Rapid spheroid clearing on a microfluidic chip.

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-1. The process is reversible upon increasing the pH, with the rate constant for the shrinkage being -0.12 min-1. 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.