In vitro quantification of the effect of mechanical loads on cells by live microscopy requires precise control of load and culture environment. Corresponding systems are often bulky, their setup and maintenance are time consuming, or the cell yield is low. Here, we show the design and initial testing of a new cell culture system that fits on standard light microscope stages. Based on the parallel plate principle, the system allows for live microscopy of cells exposed to flow-induced shear stress, features short setup time and requires little user interaction. An integrated feedback-controlled heater and a bubble trap enable long observation times. The key design feature is the possibility for quick exchange of the cultured cells. We present first test results that focus on verifying the robustness, biocompatibility, and ease of use of the device.

BACKGROUND:The application of acellular matrices, biomaterials, and polymeric scaffolds in reconstructive surgery facilitates postsurgical tissue remodeling and is increasingly used clinically in order to improve tissue healing and implant coverage. This study presents an in vivo investigation of the integration of the knitted, silk-derived surgical scaffold, SERI(®) with regard to angiogenesis and wound healing. METHODS:SERI(®) Surgical Scaffold was implanted into a full-thickness skin defect in male C57BL/6J mice (n = 45) via the dorsal skinfold chamber (DSC). Skin tissue samples were collected for histology on days 2, 5, 7, 10, 14, and 21 (n = 5 per time point) post implantation. Immunohistochemistry was performed for various angiogenic and inflammatory markers, as well as collagen deposition (CD31, VEGF, CD3, CD45, Desmin, and Sirius red). Vascular corrosion casting was used to assess the neovasculature within the silk and was visualized with scanning electron microscopy. RESULTS:We observed both early and late stages of inflammation during the healing process characterized by the infiltration of regenerating tissue by different subsets of leukocytes. Histological analysis displayed capillary-containing granulation tissue with full scaffold integration. In addition, collagen deposition within the scaffold and full skin defect was significantly increased over time. Qualitative analysis of the regenerated vasculature through corrosion casting and scanning electron microscopy revealed a complex, angiogenic network of capillaries originating from the wound bed. CONCLUSIONS:Based on these findings, SERI(®) displays the potential to be a promising resorbable bioengineered material for use in reconstructive surgery.

Malfunction of oxygen regulation in kidney and liver may lead to the pathogenesis of chronic diseases. The underlying mechanisms are poorly understood. In kidney, it is hypothesized that renal gas shunting from arteries to veins eliminates excess oxygen. Such shunting is highly dependent on the structure of the renal vascular network. The vascular tree has so far not been quantified under maintenance of its connectivity as three-dimensional imaging of the vessel tree down to the smallest capillaries, which in mouse model are smaller than 5 μm in diameter, is a challenging task. An established protocol uses corrosion casts and applies synchrotron radiation-based micro-computed tomography (SRμCT), which provides the desired spatial resolution with the necessary contrast. However, SRμCT is expensive and beamtime access is limited. We show here that measurements with a phoenix nanotomrm (General Electric, Wunstorf, Germany) can provide comparable results to those obtained with SRμCT, except for regions with small vessel structures, where the signal-to-noise level was significantly reduced. For this purpose the nanotom®m measurement was compared with its corresponding measurement acquired at the beamline P05 at PETRA III at DESY, Hamburg, Germany.

Fluorescence long-term imaging of cellular processes in three-dimensional cultures requires the control of media supply, temperature, and pH, as well as minimal photodamage. We describe a system based on a light sheet fluorescence microscope (LSFM), which is optimized for long-term, multi-position imaging of three-dimensional in-gel cell cultures. The system integrates a stable culture condition control system in the optical path of the light-sheet microscope. A further essential element is a biocompatible agarose container suitable for the LSFM, in which any cell type can be cultured in different gel matrices. The TC-LSFM allows studying any in vitro cultured cell type reacting to, dividing in, or migrating through a three-dimensional extracellular matrix (ECM) gel. For this reason we called it "tissue culture-LSFM" (TC-LSFM). The TC-LSFM system allows fast imaging at multiple locations within a millimeter-sized ECM gel. This increases the number of analyzed events and allows testing population effects. As an example, we show the maturation of a cyst of MDCK (canine kidney epithelial) cells over a period of three days. Moreover, we imaged, tracked, and analyzed MDCK cells during the first five days of cell aggregate formation and discovered a remarkable heterogeneity in cell cycle lengths and an interesting cell death pattern. Thus, TC-LSFM allows performing new long-term assays assessing cellular behavior in three-dimensional ECM-gel cultures. For example migration, invasion or differentiation in epithelial cell systems, stem cells, as well as cancer cells can be investigated.

Topographic modifications of the substrate of a cell culture have the potential to guide cell polarization and migration, through which epidermal wound healing may be accelerated. Classic topographic contact guidance is based on the interaction between cells and a supporting scaffold that interferes with the establishment of focal adhesions, thereby influencing the organization of the actin cytoskeleton. Using soft lithography techniques on PDMS, we engineered gratings with groove and ridge widths of 1 μm and a groove depth of 0.6 μm. These gratings were applied to the apical free surface of human dermal fibroblasts during in vitro wound healing. Gratings oriented perpendicularly to the wound induced a significant enhancement of cell polarization, migration speed and directionality which resulted in faster wound coverage. The apically applied texture influenced the deposition of the extracellular matrix into the wound yielding homogeneously distributed fibronectin fibers. Apical guidance was not mediated by the establishment of focal adhesions between cells and the topographically modified patch, thus allowing for removal of the latter after complete healing. Altogether, our results demonstrate an alternative guidance scheme based on the apical, adhesion-free interaction between migrating cells and an anisotropic surface topography, which leads to faster healing in an in vitro wound model.

The brain's response to external painful stimuli can be assessed through contact heat evoked cortical potentials that enable the evaluation of the integrity of pain pathways. This work aims to improve the reliability of this diagnostic procedure by decoupling the effects of heat transfer and nerve fiber conduction. It is herein shown experimentally that the latency of the N2 contact heat evoked cortical potentials component is the most stable diagnostic parameter. The contribution of heat transfer to N2 contact heat evoked cortical potentials latency was modeled as a function of the subject's pain threshold, allowing for the separation of nerve fiber pathology from thermodynamic influences.

Cell motility contributes to the formation of organs and tissues, into which multiple cells self‐organize. However such mammalian cellular motilities are not characterized in a quantitative manner and the systemic consequences are thus unknown. A mathematical tool to decipher cell motility, accounting for changes in cell shape, within a three‐dimensional (3D) cell system was missing. We report here such a tool, usable on segmented images reporting the outline of clusters (cells) and allowing the time‐resolved 3D analysis of circular motility of these as parts of a system (cell aggregate). Our method can analyze circular motility in sub‐cellular, cellular, multi‐cellular, and also non‐cellular systems for which time‐resolved segmented cluster outlines are available. To exemplify, we characterized the circular motility of lumen‐initiating MDCK cell aggregates, embedded in extracellular matrix. We show that the organization of the major surrounding matrix fibers was not significantly affected during this cohort rotation. Using our developed tool, we discovered two classes of circular motion, rotation and random walk, organized in three behavior patterns during lumen initiation. As rotational movements were more rapid than random walk and as both could continue during lumen initiation, we conclude that neither the class nor the rate of motion regulates lumen initiation. We thus reveal a high degree of plasticity during a developmentally critical cell polarization step, indicating that lumen initiation is a robust process. However, motility rates decreased with increasing cell number, previously shown to correlate with epithelial polarization, suggesting that migratory polarization is converted into epithelial polarization during aggregate development. © 2010 Wiley‐Liss, Inc.