Internal structures of scaffold-free 3D cell cultures visualized by synchrotron radiation-based micro-computed tomography
Belma SaldamliJulia HerzenFelix BeckmannJutta TübelJohannes SchauweckerRainer BurgkartPhilipp JürgensHans‐Florian ZeilhoferRobert SaderBert Müller
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Abstract:
Recently the importance of the third dimension in cell biology has been better understood, resulting in a re-orientation towards three-dimensional (3D) cultivation. Yet adequate tools for their morphological characterization have to be established. Synchrotron radiation-based micro computed tomography (SRμCT) allows visualizing such biological systems with almost isotropic micrometer resolution, non-destructively. We have applied SRμCT for studying the internal morphology of human osteoblast-derived, scaffold-free 3D cultures, termed histoids. Primary human osteoblasts, isolated from femoral neck spongy bone, were grown as 2D culture in non-mineralizing osteogenic medium until a rather thick, multi-cellular membrane was formed. This delicate system was intentionally released to randomly fold itself. The folded cell cultures were grown to histoids of cubic milli- or centimeter size in various combinations of mineralizing and non-mineralizing osteogenic medium for a total period of minimum 56 weeks. The SRμCT-measurements were performed in the absorption contrast mode at the beamlines BW 2 and W 2 (HASYLAB at DESY, Hamburg, Germany), operated by the GKSS-Research Center. To investigate the entire volume of interest several scans were performed under identical conditions and registered to obtain one single dataset of each sample. The histoids grown under different conditions exhibit similar external morphology of globular or ovoid shape. The SRμCT-examination revealed the distinctly different morphological structures inside the histoids. One obtains details of the histoids that permit to identify and select the most promising slices for subsequent histological characterization.Keywords:
Mineralized tissues
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INTRODUCTION: Cell culture experiments are typically performed as in vitro studies based on 2D seeding and characterization. Here, the procedures can be kept relatively simple. With respect to the in vivo situation, however, 2-D models are often inappropriate due to the 3-D character of living tissue in nature. Consequently, 3-D in vitro systems should better resemble the natural situation. For the 3-D in vitro studies suitable scaffolds have to be chosen and methods for 3-D characterization have to be adapted. Micro-tomography using X-rays (µCT) belongs to the most promising techniques for 3-D characterization. Using synchrotron radiation sources, the spatial resolution can be extended down to the sub-micrometer range and individual cells can be made visible. Since the samples consist mainly of light elements, the cells have to be labelled by the use of highly absorptive agents, well known from electron microscopy, to obtain enough contrast in the most frequently used absorption contrast modus. Contrary to electron microscopy, µCT does not need vacuum conditions making experiments in the hydrated state possible. The aim of the current study is the optimisation of µCT to uncover cell shape and cell distribution on porous scaffolds, which can be even opaque, in the hydrated environment. METHODS: Tomography is a technique to nondestructively reconstruct a 3-D image of a solid state from a set of filtered 2-D projections. Using the parallel beams of a synchrotron radiation source, the sample is precisely rotated stepwise to 180°, and after each step a projection is recorded. Because the contrast mechanism is X-ray absorption, one gets a representation of the local absorption coefficients. 1 This means that the constituents have to differ in X-ray absorption to allow segmentation. The samples, rat tendon fibroblasts seeded on texturized poly-ethylene-teraphtalate (PET) multifilament yarns (30 filaments with a diameter of 20 µm each) embedded in a hydrated matrix, were held in low absorbing glass or plastic containers. Since the fibroblasts show low X-ray absorption as the surrounding embedding and scaffold material it is inevitable to stain the cells with a higher absorbing contrast agent. For first experiments, OsO4 has been used. The sample preparation
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We present a method in which a precise region of interest within an intact organism is spatially mapped in three dimensions by non-invasive micro-computed X-ray tomography (micro-CT), then further evaluated by light microscopy (LM) and transmission electron microscopy (TEM). Tissues are prepared as if for TEM including osmium fixation, which imparts soft tissue contrast in the micro-CT due to its strong X-ray attenuation. This method may therefore be applied to embedded, archived TEM samples. Upon selection of a two-dimensional (2-D) projection from a region of interest (ROI) within the three-dimensional volume, the epoxy-embedded sample is oriented for microtomy so that the sectioning plane is aligned with the micro-CT projection. Registration is verified by overlaying LM images with 2-D micro-CT projections. Structures that are poorly resolved in the micro-CT may be evaluated at TEM resolution by observing the next serial ultrathin section, thereby accessing the same ROI by all three imaging techniques. We compare white adipose tissue within the forelimbs of mice harboring a lipid-altering mutation with their littermate controls. We demonstrate that individual osmium-stained lipid droplets as small as 15 µm and separated by as little as 35 µm may be discerned as separate entities in the micro-CT, validating this to be a high-resolution, non-destructive technique for evaluation of fat content.
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The aim of this work was to introduce high-resolution computed tomography (micro-CT) for scaffolds made from soft natural biomaterials, and to compare these data with the conventional techniques scanning electron microscopy and light microscopy. Collagen-based scaffolds were used as examples. Unlike mineralized tissues, collagen scaffolds do not provide enough X-ray attenuation for micro-CT imaging. Therefore, various metal-based contrast agents were applied and evaluated using two structurally distinct scaffolds, one with round pores and one with unidirectional lamellae. The optimal contrast techniques for obtaining high-resolution three-dimensional images were either a combination of osmium tetroxide and uranyl acetate, or a combination of uranyl acetate and lead citrate. The data obtained by micro-CT analysis were in line with data obtained by light and electron microscopy. However, small structures (less than a few mum) could not be visualized due to limitation of the spot size of the micro-CT apparatus. In conclusion, reliable three-dimensional images of scaffolds prepared from soft natural biomaterials can be obtained using appropriate contrast protocols. This extends the use of micro-CT analysis to soft materials, such as protein-based biomaterials.
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