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Proc Natl Acad Sci U S A. 2016 Dec 27;113(52):14915-14920. doi: 10.1073/pnas.1609569114. Epub 2016 Dec 12.

Clarifying intact 3D tissues on a microfluidic chip for high-throughput structural analysis.

Author information

1
Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, Canada M5S 3G9.
2
Department of Physiology, University of Toronto, Toronto, ON, Canada M5S 1A8.
3
Toronto General Research Institute, University Health Network, Toronto, ON, Canada M5G 2M9.
4
Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, Canada M5S 3G9; warren.chan@utoronto.ca.
5
Department of Chemistry, University of Toronto, Toronto, ON, Canada M5S 3H6.
6
Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada M5S 3E1.
7
Department of Chemical Engineering, University of Toronto, Toronto, ON, Canada M5S 3E5.
8
Department of Material Science and Engineering, University of Toronto, Toronto, ON, Canada M5S 3E1.

Abstract

On-chip imaging of intact three-dimensional tissues within microfluidic devices is fundamentally hindered by intratissue optical scattering, which impedes their use as tissue models for high-throughput screening assays. Here, we engineered a microfluidic system that preserves and converts tissues into optically transparent structures in less than 1 d, which is 20× faster than current passive clearing approaches. Accelerated clearing was achieved because the microfluidic system enhanced the exchange of interstitial fluids by 567-fold, which increased the rate of removal of optically scattering lipid molecules from the cross-linked tissue. Our enhanced clearing process allowed us to fluorescently image and map the segregation and compartmentalization of different cells during the formation of tumor spheroids, and 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. We further developed an image analysis algorithm that automates the analysis of the vasculature connectivity, volume, and cellular spatial distribution of 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.

KEYWORDS:

3D imaging; CLARITY; computational analysis; fluorescence imaging; microfluidic

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