Non-Invasive and Continuous Monitoring of Differentiation Process into Cardiomyocytes By 2D-SPR Observation

Abstract

Introduction Two-dimensional surface plasmon resonance (2D-SPR) measurement method has the advantages of noninvasive and real-time for observation of intracellular reactions. We have already applied this attractive method to monitor cell differentiation noninvasively.P19CL6, mouse embryonal carcinoma cells were cultured in a medium containing DMSO and 5-azacytidine to induce cardiac differentiation. 2D-SPR observation of the cells was then performed every two days to monitor intracellular refractive index change. The resonance angle of cell regions increased as the differentiation progressed. However, in previous study, cells in differentiation process were once detached from the well plate with trypsin treatment and replated on a gold chip and 2D-SPR measurement was done after one day. Therefore, we considered that the cells might be damaged by trypsin treatment exactly. Here, we aimed to realize noninvasive and continual 2D-SPR observation of the cells during cardiac differentiation process. In this experiment, the sensor chip chamber was covered with a gas permeable membrane and cardiac differentiation process from day 4 to day 14 was continually observed with a high resolution of SPR imager. Experimental method In this experiment, P19CL6 cells were cultured on a well plate during the period for formation of embryoid bodies from day 0 to day 4 in the presence of DMSO, and cardiac differentiation was induced on a gold chip from day 4 to day 14 in the presence of DMSO and 5-Azacitidine.The gold sensor chip chamber was covered with a gas permeable membrane to avoid contamination. 2D-SPR observations were carried out every two days to obtain the SPR images of cell regions and to monitor how the resonance angle changes at the cell regions during cardiac differentiation process. Result and discussion It was demonstrated that cardiac differentiation of P19CL6 cells on a gold chip was possible and the SPR images of cell regions were continually observed from day 4 to day 14 without contamination. Though the average of the resonance angles at cell regions decreased from day 4 to day 8, but it changed to increase from day 8 to day 12 and reached the maximum on day 12 and day 14. It is considered that intracellular proteins may be reformed to differentiate from embryonic cells into cardiomyocytes from the day 4 to day 8. The cells became cardiomyocyte-like on the day 10 and beating could be observed on a well plate from day 12.From these results we conclude that continual observation with a SPR imager is very promising method for noninvasive monitoring of cardiac differentiation. Figure 1