Tomography studies of human foreskin fibroblasts on polymer yarns

Cell culture experiments are usually performed as in vitro studies based on 2D seeding and characterization (light microscopy). With respect to the in vivo situation, however, 2D studies are often inappropriate due to the 3D character of living tissue in nature. Textiles with their versatile 3D structures are chosen as suitable scaffolds in tissue engineering for 3D in vitro studies. Micro-computed tomography using X-rays (μCT) belongs to the most promising techniques for isotropic, noninvasive 3D characterization. Using synchrotron radiation (SRμCT) the spatial resolution can be extended to the sub-micrometer range well below cell size. μCT does not need vacuum conditions making experiments in the hydrated state possible, as we show by data from SRμCT acquired at second and third-generation synchrotron sources. We seeded human foreskin fibroblasts on polymer multifilament yarns. These composites, embedded in a hydrogel or fluid, are held in thin-walled glass capillaries. Since the composites consist of light elements, the cells have to be labeled for visualization by the use of highly absorptive agents, osmium and gold. In order to hold the label concentration as low as possible, we present a way to choose the photon energy for which the minimum concentration is reached. Differences in threshold selection for second- and third-generation synchrotron sources are pointed out, revealing the advantages of both types with respect to quantitative analysis. The study is based on appropriate staining methods and protocols developed in our laboratory. With the results we demonstrate that SRμCT yields images similar to established electron and light microscopy but uncovers also the microstructure in 3D space.