Continuous Monitoring of Differentiation Process of Neuronal Progenitor Cells By Two-Dimensional Surface Plasmon Resonance Observation at Single Cell Level
Authors: Gokura Hikari , Shinohara Hiroaki , Suga Minoru 
Published: December 2020

Introduction
In our laboratory, we have examined to monitor intracellular events noninvasively by the two-dimensional surface plasmon resonance (2D-SPR) observation. In 2D-SPR observation, the refractive index change of the cell bottom can be observed as local changes in reflected light intensity. In previous study, we applied 2D-SPR observation to monitor the differentiation process of neuronal progenitor cells and found that the reflected light intensity at cell regions vibrated over time after neurite outgrowth. This finding suggested that it may be possible to evaluate neuronal differentiation very simply by monitoring of the SPR vibration phenomenon at the single cell region and analyzing the vibration frequency.
However, in previous studies SPR observation before and after differentiation was done to the cells which were re-inoculated on a gold sensor chip for observation before one day. Therefore continuous observation of the differentiation process of the same progenitor cells have not been done yet. In this study, PC12 (Rat adrenal pheochromocytoma) as a neuronal progenitor cell model were cultured on a gold chip, and continual 2D-SPR observation of same cell regions was examined in differentiation process into neuron-like cells by NGF administration.
Experimental method
400 μl of cell suspension of PC12 cells in DMEM medium containing 5% FBS and 10% HS was seeded in a sensor chamber in which PDMS well was adhered on a gold chip, and incubated overnight in an CO2 incubator. The next day, the medium was removed and replaced with a DMEM medium (containing 1% HS and 100 ng / ml NGF). At this time, the gas permeable membrane sheet was attached over the cell culture chamber. The gold chip chamber in which the cells were cultured was observed every day until the day 4 with a high resolution 2D-SPR instrument (model 2D-SPR04B, NTT-AT Japan). The day of the medium changed was set on the day 0. The sensor chamber was maintained in the incubator except 2D-SPR observation. A phase-contrast microscope was used to observe how PC12 cells adhered to the gold chip and differentiated into neuron-like cells by NGF administration and following neurite extension. At the same time, 2D-SPR observation of same PC12 cell regions on a gold chip was done to measure resonance angle and to monitor the reflected light intensity of the same cell region over time.
Result and discussion
Fig. 1 shows the time-course of the reflection light intensity at several cell regions on day 1 and day 2. By comparing the SPR signal on day1 and day2 it was demonstrated that the SPR signal at the cell region on day 2 showed significant vibration. The neurite outgrowth in PC12 cells on the day 2 was observed clearly. It is well known that polymerization and depolymerization of tubulin and actin filament, which are cytoskeletons, are actively performed for neurite out growth. Therefore we speculated that the SPR signal vibration of the reflection intensity might be due to the refractive index change by polymerization and depolymerization of the cytoskeleton closed to the cell bottom. In the presentation, we also mention the vibration frequency which way play a marker for differentiation evaluation, by the Fourier analysis of this vibration phenomenon.
At the present time, 2D-SPR could not be observed after day 3 because the cell adhesion was weakened and cells were detached from the gold chip. We are now trying the improvement of cell adhesion and longer continuous monitoring of neuronal differentiation process by 2D-SPR observation.

Figure 1