It is still unclear how the activity of sympathetic and parasympathetic neurons influences the activity of cardiomyocytes in culture. We developed a device for co-culturing sympathetic neurons, parasympathetic neurons, and cardiomyocytes using micro-fabrication techniques. Morphological connections between each type of autonomic neuron and the cardiomyocytes were observed by immunostaining. The inter-beat-interval (IBI) of the cardiomyocytes was modulated after electrically stimulating each type of autonomic neuron. Modulation of the IBI was blocked by the addition of pharmacological blockers to the culture medium. These results suggest that the co-culture device can be utilized to understand how the activity of sympathetic neurons and parasympathetic neurons influences the activity of cardiomyocytes in the culture environment.

Hydrogel patterning methods are widely used for cell patterning because they offer better long-term stability than protein patterning methods such as micro-contact printing, but conventional hydrogel patterning methods require special apparatuses such as a laser or an electron beam lithography system or they have complicated chemical operations which prevent their practical use in biological laboratories. A simple method was developed to cast a hydrogel solution without external power sources using a polydimethylsiloxane (PDMS) mold with micro-channels. This study employed "the accumulation of vacuum pressure" in a degassed lump of PDMS as a driving force for introducing agarose solution into the micro-channels. Sufficient vacuum pressure could be accumulated within 1 h in the PDMS elastomer that was acting as a vacuum tank, and 2 w/v% agarose solution could be aspirated into the micro-channels with widths from 100 to 2000 mm and a height of 19 mm, fully filling them. After the gelation and dehydration of agarose solution in the micro-channels, the patterns of agarose gel on the channels were successfully cast with a 90%-width accuracy. By using the repellency of agarose gel toward cell adhesion, patterned cultures of myoblasts and cortical neurons were successfully prepared. This technique is expected to be useful in repellency-guided cell patterning for various types of cells, with applications to cell-cell interactions and axon guidance.