Pancreatic islets are heavily vascularized in vivo with each insulin secreting beta-cell associated with at least one endothelial cell (EC). This structure is maintained immediately post-isolation; however, in culture the ECs slowly deteriorate, losing density and branched morphology. We postulate that this deterioration occurs in the absence of blood flow due to limited diffusion of media inside the tissue. To improve exchange of media inside the tissue, we created a microfluidic device to culture islets in a range of flow-rates. Culturing the islets from C57BL6 mice in this device with media flowing between 1 and 7 ml/24 hr resulted in twice the EC-density and -connected length compared to classically cultured islets. Media containing fluorescent dextran reached the center of islets in the device in a flow-rate-dependant manner consistent with improved penetration. We also observed deterioration of EC morphology using serum free media that was rescued by addition of bovine serum albumin, a known anti-apoptotic signal with limited diffusion in tissue. We further examined the effect of flow on beta-cells showing dampened glucose-stimulated Ca2+-response from cells at the periphery of the islet where fluid shear-stress is greatest. However, we observed normal two-photon NAD(P)H response and insulin secretion from the remainder of the islet. These data reveal the deterioration of islet EC-morphology is in part due to restricted diffusion of serum albumin within the tissue. These data further reveal microfluidic devices as unique platforms to optimize islet culture by introducing intercellular flow to overcome the restricted diffusion of media components.

No abstract

In situ atomic force microscopy (AFM) is an exceedingly powerful and useful technique for characterizing the structure and assembly of proteins in real-time, in situ, and especially at model membrane interfaces, such as supported planar lipid bilayers. There remains, however, a fundamental challenge with AFM-based imaging. Conclusions are inferred based on morphological or topographical features. It is conventionally very difficult to use AFM to confirm specific molecular conformation, especially in the case of protein-membrane interactions. In this case, a protein may undergo subtle conformational changes upon insertion in the membrane that may be critical to its function. AFM lacks the ability to directly measure such conformational changes and can, arguably, only resolve features that are topographically distinct. To address these issues, we have developed a platform that integrates in situ AFM with attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. This combination of tools provides a unique means of tracking, simultaneously, conformational changes, not resolvable by in situ AFM, with topographical details that are not readily identified by conventional spectroscopy. Preliminary studies of thermal transitions in supported lipid bilayers and direct evidence of lipid-induced conformational changes in adsorbed proteins illustrates the potential of this coupled in situ functional imaging strategy.

invasion of hosts and disease initiation and progression. In order to monitor the pathogen invasion process, most often one mixes a solution containing pathogens into a culture of host cells, and then hopes the host cell being monitored is invaded by a pathogen. These chance encounters have been monitored using fluorescence microscopy, allowing for example fluorescent effector proteins to be monitored as they are injected into a host cell (Enninga, Mounier et al. 2005; Schlumberger, Muller et al. 2005). Such ''mix and hope'' strategies are simple, direct, and necessary for initial studies. However, they do not provide a path to obtain large sample sizes, control of timing of pathogen invasion, or a way to determine how many bacteria are required to defeat host defenses. We present recent results from using optoelectronic tweezers (OET) (Chiou, et al. 2005) as a tool for manipulating single pathogenic bacteria, opening a promising route for controlled initiation of HP interactions. Optoelectronic trapping uses laser-excited carriers inside a thin film of amorphous or crystalline Si to create a non-uniform electric field. These carriers, coupled with an electric field modulated in the MHz range produces strong field gradients. The object being trapped is polarized by the electric field, and gradients in the electric field create a potential well which traps the object. This trapping is obtained at extremely low optical intensities (<1W/cm^2) and does not require functionalization of the pathogen in order to facilitate delivery to the host, opening up novel possibilities for massively parallel studies of HP interactions. Human mesenchymal stem cells (hMSCs) express markers of different lineages when grown on matrices of an elasticity that mimics various tissues of mesenchymal origin. Of particular interest to our group is the possibility of using this approach to drive hMSCs into skeletal muscle lineage for repair of damaged skeletal muscle. In order to identify and determine the necessary in vitro culture conditions that will lead to optimal commitment of cells to the muscle lineage in vivo, we have been developing biophysical techniques to allow stem cell precommitment to a specific lineage prior to implantation in a diseased tissue by controlling matrix stiffness under in vitro culture conditions. This technology has led to a highly reproducible approach to optimizing stem cell fate for human transplantation. The physical nature of a cell's microenvironment -including elasticity of the surrounding tissue -appears to exert a significant influence on cell morphology, cytoskeleton and gene expression. Numerous gel systemsparticularly polyacrylamide gels -have tunable elasticity that can be adjusted over several orders of magnitude from extremely soft to stiff, mimicking the elasticity of a wide range of tissues by controlling the extent of polymer cross-linking. Here, we use cross-linked polyacrylamide hydrogels that mimic the true in-vivo muscle-like elasticity (10-15 kPa) to induce hMSCs to differentiate into my...