Takeaki Kawashima

Micropatterning contractile C2C12 myotubes embedded in a fibrin gel

Contractile C2C12 myotube line patterns embedded in a fibrin gel have been developed to afford a physiologically relevant and stable bioassay system. The C2C12 myotube/fibrin gel system was prepared by transferring a myotube monolayer from a glass substrate to a fibrin gel while retaining the original line patterns of myotubes. To endow the myotubes with contractile activity, a series of electrical pulses was applied through a pair of carbon electrodes placed at either side of a fibrin gel separately. The frequency and magnitude of myotube contraction were functions of the pulse frequency and duration, respectively. We found that the myotubes supported by an elastic fibrin gel maintained their line patterns and contractile activities for a longer period of time (1 week) than myotubes adhered on a conventional culture dish. Biotechnol. Bioeng. 2010;105: 1161–1167.

A scalable controlled-release device for transscleral drug delivery to the retina.

A transscleral drug-delivery device, designed for the administration of protein-type drugs, that consists of a drug reservoir covered with a controlled-release membrane was manufactured and tested. The controlled-release membrane is made of photopolymerized polyethylene glycol dimethacrylate (PEGDM) that contains interconnected collagen microparticles (COLs), which are the routes for drug permeation. The results showed that the release of 40-kDa FITC-dextran (FD40) was dependent on the COL concentration, which indicated that FD40 travelled through the membrane-embedded COLs. Additionally, the sustained-release drug formulations, FD40-loaded COLs and FD40-loaded COLs pelletized with PEGDM, fine-tuned the release of FD40. Capsules filled with COLs that contained recombinant human brain-derived neurotrophic factor (rhBDNF) released bioactive rhBDNF in a manner dependent on the membrane COL concentration, as was found for FD40 release. When capsules were sutured onto sclerae of rabbit eyes, FD40 was found to spread to the retinal pigment epithelium. Implantation of the device was easy, and it did not damage the eye tissues. In conclusion, our capsule is easily modified to accommodate different release rates for protein-type drugs by altering the membrane COL composition and/or drug formulation and can be implanted and removed with minor surgery. The device thus has great potential as a conduit for continuous, controlled drug release.

Generation of Patterned Cell Co-Cultures in Silicone Tubing Using a Microelectrode Technique and Electrostatic Assembly

We report a method for producing patterned cell adhesion inside silicone tubing. A platinum needle microelectrode was inserted through the wall of the tubing and an oxidizing agent electrochemically generated at the inserted electrode. This agent caused local detachment of the anti-biofouling heparin layer from the inner surface of the tubing. The cell-adhesive protein fibronectin selectively adsorbed onto the newly exposed surface, making it possible to initiate a localized cell culture. The electrode could be readily set in place without breaking the tubular structure and, importantly, almost no culture solution leaked from the electrode insertion site after the electrode was removed. Ionic adsorption of poly-L-lysine at the tubular region retaining a heparin coating was used to switch the heparin surface from cell-repellent to cell-adhesive, thereby facilitating the adhesion of a second cell type. The combination of the electrode-based technique with electrostatic deposition enabled the formation of patterned co-cultures within the semi-closed tubular structure. The controlled co-cultures inside the elastic tubing should be of value for cell-cell interaction studies following application of chemical or mechanical stimuli and for tissue engineering-based bioreactors.