In vitro bioassay systems with cultured living cells have been developed extensively as an alternative to animal experiments to quantify the physiological functions under simplified and controlled conditions. The bioassay systems integrated with an electrical stimulation system have enabled to control electrophysiological activities of the cells such as neural, cardiac, and skeletal muscle cells. Especially, the systems using skeletal muscle cells are one of the promising device to reveal the complex mechanisms of type 2 diabetes and obesity because these diseases are associated with a disorder of insulin- or contraction-induced glucose metabolism in a skeletal muscle cell in vivo. In addition, recent lines of evidence have suggested that the skeletal muscle functions as an endocrine organ that secretes a number of cytokines (myokines) in response to exercise in order to regulate the physiological activities of the whole body including glucose metabolism of the muscle itself. Three-dimensionally reconstructed skeletal muscle cells have been proven to exhibit in vivo-like structural and genetic characteristics, and therefore, various three dimensional culture technique has been developed. On the other hand, extracellular electrical stimulation using a pair of electrodes immersed in both sides of a culture medium still has technical problems concerning limitations on the current value, which can be applied without a faradic reaction causing perturbation of culture conditions such as pH value. Additionally, in the conventional electrode configuration, most of the applied current tends to pass only through the culture medium surrounding the insulative living cells, which cannot cause cell depolarization effectively. For reliable and reproducible bioassay, it is necessary to develop the low invasive electrical stimulation culture system in which the applied current can pass through the cells. In this study, we developed the in vitro bioassay systems with three dimensional skeletal muscle fiber enclosing a stretchable and biocompatible electrode wire composed of conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT) and polyurethane (PU). The fibrous PEDOT has much capacity in its electrical double layer even in a composite form with PU that can apply current charge required for excitation of the surrounding skeletal muscle cells without any faradaic reaction. The electric current can pass through the cells surrounding the electrode wire, resulting in their quantitative depolarization, which was demonstrated by visualizing intracellular Ca2+ transients upon applied electrical stimulation with a Ca2+-sensitive fluorescent dye. PEDOT/PU wire was fabricated by twisting the PEDOT/PU strip (30 mm x 3 mm, 3 μm in thickness) cut out from the film. The PEDOT/PU film was prepared by spin-coating a precursor solution of EDOT monomer, pTSFe(III) dopant, and PU, followed by thermal polymerization of PEDOT on a glass substrate. Cyclic voltammetry measured in 0.1 M KCl solution suggested that maximum charge storing in an electric double layer of the PEDOT/PU wire was 75 μC. The wire was centrally set into the PDMS chamber pretreated with cell repulsive polymer solution. The C2C12 myoblast cells suspended in the mixture of collagen and Matrigel were poured into the PDMS chamber and incubated for 30min at 37 oC to facilitate gelation of the mixture. During three days of culture, the seeded cells self-assembled around the PEDOT/PU wire contracting the collagen/Matrigel matrix, resulting in a fiber form of myoblast cells/collagen/Matrigel matrix fiber around the PEDOT/PU wire. The cells were continuously cultured in the medium for additional seven days to induce differentiation of the myoblasts into the myotubes. Fluorescent Live/Dead imaging showed that almost all the myotubes around the PEDOT/PU electrode wire in alive suggesting non-citotoxity of the PEDOT/PU wire itself. The myotubes were aligned to longitudinal direction of the wire, which is similar structure as in vivo skeletal muscle tissue. Upon application of the constant current pulses to the PEDOT/PU electrode wire against a carbon electrode set outside of the myotubes/collagen/Matrigel matrix fiber, the cells exhibited twitch and tetanus contraction depending on the applied electric pulse frequency. The threshold current charge evaluated by visualizing intracellular Ca2+ transients upon applied electrical stimulation was 1.0 μC which value was much smaller than maximum charge derived from the electric double layer of PEDOT/PU wire, 75 μC. This suggested low invasive electric stimulation of the PEDOT/PU electrode even in close to the surrounding cells. Additional utility of embedding the electrode into the three dimensional skeletal muscle fiber was successfully demonstrated by selective electrical stimulation of the skeletal muscle fiber arrays in the same culture medium, that is beneficial for high-contrast comparison of the contraction effect on the skeletal muscle cellular functions such as contraction-dependent myokine secretion activity and contraction-dependent control of glucose metabolism in type 2 diabetes.
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Nov 1, 2016
Jun Ho Kim + 5
Open Access
