Rapid spheroid clearing on a microfluidic chip

Abstract

Spheroids are three-dimensional (3D) cell cultures that aim to bridge the gap between the use of whole animals and cellular monolayers. Microfluidics is regarded as an enabling technology to actively control the chemical environment of 3D cell cultures. Although a wide variety of platforms have been developed to handle spheroid cultures, the development of analytical systems for spheroids remains a major challenge. In this study, we engineered a microfluidic large-scale integration (mLSI) chip platform for tissue-clearing and imaging. To enable handling and culturing of spheroids on mLSI chips, with diameters within hundreds of microns, we first developed a general rapid prototyping procedure, which allows scaling up of the size of pneumatic membrane valves (PMV). The presented prototyping method makes use of milled poly(methylmethacrylate) (PMMA) molds for obtaining semi-circular microchannels with heights up to 750 μm. Semi-circular channel profiles are required for the functioning of the commonly used PMVs in normally open configuration. Height limits to tens of microns for this channel profile on photolithographic molds have hampered the application of 3D tissue models on mLSI chips. The prototyping technique was applied to produce an mLSI chip for miniaturization, automation, and integration of the steps involved in the tissue clearing method CLARITY, including spheroid fixation, acrylamide hydrogel infiltration, temperature-initiated hydrogel polymerization, lipid extraction, and immuno-fluorescence staining of the mitochondrial protein COX-IV, and metabolic enzyme GAPDH. Precise fluidic control over the liquids in the spheroid culturing chambers allowed implementation of a local hydrogel polymerization reaction, exclusively within the spheroid tissue. Hydrogel-embedded spheroids undergo swelling and shrinkage depending on the pH of the surrounding buffer solution. A pH-jump from 8.5 to 5.5 shrinks the hydrogel-embedded spheroid volume by 108% with a rate constant of 0.36 min. The process is reversible upon increasing the pH, with the rate constant for the shrinkage being -0.12 min. Repetitive cycling of the pH induces an osmotic flow within the hydrogel-embedded spheroid. Thirty cycles, performed in a total time interval of 10 minutes on-chip, reduced the clearing time of a hydrogel-embedded spheroid (with a diameter of 200 μm) from 14 days to 5 hours. Therefore, we developed a physicochemical method to decrease the clearing time of hydrogel-embedded tissues. While the osmotic pump mechanism is an alternative to electrophoretic forces for decreasing tissue clearing times, the integration of the CLARITY method on chip could enable high throughput imaging with 3D tissue cultures.

Rapid Clearing for High Resolution 3D Imaging of Ex Vivo Pancreatic Cancer Spheroids

“…Recently developed tissue clearing methods have been used to produce high quality images [ 25 ], but most of the commercially available protocols last about 72 h and are extremely expensive. Some studies attempted to adapt tissue clearing procedures for spheroid and organoid protocols [ 17 , 18 , 19 , 20 ], however, these studies used light sheet microscopy and not a standard confocal microscope, and they required long procedures or use of specific designated microchips.…”

“…We, as well as others, have shown that spheroids can deposit dense ECM and thus be used ex vivo as a model for the pancreatic tumor [ 13 , 14 , 15 , 16 ]. Previous attempts to adapt optic clearing protocols for spheroids and organoids typically required either the use of light sheet microscopes, special flow chip devices, physical sectioning, or long procedures [ 17 , 18 , 19 , 20 ].…”

Influence of ClearT and ClearT2 Agitation Conditions in the Fluorescence Imaging of 3D Spheroids

“…For that purpose, the clearing solutions promote the homogenization of the refractive index (RI) through the spheroids and reduce the light scattering effect [ 15 , 23 ]. In the literature, several optical clearing methods have been used for improving the spheroids analysis, namely Clear T [ 19 ], Clear T2 [ 18 , 20 , 21 ], SeeDB [ 14 , 18 ], FocusClear ™ [ 22 ], BABB [ 24 , 25 , 26 , 27 ], Scale [ 14 , 18 ], CUBIC [ 28 ], and CLARITY [ 29 , 30 ]. Among these, Clear T and Clear T2 methods are organic-solvent and detergent free, the solutions are easy to handle, and the protocols are not very time-consuming.…”

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

“…The PACT and PARS methods use lower concentrations of fixatives and hydrogel monomers in conjugation with higher SDS concentrations (8% (w/v)), to facilitate and increase the passive diffusion of SDS into the sample and thus obtain a greater clearing without using the electrophoresis system. Furthermore, the authors also investigated the influence of the pH value in the media surrounding the hydrogel-embedded spheroids on the swelling and shrinkage of the gel(Silva Santisteban et al, 2018). In the PACT method, sample-hydrogel hybrid is simply merged with an SDS solution, while in the PARS method the removal of the lipids is performed by perfusing continually the sample with the SDS solution(Yang et al, 2014b).CLARITY-based methods were also used to clear spheroids.…”

“…The results demonstrated that the clearing time of the spheroids off-chip took 10-14 days, while this process was reduced to 2 days when a microfluidic system was used. Furthermore, the authors also investigated the influence of the pH value in the media surrounding the hydrogel-embedded spheroids on the swelling and shrinkage of the gel(Silva Santisteban et al, 2018). These changes lead to the creation of osmotic pressure and thus facilitate the lipids extraction from the spheroids.…”