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

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.

Optical clearing methods: An overview of the techniques used for the imaging of 3D spheroids

“…Moreover, increasing the acrylamide concentration decreases the extent of tissue shrinkage (e.g., 80-90% of the spheroids initial volume was maintained when used 8% (w/v) acrylamide). On the other hand, SDS solution flow led to the lipophilic dye (DiO) fluorescence loss but simultaneously did not affect the spheroids stain with Transferrin-647 as well as facilitated the spheroids staining with DAPI and Phalloidin-488 (Chen et al, 2016). The results obtained also demonstrated that the imaging depth was increased by 150% for MCF-7 spheroids (labeled with CellTracker™ Red), and 250% for U87MG-GFP spheroids, when compared to the noncleared spheroids.…”

“…On the other hand, SDS solution flow led to the lipophilic dye (DiO) fluorescence loss but simultaneously did not affect the spheroids stain with Transferrin-647 as well as facilitated the spheroids staining with DAPI and Phalloidin-488 (Chen et al, 2016). On the other hand, SDS solution flow led to the lipophilic dye (DiO) fluorescence loss but simultaneously did not affect the spheroids stain with Transferrin-647 as well as facilitated the spheroids staining with DAPI and Phalloidin-488 (Chen et al, 2016).…”

“…The original clear lipid-exchanged acrylamide-hybridized rigid imaging/immunostaining/in situ hybridization-compatible tissue-hydrogel (CLARITY) method (also known as active CLARITY) was proposed in 2013 by Chung et al (2013) (Chen et al, 2016).…”

“…Additionally, both methods usually use RIMS (Refractive Index Matching Solution composed mainly of Histodenz or Sorbitol), which is a more economical RI-matching solution than FocusClear™(Yang et al, 2014b). Nevertheless, the authors observed that the spheroids shrank to 150 μm in diameter after the clearing process (Silva Santisteban et al, 2018).Chen and co-workers also investigated the application of microfluidic technology to clear whole intact spheroids (e.g., MCF-7 spheroids with 250 µm of diameter and GFP-expressing U87MG spheroids with a diameter of 450 µm) using a CLARITY-based method and perform imaging by CLSM(Chen et al, 2016). Silva Santisteban, Rabajania, Kalinina, Robinson, and Meier (2018) used CLARITY and a microfluidic system to clear an image human-derived adipose stem cells spheroids (200 ± 50 μm in diameter) by CLSM.…”

Cancer-on-a-chip systems at the frontier of nanomedicine

“…To address this issue, Chan and colleagues recently developed an imaging-on-a-chip system to achieve optical clearing and high-resolution imaging of optically transparent structures of intact tumor spheroids [72]. With the built-in fluid exchange capability of this microfluidic chip, multiple tumor spheroids could be cleared within 1 day by removing the scattering lipid molecules post crosslinking, allowing for high-throughput tissue imaging of intact organoids, analyses of tumor biomarkers and microstructures, and, potentially, investigations of nanomedicine-cancer organoid interactions.…”