of liver specific genes not or only slightly expressed in standard monolayer culture. The system might also be useful as a stem cell cultivation system since first differentiation experiments with stem cell lines were promising.

We describe a chip-based platform for the three-dimensional cultivation of cells in micro-bioreactors. One chip can house up to 10 Mio. cells that can be cultivated under precisely defined conditions with regard to fluid flow, oxygen tension etc. in a sterile, closed circulation loop.

Chip baseado em Cultura de Células tridimensional em perfundidos Micro-biorreatores

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from ...

Biologia

Interview: Bioreactors and Surfaced-Modified 3D-Scaffolds for Stem Cell Research

A Nature Editorial in 2003 asked the question "Good-bye, flat biology?" What does this question imply? In the past, many in vitro culture systems, mainly monolayer cultures, often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.
The last generation of CellChips -- micro-thermoformed containers -- a specific technology was developed, which offers the additional possibility to modify the whole surface of the 3D formed containers. This allows a surface-patterning on a submicron scale with distinct signalling molecules. Sensors and signal electrodes may be incorporated. Applications range from basic research in cell biology to toxicology and pharmacology. Using biodegradable polymers, clinical applications become a possibility. Furthermore, the last generation of micro-thermoformed chips has been optimized to allow for cheap mass production.

In the past many in vitro culture systems -- mainly monolayer cultures -- often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.

Entrevista: biorreatores e apareceu-Modified 3D-andaimes para Investigação em Células Estaminais

A Nature Editorial in 2003 asked the question  "Good-bye, flat biology?"  What does this question imply? In the past, many in vitro culture systems,  ...

Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address the requirements (e.g., mechanics and vascularity) of more intricate organs and tissues. Electrospinning is a popular technique to create fibrous scaffolds that mimic the architecture and size scale of the native extracellular matrix. These fibrous scaffolds are also useful as cell culture substrates since the fibers can be used to direct cellular behavior, including stem cell differentiation (see extensive reviews by Mauck et al. and Sill et al. for more information). In this article, we describe the general process of electrospinning polymers and as an example, electrospin a reactive hyaluronic acid capable of crosslinking with light exposure (see Ifkovits et al. for a review on photocrosslinkable materials). We also introduce further processing capabilities such as photopatterning and multi-polymer scaffold formation. Photopatterning can be used to create scaffolds with channels and multi-scale porosity to increase cellular infiltration and tissue distribution. Multi-polymer scaffolds are useful to better tune the properties (mechanics and degradation) of a scaffold, including tailored porosity for cellular infiltration. Furthermore, these techniques can be extended to include a wide array of polymers and reactive macromers to create complex scaffolds that provide the cues necessary for the development of successful tissue engineered constructs.

The process of electrospinning polymers for tissue engineering and cell culture is addressed in this article. Specifically, the electrospinning of photoreactive macromers with additional processing capabilities of photopatterning and multi-polymer electrospinning is described.

Eletrofiação Andaimes Polymer fibrosos para Engenharia de Tecidos e Cultura de Células

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

Isolamento de células epiteliais mamárias de tridimensional Mixed-célula esferoidal co-cultura

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

Duas e três dimensões imagens de células vivas de Proteínas de DNA resposta a danos

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

Rato Fetal Whole Intestino Sistema de Cultura para<em&gt; Ex Vivo</em&gt; Manipulação de vias de sinalização e tridimensional Imagens ao vivo de Desenvolvimento Villus

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

FRET Imaging no hidrogéis tridimensionais

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine system, but the complex structural relationships and three-dimensional (3D) vasculature architectures of the ovary are not well described. In order to visualize the 3D connections and architecture of blood vessels in the intact ovary, the first important step is to make the ovary optically clear. In order to avoid tissue shrinkage, we used the hydrogel fixation-based passive CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/ Immunostaining/In situ-hybridization-compatible Tissue Hydrogel) protocol method to clear an intact ovary. Immunostaining, advanced multiphoton confocal microscopy, and 3D image-reconstructions were then used for the visualization of ovarian vessels and follicular capillaries. Using this approach, we showed a significant positive correlation (P &#60;0.01) between the length of the follicular capillaries and volume of the follicular wall.

Here we present an adaptation of the passive CLARITY and 3D reconstruction method for visualization of the ovarian vasculature and follicular capillaries in intact mouse ovaries.

Reconstrução tridimensional da arquitetura Vascular do ovário passiva Mouse clareza-limpo

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo-Clear: Um método de compensação por imagem tridimensional do tecido adiposo tecido

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.

The entire 3D structure and cellular content of organoids, as well as their phenotypic resemblance to the original tissue can be captured using the single-cell resolution 3D imaging protocol described here. This protocol can be applied to a wide range of organoids varying in origin, size and shape.

Resolução unicelular imagens tridimensionais de organoides intactos

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development ...