Topography and Permeability Analyses of Vasculature-on-a-Chip Using Scanning Probe Microscopies.

Microphysiological systems (MPS) or organs-on-chips (OoC) can emulate the physiological functions of organs in vitro and are effective tools for determining human drug responses in preclinical studies. However, the analysis of MPS has relied heavily on optical tools, resulting in difficulties in real-time and high spatial resolution imaging of the target cell functions. In this study, the role of scanning probe microscopy (SPM) as an analytical tool for MPS is evaluated. An access hole is made in a typical MPS system with stacked microchannels to insert SPM probes into the system. For the first study, a simple vascular model composed of only endothelial cells is prepared for SPM analysis. Changes in permeability and local chemical flux are quantitatively evaluated during the construction of the vascular system. The morphological changes in the endothelial cells after flow stimulation are imaged at the single-cell level for topographical analysis. Finally, the possibility of adapting the permeability and topographical analysis using SPM for the intestinal vascular system is further evaluated. It is believed that this study will pave the way for an in situ permeability assay and structural analysis of MPS using SPM.

Micropipet-Based Navigation in a Microvascular Model for Imaging Endothelial Cell Topography Using Scanning Ion Conductance Microscopy.

Scanning ion conductance microscopy (SICM) has enabled cell surface topography at a high resolution with low invasiveness. However, SICM has not been applied to the observation of cell surfaces in hydrogels, which can serve as scaffolds for three-dimensional cell culture. In this study, we applied SICM for imaging a cell surface in a microvascular lumen reconstructed in a hydrogel. To achieve this goal, we developed a micropipet navigation technique using ionic current to detect the position of a microvascular lumen. Combining this navigation technique with SICM, endothelial cells in a microvascular model and blebs were visualized successfully at the single-cell level. To the best of our knowledge, this is the first report on visualizing cell surfaces in hydrogels using a SICM. This technique will be useful for furthering our understanding of the mechanism of intravascular diseases.

Fabrication of three-dimensional calcium alginate hydrogels using sacrificial templates of sugar.

Hydrogels are receiving increasing attention in bioapplications. Among hydrogels, calcium alginate (Ca-alginate) hydrogels are widely used for their biocompatibility, low toxicity, low cost, and rapid fabrication by simple mixing of Ca2+ and sodium alginate (Na-alginate). For bioapplications using hydrogels, it is necessary to construct designed hydrogel structures. Although several methods have been proposed for fabricating designed hydrogels, a simple and low-cost method is desirable. Therefore, we developed a new method using sacrificial templates of sugar structures to fabricate three-dimensional (3D) designed Ca-alginate hydrogels. In this method, Na-alginate solution is mixed with molten sugar, and the resulting highly viscous material used to mold 3D sugar structures as sacrificial templates. Since sugar constructs are easily handled compared to hydrogels, sugar templates are useful for preparing 3D constructs. Finally, the sugar and Na-alginate structure is immersed in a CaCl2 solution to simultaneously dissolve the template and form the Ca-alginate hydrogel. The resulting hydrogel takes the shape of the sugar template. By stacking and fusing various sugar structures, such as fibers and blocks, 3D designed Ca-alginate hydrogels can be successfully fabricated. This simple and low-cost method shows excellent potential for application to a variety of bioapplications.

Electrochemical fabrication of fibrin gels via cascade reaction for cell culture.

We present a new strategy for fabricating fibrin gels by electrochemically controlling a cascade reaction and its application in cell culture. In this strategy, Ca2+ is released from CaCO3 particles via electrochemical acidification. In the presence of the released Ca2+, the activated cascade reaction produces fibrin gels.

Electrodeposition-based rapid bioprinting of 3D-designed hydrogels with a pin art device.

Three-dimensional (3D) designed hydrogels are receiving considerable attention for use in tissue engineering. Herein, we present a novel method for bioprinting 3D hydrogels by electrodeposition with a pin art device. The device consists of a metal substrate and an array of electrode pins that can slide independently. To fabricate a 3D-hydrogel, pins are pushed from the rear with a 3D object to generate a 3D extruded-pin relief of the object; the extruded pins are then inserted into a chitosan/gelatin hydrogel. Due to H+ consumption at these pins, which collectively act as a cathode, the protonated amino groups of the chitosan become deprotonated, which results in the electrodeposition of the chitosan bound to the gelatin onto the extruded pins. The untreated hydrogel is removed by heating to provide the 3D-designed chitosan/gelatin hydrogel. As a proof of concept, hydrogels of various shapes were fabricated. In addition, cells were successfully cultured in a hydrogel, highlighting its biocompatibility. This method is useful for constructing 3D artificial tissue consisting of hydrogels and cells.

Electrochemical Imaging of Cell Activity in Hydrogels Embedded in Grid-shaped Polycaprolactone Scaffolds Using a Large-scale Integration-based Amperometric Device.

Tissue engineering requires analytical methods to monitor cell activity in hydrogels. Here, we present a method for the electrochemical imaging of cell activity in hydrogels embedded in printed polycaprolactone (PCL) scaffolds. Because a structure made of only hydrogel is fragile, PCL frameworks are used as a support material. A grid-shaped PCL was fabricated using an excluder printer. Photocured hydrogels containing cells were set at each grid hole, and cell activity was monitored using a large-scale integration-based amperometric device. The electrochemical device contains 400 microelectrodes for biomolecule detection, such as dissolved oxygen and enzymatic products. As proof of the concept, alkaline phosphatase and respiration activities of embryonic stem cells in the hydrogels were electrochemically monitored. The results indicate that the electrochemical imaging is useful for evaluating cells in printed scaffolds.

Hydrogel electrodeposition based on bipolar electrochemistry.

Bipolar electrochemistry has attracted great interest for applications based on sensing, electrografting, and electrodeposition, because the technique enables electrochemical reactions to be induced at multiple bipolar electrodes (BPEs) with only a single power supply. However, there are only a few reports on the biofabrication of hydrogels using BPEs. In this study, we applied bipolar electrochemistry to achieve the electrodeposition of calcium-alginate hydrogels at specified target areas, which is possible because of the use of water electrolysis to obtain acidification at the anodic pole. This scheme was used to successfully fabricate an array of hydrogel deposits at a BPE array. In addition, hydrogels were successfully fabricated either at only the target BPEs or only the target areas of BPEs by repositioning the driving electrodes. Furthermore, a hydrogel was drawn on a large BPE as a canvas by using small driving electrodes. As a demonstration of the electrodeposited hydrogels for bioapplications, mammal cells were cultured in the hydrogels. Because the amount and shape of the hydrogel deposits can be controlled by using the bipolar system, the system we developed can be used for biosensors and cell culture platforms.