Assembly of skeletal muscle cells on a Si-MEMS device and their generative force measurement.

We have fabricated a simple Si-MEMS device consisting of a microcantilever and a base to measure active tension generated by skeletal muscle myotubes derived from murine myoblast cell line C2C12. We have developed a fabrication process for integration of myotubes onto the device. To position myotubes over the gap between the cantilever and the base without damage due to mechanical peeling or the use of an enzymatic reaction, we cultured myotubes on poly-N-isopropylacrylamide (PNIPAAm) as a sacrifice layer. By means of immune staining of alpha-actinin, it was confirmed that a myotube micropatterned onto the device bridged the gap between the cantilever and the base. After 7d differentiation, the myotube was actuated by electrical stimulation. The active tension generated by the myotube was evaluated by measuring the bending of the cantilever using image processing. On twitch stimulation, the myotube on the device contracted and generated active tension in response to the electrical signals. On tetanus tension measurement, approximately 1.0 microN per single myotube was obtained. The device developed here can be used in wide area of in vitro skeletal muscle studies, such as drug screening, physiology, regenerative medicine, etc.

Quantification of structural properties of cell-sized individual liposomes by flow cytometry.

We describe a new high-throughput method of quantifying the structural properties of individual cell-sized liposomes. An internal aqueous solution of liposomes was labeled with a green fluorescent marker and the membrane with a red marker. The double-labeled liposomes were analyzed using flow cytometry, and the internal aqueous volume and lipid membrane volume of each liposome were measured. The experimental results indicate that both the internal aqueous and lipid membrane volumes positively correlate with the intensity of forward-scatter (FS) and side-scatter (SS) signals in a logarithmic scale. In addition, liposomes in 18 small areas gated by log(FS) and log(SS) were sorted by fluorescence-activated cell sorting (FACS), and observed by optical microscopy. Structural characteristics observed in the microscopy images of heterogeneous liposomes correlated with FACS data. Because this method does not employ any particular assumption about the shape and structure of liposomes, flow cytometry is a powerful tool for estimating the internal and membrane volumes of individual cell-sized liposomes with heterogeneous shapes and structures.