RESUMO
Zebrafish xenografts are an established model in cancer biology, with a steadily rising number of models and users. However, as of yet, there is no platform dedicated to standardizing protocols and sharing data regarding zebrafish xenograft phenotypes. Here, we present the Xenograft Phenotype Interactive Repository (XePhIR, https://www.xephir.org) as an independent data-sharing platform to deposit, share and repurpose zebrafish xenograft data. Deposition of data and publication with XePhIR will be done after the acceptation of the original publication. This will enhance the reach of the original research article, enhance visibility and do not interfere with the publication or copyrights of the original article. With XePhIR, we strive to fulfill these objectives and reason that this resource will enhance reproducibility and showcase the appeal and applicability of the zebrafish xenograft model. Database URL: https://www.xephir.org.
Assuntos
Peixe-Zebra , Animais , Bases de Dados Factuais , Xenoenxertos , Humanos , Fenótipo , Reprodutibilidade dos Testes , Peixe-Zebra/genéticaRESUMO
During bioprinting, cells are suspended in a viscous bioink and extruded under pressure through small diameter printing needles. The combination of high pressure and small needle diameter exposes cells to considerable shear stress, which can lead to cell damage and death. Approaches to monitor and control shear stress-induced cell damage are currently not well established. To visualize the effects of printing-induced shear stress on plasma membrane integrity, we add FM 1-43 to the bioink, a styryl dye that becomes fluorescent when bound to lipid membranes, such as the cellular plasma membrane. Upon plasma membrane disruption, the dye enters the cell and also stains intracellular membranes. Extrusion of alginate-suspended NIH/3T3 cells through a 200µm printing needle led to an increased FM 1-43 incorporation at high pressure, demonstrating that typical shear stresses during bioprinting can transiently damage the plasma membrane. Cell imaging in a microfluidic channel confirmed that FM 1-43 incorporation is caused by cell strain. Notably, high printing pressure also impaired cell survival in bioprinting experiments. Using cell types of different stiffnesses, we find that shear stress-induced cell strain, FM 1-43 incorporation and cell death were reduced in stiffer compared to softer cell types and demonstrate that cell damage and death correlate with shear stress-induced cell deformation. Importantly, supplementation of the suspension medium with physiological concentrations of CaCl2greatly reduced shear stress-induced cell damage and death but not cell deformation. As the sudden influx of calcium ions is known to induce rapid cellular vesicle exocytosis and subsequent actin polymerization in the cell cortex, we hypothesize that calcium supplementation facilitates the rapid resealing of plasma membrane damage sites. We recommend that bioinks should be routinely supplemented with physiological concentrations of calcium ions to reduce shear stress-induced cell damage and death during extrusion bioprinting.
Assuntos
Bioimpressão , Alginatos , Animais , Bioimpressão/métodos , Cálcio , Suplementos Nutricionais , Camundongos , Impressão Tridimensional , Engenharia Tecidual/métodos , Alicerces TeciduaisRESUMO
Materials made of recombinant spider silk proteins are promising candidates for cardiac tissue engineering, and their suitability has so far been investigated utilizing primary rat cardiomyocytes. Herein, we expanded the tool box of available spider silk variants and demonstrated for the first time that human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes attach, contract, and respond to pharmacological treatment using phenylephrine and verapamil on explicit spider silk films. The hiPSC-cardiomyocytes contracted for at least 14 days on films made of positively charged engineered Araneus diadematus fibroin 4 (eADF4(κ16)) and three different arginyl-glycyl-aspartic acid (RGD)-tagged spider silk variants (positively or negatively charged and uncharged). Notably, hiPSC-cardiomyocytes exhibited different morphologies depending on the spider silk variant used, with less spreading and being smaller on films made of eADF4(κ16) than on RGD-tagged spider silk films. These results indicate that spider silk engineering is a powerful tool to provide new materials suitable for hiPSC-based cardiac tissue engineering.
RESUMO
Nano- and micro-scale topographical features play a critical role in the induction and maintenance of various cellular properties and functions, including morphology, adhesion, gene regulation, and cell-to-cell communication. In addition, recent studies have indicated that the structure and function of heart tissue are also sensitive to mechanical cues at the nano- and micro-scale. Although fabrication methods exist for generating topographical features on polymeric scaffolds for cell culture, current techniques, especially those with nano-scale resolution, are typically complex, prohibitively expensive and not accessible to most biology laboratories. Here, we present a simple and tunable fabrication method for the production of patterned electrospun fibers that simulate the complex anisotropic and multi-scale architecture of cardiac tissue, to promote cardiac cell alignment. This method is based on the combination of electrospinning and soft lithography techniques, in which electrospun fibers, based on a blend of poly(glycerol sebacate) and poly(caprolactone), were collected on a patterned Teflon-coated silicon wafer with imprinted topographical features. Different surface topographies were investigated, such as squares and grooves, with constant or different interspatial distances. In vitro cell culture studies successfully demonstrated the alignment of both C2C12 myoblasts and neonatal rat cardiomyocytes on fabricated electrospun patterned surfaces. C2C12 cells were cultured over a period of 72h to study the effect of topographical cues on cell morphology. Cells attached within the first 8h after seeding and after 24h most of the cells started to align responding to the topographical cues. Similarly, cardiomyocytes responded to the topographical features by aligning themselves and by expressing Connexin 43 along cellular junctions. Summarizing, we have developed a new method with the potential to significantly promote cardiac tissue engineering by fabricating electrospun fibers with defined topographical features to guide and instruct donor and/or host cells.
Assuntos
Glicerol/análogos & derivados , Coração/fisiologia , Miócitos Cardíacos/citologia , Poliésteres/química , Engenharia Tecidual/métodos , Animais , Animais Recém-Nascidos , Materiais Biocompatíveis/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Células Cultivadas , Conexina 43/metabolismo , Decanoatos , Glicerol/química , Coração/efeitos dos fármacos , Teste de Materiais , Camundongos , Impressão Molecular , Miócitos Cardíacos/efeitos dos fármacos , Polímeros , Ratos Sprague-Dawley , Propriedades de SuperfícieRESUMO
Cardiomyocytes withdraw from the cell cycle in the early neonatal period, rendering the adult heart incapable to regenerate after injury. In the present study, we report the establishment of a cell-free system to investigate the control of cell cycle reentry in mammalian ventricular cardiomyocyte nuclei and to specifically address the question of whether nuclei from terminally differentiated cardiomyocytes can be stimulated to reenter S phase when incubated with extracts from S-phase cells. Immobilized cardiomyocyte nuclei were incubated with nuclei and cytoplasmic extract of synchronized H9c2 muscle cells or cardiac nonmyocytes. Ongoing DNA synthesis was monitored by biotin-16-dUTP incorporation as well as proliferating cell nuclear antigen expression and localization. Nuclei and cytoplasmic extract from S-phase H9c2 cells but not from H9c2 myotubes induced DNA synthesis in 92% of neonatal cardiomyocyte nuclei. Coincubation in the presence of cycloheximide indicated that de novo translation is required for the reinduction of S phase. Similar results were obtained with adult cardiomyocyte nuclei. When coincubated with both cytoplasmic extract and nuclei or nuclear extracts of S-phase cells, >70% of adult cardiomyocyte nuclei underwent DNA synthesis. In conclusion, these results demonstrate that postmitotic ventricular myocyte nuclei are responsive to stimuli derived from S-phase cells and can thus bypass the cell cycle block. This cell-free system now makes it feasible to analyze the molecular requirements for the release of the cell cycle block and will help to engineer strategies for regenerative growth in cardiac muscle.