Electrochemiluminescence Imaging for High Throughput Analysis of Spheroids

Introduction

Three-dimensional cultured cells, such as spheroids are believed to be promising models for drug screening and regenerative medicine because they effectively mimic the cellular response and metabolic activity in physiological contexts. To further study their functions for applications in clinical contexts, high-throughput analysis of living spheroids is required. To electrochemically evaluate cellular activity of spheroids, scanning electrochemical microscopy (SECM) has been widely used¹; with the shortcomings of low through put and complicated control of scanning a probe. Another strategy utilized electrode array devices to this end², although the spatial resolution is limited by the numbers and sizes of integrated electrodes. Here, we present electrochemiluminescence (ECL) imaging system for evaluation of cellular activity of 3D cell spheroids. ECL is an optical technique that utilizes electrochemical reaction to produce luminescence, and widely used in bioanalytical field. The unique features of ECL such as simple setups and temporal controllability of light emission are favorable for analysis of cells and molecules derived from cells. However, there is few studies which apply ECL for imaging of 3D cell spheroids. L-012, a luminol analog was used for ECL luminophore, which emits strong luminescence in the presence of H₂O₂. In the proposed system, H₂O₂ was generated in situ by electrochemical reduction of O₂, former to the electrochemical oxidation of L-012. Owing to the electrochemical reduction of O₂ to H₂O₂, efficient luminescence of L-012 was obtained. Moreover, as living cell spheroids consume O₂ to maintain cellular activity, the electrochemical reduction of O₂ is hindered at the peripheral environment of a spheroid, resulting with decrease in luminescence around the spheroid. Thus, the distribution of ECL suppression indicates the respiratory activity of a spheroid. To demonstrate feasibility of the proposed ECL system, the respiratory activity of mesenchymal stem cell (MSC) spheroids was measured either with ECL imaging and SECM. Finally, time-lapse and multiple imaging of the cellular activity of the living MSC spheroids were demonstrated in the ECL imaging.

Experimental

The ECL system was composed of an Au substrate as a working electrode, an Ag/AgCl and a Pt wire as a reference and counter electrode, respectively. A chamber made of polydimethylsiloxane (PDMS) was bonded on the Au substrate. The electrodes were connected to a potentiostat and mounted on a stereomicroscope. To demonstrate ECL imaging of cell activities, human mesenchymal stem cells (hMSCs) were prepared. For fabrication of spheroids, the MSCs were seeded at 2×10⁵ cells per well in a 96-well U-bottom plate in DMEM. MSC spheroids were formed within 48 h and the half of the spheroids were induced to chondrogenic differentiation by commercial differentiation medium subsequently. For ECL imaging, 200 mM L-012 solution (PBS, pH 7.4) was introduced to the chamber, and spheroids were settled on the substrate using a micropipette. A potential of -0.3 V was applied for 30 s to generate H₂O₂, followed by +0.7 V for 30 s to oxidize L-012. During the potential apply, ECL images were taken by a digital camera with 30 s of exposure time. For the time-lapse imaging, multiple spheroids were introduced on the Au electrode at once and ECL imaging was conducted 2–60 min after they settled.

Results and discussion

In the ECL images of MSC spheroids, the bodies of the spheroids displayed bright emission, possibly due to the compacted spherical structure of the spheroids that scattered light from ECL. At the living spheroid, the circumference of the spheroid became dark, indicating that the ECL was suppressed due to O₂ consumption of the spheroid. In fact, there was no suppression of ECL around fixed spheroids as there was no respiratory activity in those cells. Interestingly, in comparison with the control and differentiation induced spheroids, the decline of the ECL intensity was larger in the latter spheroid indicating inherent respiratory activity among induction of the differentiation. With the ECL images, the respiratory activity of the spheroids was calculated by quantifying the distribution of ECL intensity around the spheroid. The results corresponded well with that measured by SECM. The time-lapse ECL imaging of multiple MSC spheroids displayed subtle changes in the respiratory activity of the spheroids during observation. When the spheroids were removed from the electrode after 60 min, the area right under the spheroids remained slightly dark, indicating cellular components or metabolites might have adsorbed on the electrode surface. Overall, the ECL system enabled simultaneous analysis of cellular activity of multiple spheroids with good resolution comparable with that of SECM, while the measurement time was highly improved with a single flat electrode for ECL reaction.

References

1. Kaoru Hiramoto et al., Electrochim. Acta, 340, 135979, (2020)

2. Kosuke Ino et al., Electrochim. Acta, 268, 554-561, (2018)