Perspective: Extending the Utility of Three-Dimensional Organoids by Tissue Clearing Technologies

Abstract

An organoid, a self-organizing organ-like tissue developed from stem cells, can exhibit a miniaturized three-dimensional (3D) structure and part of the physiological functions of the original organ. Due to the reproducibility of tissue complexity and ease of handling, organoids have replaced real organs and animals for a variety of uses, such as investigations of the mechanisms of organogenesis and disease onset, and screening of drug effects and/or toxicity. The recent advent of tissue clearing and 3D imaging techniques have great potential contributions to organoid studies by allowing the collection and analysis of 3D images of whole organoids with a reasonable throughput and thus can expand the means of examining the 3D architecture, cellular components, and variability among organoids. Genetic and histological cell-labeling methods, together with organoid clearing, also allow visualization of critical structures and cellular components within organoids. The collected 3D data may enable image analysis to quantitatively assess structures within organoids and sensitively/effectively detect abnormalities caused by perturbations. These capabilities of tissue/organoid clearing and 3D imaging techniques not only extend the utility of organoids in basic biology but can also be applied for quality control of clinical organoid production and large-scale drug screening.

CLARITY and Light-Sheet microscopy sample preparation in application to human cerebral organoids

“…Based on the various tissue clearance techniques analysis, we settled on the use of hydrogel-tissue chemistry as a clearing agent. Generally hydrophobic and hydrophilic reagent-based protocols are applied to the investigation of spheroids or hollow organoids such as intestinal organoids (Susaki, Takasato, 2021), while hydrogel reagents are used for the clarifying of human iPSC-derived retinal organoids (Cora et al, 2019) and iPSC-derived cerebral organoids (Renner M. et al, 2017;Sakaguchi et al, 2019;Albanese et al, 2020). Hydrogel-tissue chemistry-based protocols maximize the preservation of the internal structure of organoids and allow to achieve high optical resolution and low background at fluorescent microscopy.…”

“…To date, a number of protocols (more than a dozen) have been developed for making tissue transparent: SeeDB (Ke et al, 2013), ScaleA2 (Hama et al, 2011), uDISCO (Pan et al, 2016), CLARITY (Chung, Deisseroth, 2013), CUBIC (Susaki et al, 2015) and others. In general, all protocols can be divided into three groups, depending on the chemicals used for tissue clearance: organic solvents (hydrophobic reagent)-based protocols (BABB, 3DISCO, ECi method), hydrophilic reagent-based protocols (ClearT, Scale, FUnGI, Fructoseglycerol, CUBIC and other) and hydrogel-tissue chemistry-based protocol (CLARITY, SWITCH and SHIELD) (Ueda et al, 2020;Susaki, Takasato, 2021). Some of them have different advantages like quality and speed of clearing or simplicity of the procedure.…”

“…Various techniques have been used for organoid tissue clearing and several studies have compared different clarifying methods which could be applied to mini-organs (Susaki, Takasato, 2021). Some techniques, such as the hydrophilic clearing protocols (ClearT2 and ScaleS) are most acceptable for clearing small spheroids such as neurospheres (Boutin, Hoffman-Kim, 2015) or cancer cell spheroids (Boutin et al, 2018).…”

“…It should be noted that these protocols are very convenient and take only three days without application of harmful chemicals (Dekkers et al, 2019;van Ineveld et al, 2020). For complex and dense brain organoids, stronger clearing protocols including delipidation procedure are usually used (Susaki, Takasato, 2021). Applying organic solvent-based methods like 2Eci (2nd generation Ethyl cinnamate-based clearing method) (Masselink et al, 2019;Goranci-Buzhala et al, 2020) or BABB method for midbrain organoids (Renner H. et al, 2020) can get a relatively quick (within a few days) result.…”

“…That is due to the tissue hydrogel scaffold preparation by cross-linking hydrogel monomers to native biomolecules (Gradinaru et al, 2018). The creation of such a polymer frame in the brain organoids allows combining these protocols with additional procedures with sodium dodecyl sulfate and physical electrophoresis, as well as with high-resolution imaging of Expansion Microscopy with a general microscopy setup (Wassie et al, 2019;Susaki, Takasato, 2021).…”