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On-chip clearing for live imaging of 3D cell cultures

Tingting Yu,Tingting Yu,Xiang Zhong,Xiang Zhong,Qihang Yang,Qihang Yang,Chao Gao,Chao Gao,Wenyue Chen,Wenyue Chen,Xiang Liu,Xiang Liu,Zhang Liu,Zhang Liu,Tingting Zhu,Tingting Zhu,Dongyu Li,Dongyu Li,Peng Fei,Peng Fei,Zaozao Chen,Zaozao Chen,Zhongze Gu,Zhongze Gu,Dan Zhu,Dan Zhu

Three-dimensional (3D) cell cultures provide an important model for various biological studies by bridging the gap between two-dimensional (2D) cell cultures and animal tissues. Microfluidics has recently provided controllable platforms for handling and analyzing 3D cell cultures. However, on-chip imaging of 3D cell cultures within microfluidic devices is hindered by the inherent high scattering of 3D tissues. Tissue optical clearing techniques have been used to address this concern but remain limited to fixed samples. As such, there is still a need for an on-chip clearing method for imaging live 3D cell cultures. Here, to achieve on-chip clearing for live imaging of 3D cell cultures, we conceived a simple microfluidic device by integrating a U-shaped concave for culture, parallel channels with micropillars, and differentiated surface treatment to enable on-chip 3D cell culture, clearing, and live imaging with minimal disturbance. The on-chip tissue clearing increased the imaging performance of live 3D spheroids with no influence on cell viability or spheroid proliferation and demonstrated robust compatibility with several commonly used cell probes. It allowed dynamic tracking of lysosomes in live tumor spheroids and enabled quantitative analysis of their motility in the deeper layer. Our proposed method of on-chip clearing for live imaging of 3D cell cultures provides an alternative for dynamic monitoring of deep tissue on a microfluidic device and has the potential to be used in 3D culture-based assays for high-throughput applications.