Bump, Steven (2015)
Microscopic characterization of functionalized paper as a platform for 3D cell cultures.
Technische Universität Darmstadt
Dissertation, Erstveröffentlichung

Kurzbeschreibung (Abstract)

To achieve an understanding and complete description of the functional properties of three dimensional (3D) cell culture systems, a large set of parameters is required, which clearly contrasts this cell cultivation approach from traditional two dimensional (2D), planar cultivation techniques . As an alternative to describe the characteristics of a 3D cell culture system by its physicochemical properties (e. g. stiffness, porosit y, level of crosslinking), the behavior of the cultivated cells can be used as a read-out parameter to characterize the 3D cultivation system . In this work, the cellular parameters membrane dynamics, actin fiber morphology and migration were used to investigate the differences between classic, planar and a collagen based three dimensional cell culture system

Membrane dynamics – assessed by FRAP measurements of CAAX -mCherry – as well as actin formation – visualized by in vitro staining with LifeAct -tagRFP

– showed distinct differences when investigated in planar or three dimensional systems. FRAP experiments with CAAX - mCherry showed, that even though the overall membrane composition does not appear to be different, mobility of the membrane is significantl y higher in three dimensional cell culture systems than in two dimensional . A view at the actin cytoskeleton revealed the already established difference: stress fibers and cortical actin are more pronounced in planar cell culture systems compared to cells c ultivated in three dimensional systems. Interestingly, cells originall y seeded in collagen hydrogels which migrated towards the glass surface show features in actin cytoskeleton formation resembling both culture conditions: both, actin stress fibers within the cell body as well as cortical actin are visible in those parts of the cells directly contacting the glass surface .

The observed migration towards the glass surface gave rise to the investigation of this behavior. Migration in response to mechanical si gnals is termed durotaxis . Cells cultivated in collagen hydrogels or collagen hydrogels supported by cellulose sheets over a period of time were microscopicall y investigated to determine the distribution of cells . Cell distribution in unsupported collagen hydrogels was clearly in favor of hydrogel regions in close proximity to the glass surface. By applying supporting material in form of cellulose sheets, the cell culture was freely floating in the culture medium, resulting in an even distribution throughout the entire thickness of the cell culture system .

As 3D cell culture systems make it more challenging to perform high quality imaging due to the inherent scattering and loss of intensity with increasing optical penetration, a post imaging processing tool set was evaluated and benchmarked in order to counteract these image corrupting effects and improve the image quality. This in turn also improves the compatibility of cellulose sheets with the commonly used tool set in life sciences: fluorescence microscopy. Special emphasis was put on the identification of a serviceable and performance -linked deconvolution setup . A GPU based CUDA Deconvolution plugin showed the best time performance but ultimately failed to produce the same quality level of image restoration as the three tested CPU based deconvolution applications. Among these three, the commercial HyugensPro software showed the best results in terms of increasing the contrast . The Iterative Deconvolution 3D plugin comes close to producing comparable results to the HuygensPro software, however, the time consumption for this application is up to 10 times larger. Finally, the plugin Deconvolution Lab showed reasonably satisfying results in terms of image restoration quality, while performing deconvolution slightly faster than HuygensPro.

Finally, cellulose sheets are used for the cultivation of cells in 3D as an example of for paper as a versatile platform for the development of functional devices . Therefore a method is required that delivers spatially resolved, quantitative, sensitive, and, most importantly, also dynamic measurements . Optical microscopy has long been recognized as a method to characterize the heterogeneous and complex structure of paper. With fluorescence detection, the functionality has even been extended to provide chemical selectivity, e . g. to determine the distribution of secondary modifications like coatings and fillers throughout a sheet of paper. Here it is shown that quantitative widefield and confocal fluorescence microscopy are versatile methods to meet this set of demands. Confocal microscopy was used to achieve a detailed view of the interface between a hyd rophobic and rhodamine labeled polymer and a FITC labeled dextran solution. Furthermore, confocal microscopy revealed that the spatial propagation of the FITC labeled dextran solution occurs along the surface of the cellulose fibers, instead of the inter -fibers space. Widefield fluorescent microscopy was subsequently used for dynamic investigations of this spatial propagation.

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