HyClear: A Novel Tissue Clearing Solution for One-Step Clearing of Microtissues

3-D cell cultures are being increasingly used as in vitro models are capable of better mimicry of in vivo tissues, particularly in drug screenings where mass transfer limitations can affect the cancer biology and response to drugs. Three-dimensional microscopy techniques, such as confocal and multiphoton microscopy, have been used to elucidate data from 3-D cell cultures and whole organs, but their reach inside the 3-D tissues is restrained by the light scattering of the tissues, limiting their effective reach to 100-200 mu m, which is simply not enough. Tissue clearing protocols, developed mostly for larger specimens usually involve multiple steps of viscous liquid submersion, and are not easily adaptable for much smaller spheroids and organoids. In this work, we have developed a novel tissue clearing solution tailored for small spheroids and organoids. Our tissue clearing protocol, called HyClear, uses a mixture of DMSO, HPG and urea to allow for one-step tissue clearing of spheroids and organoids, and is compatible with high-throughput screening studies due to its speed and simplicity. We have shown that our tissue clearing agent is superior to many of the commonly used tissue clearing agents and allows for elucidating better quality data from drug screening experiments.

Automated summary

Three-dimensional cell cultures are increasingly used as in vitro models to mimic in vivo tissues for drug screening. However, the penetration of three-dimensional microscopy techniques is limited by the light scattering of tissues. Traditional tissue clearing protocols are not suitable for small spheroids and organoids. In this study, a novel tissue clearing solution called HyClear was developed for small spheroids and organoids. This protocol allows for one-step tissue clearing and is compatible with high-throughput screening studies.