Clarifying Intact 3D Tissues on a Microfluidic Chip for High-Throughput Structural Analysis

Chen, Yih Yang 1 ;  Pamuditha N. Silva 1 ;  Abdullah Muhammad Syed 1 ;  Shrey Sindhwani 1 ;  Jonathan V. Rocheleau 1, 2, 3 ;  Warren C. W. Chan 1, 4, 5, 6, 7

1. Institute of Biomaterials and Biomedical Engineering, University of Toronto, To; 2. Department of Physiology, University of Toronto; 3. Toronto General Research Institute, University Health Network; 4. epartment of Chemistry, University of Toronto; 5. Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto; 6. Department of Chemical Engineering, University of Toronto; 7. Department of Material Science and Engineering, University of Toronto

Structural analysis of microscale three-dimensional tissues (3D microtissues) in high-throughput is becoming increasingly important in drug discovery, regenerative medicine, and other biomedical areas because they recapitulate many in vivo biological features not present in 2D models. This can be done by using microfluidic technology to control and apply external forces to on-chip 3D microtissues, and imaging these organ-on-chip systems with confocal microscopy. However, the high cellular density of 3D microtissues scatters light, which impedes its penetration and fundamentally limits the ability to characterize the entire tissue construct. We developed an on-chip strategy to rapidly clarify, image, and analyze whole intact microtissues without compromising internal structures. Our technique removes the imaging depth limit, allowing accurate analysis and characterization of entire tissues in microfluidic chips achievable in less than 1 day, which is 20× faster than current passive clearing approaches. This accelerated clearing was achieved because our microfluidic system enhanced the exchange of interstitial fluids by 567-fold, allowing us to rapidly treat the 3D microtissue with solutions that preserve its structure and clarify it to allow total light penetration. Our enhanced clearing process allowed us to fluorescently image and map the segregation and compartmentalization of different cell types during the formation of tumor spheroids, and demonstrate our platform’s ability to reveal gradients of cell viability within the tumour spheroids as a proof-of-concept for drug screening applications. We then used our technique to track the degradation of vasculature over time within extracted murine pancreatic islets in static culture, which may have implications on the efficacy of beta-cell transplantation treatments for type 1 diabetes. To quantify the degradation of islet vasculature, we developed an image analysis algorithm that automates the analysis of vasculature connectivity, volume, and cellular spatial distribution within the intact tissue. Our technique allows whole tissue analysis in microfluidic systems, and has implications in the development of organ-on-a-chip systems, high-throughput drug screening devices, and in regenerative medicine.