3D Tissue Cell Research Articles

A three-dimensional A549 cell culture model to study respiratory syncytial virus infections

BackgroundRespiratory syncytial virus (RSV) is a primary cause of morbidity and mortality worldwide, affecting infants, young children, and immune-compromised patients; however, currently no vaccine is available for prevention of RSV infections. The overwhelming majority of our knowledge of how RSV causes infection is based upon studies that have been carried out using traditional 2D methods, with cells cultured on flat plastic dishes. Although these simplified culture systems are essential to gain an insight into the fundamentals of host-pathogen interactions, cells in 2D are not exposed to the same conditions as cells in 3D tissues in the body and are therefore a poor representation of thein vivo microenvironment. In this study, we aim to develop the first 3D culture model for RSV infection using A549 cells to test its utility for RSV pathogenesis. MethodsTo generate spheroids, A549 cells were cultured using ultra-low attachment plates to generate 25 × 103 cell spheroids. The viability of the spheroids was assessed by trypan blue exclusion assay and flow cytometry showing prominent live cells throughout the spheroids confirming high viability over seven days of incubation. ResultsImmunostaining of A549 spheroids inoculated with RSV, showed time-dependent dissemination of the viral antigen RSV-F within the spheroid, resulting in syncytia formation and a 3-fold increase in mucin secretion compared to the uninfected cells. Additionally, RSV successfully replicated in the spheroids producing infectious virus as early as day one post-inoculation and was sustained for up to 7 days post-inoculation. ConclusionsResults show that A549 spheroids are susceptible and permissive for RSV since they exhibit the characteristics of RSV infection including syncytia formation and mucin overexpression, suggesting that A549 spheroids can be used a promising model for studying RSV in vitro.

Elastic Fully Three‐dimensional Microstructure Scaffolds for Cell Force Measurements

2010 WILEY-VCH Verlag Gm Mechanical interactions between cells and their environment play an important role in regulating many cellular functions, such as migration, differentiation, and proliferation. They are also involved in complex processes like tissue formation during development of multicellular organisms. During the last two decades, several methods have been developed to visualize and measure traction forces produced by single cells. Initial experiments were performed by growing fibroblasts on a thin silicon rubber substrate that was deformed by forces exerted by migrating cells. Subsequently, elastic polyacrylamide substrates embedded with fluorescent latex beads were used to get an insight into the force vectors exerted by the cells. These methods were further improved by producing regular arrays of traceable markers in the elastic substrates allowing a quantification of traction forces exerted by adhering cells. A different approach has used arrays of elastic posts positioned on a flat 2D substrate, coated with extracellular-matrix (ECM)molecules. Because each post effectively acts like a harmonic spring, local traction forces at multiple cell adhesion sites could then be measured by simply monitoring post deflections. A modification of this assay was recently applied to measure contraction forces of single cardiomyocytes. Although these different approaches have provided valuable insight into our understanding of cellular forces, they have the disadvantage that the cells are forced into a 2D growth pattern, which significantly differs from the in vivo situation. Therefore, the development of in vitro model systems that capture more of the complexity present in 3D tissue scaffolds are highly desirable. Technologies for fabricating macroscopic tissue architectures in the millimeter and micrometer range have become available only during recent years. In contrast, methods to reliably produce flexible 3D cellular environments in the micrometer to nanometer range are still missing. Ideally, these scaffolds should have a controllable distribution of cell-substrate contact sites and an adjustable stiffness. In this Communication, we show that such biocompatible scaffolds can be fabricated by means of direct laser writing (DLW) and subsequent surface functionalization. We furthermore demonstrate that these scaffolds can be rhythmically deformed by single beating cardiomyocytes. In addition, we quantitatively evaluate the involved contraction forces by calibrating these structures using atomic force microscopy (AFM). In particular, with the method presented here, beamlike structures with defined geometries and physiologically relevant stiffness values can be produced; thus, these scaffolds structurally and mechanically mimic large macromolecules surrounding cells in 3D tissues, such as collagen fibrils. In essence, DLW (see the Experimental section) can be thought of as a pencil of light in three dimensions. Any connected structure envisioned on a computer can easily be implemented by scanning a photoresist relative to the focus of a femtosecond-laser beam (Fig. 1a). After development of a negative resist, sufficiently exposed regions correspond to the remaining polymer (Fig. 1b). Lithography of 3D polymeric templates by DLW with lateral feature sizes down to the 100 nm range has become routine in the field of nanophotonics. Along these lines, fully 3D (Fig. 1c) scaffolds can be fabricated. Sample footprints of mm up to cm dimensions can be realized by combining 300mm 300mm areas that are accessible within the piezo scanning range. The height of the structures is limited only by the working distance of the microscope lens (80mm in our case). Typical writing times for the structures shown in this Communication are less than 10min. Here, we employ the commercially available resist system Ormocomp (obtained from Micro Resist Technology GmbH). Ormocomp is a member of Ormocer, a class of inorganic (Si O Si)–organic hybrid polymer systems, previously developed at the Fraunhofer Institute for Silica Research (Wurzburg, Germany). Its detailed chemical composition is proprietary. With Ormocomp as a photoresist, complex fully 3D structures (Fig. 1a–c) can be easily manufactured and, after functionalization with fibronectin, directly used for cell culture (Fig. 1d). To demonstrate that single cells can lead to noticeable deformation of the 3D Ormocomp scaffolds, primary cardiomyocytes are isolated from chicken embryos (see the