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

Cerebral organoids are three-dimensional cell-culture systems that represent a unique experimental model reconstructing early events of human neurogenesis in vitro in health and various pathologies. The most commonly used approach to studying the morphological parameters of organoids is immunohistochemical analysis; therefore, the three-dimensional cytoarchitecture of organoids, such as neural networks or asymmetric internal organization, is difficult to reconstruct using routine approaches. Immunohistochemical analysis of biological objects is a universal method in biological research. One of the key stages of this method is the production of cryo- or paraffin serial sections of samples, which is a very laborious and time-consuming process. In addition, slices represent only a tiny part of the object under study; three-dimensional reconstruction from the obtained serial images is an extremely complex process and often requires expensive special programs for image processing. Unfortunately, staining and microscopic examination of samples are difficult due to their low permeability and a high level of autofluorescence. Tissue cleaning technologies combined with Light-Sheet microscopy allows these challenges to be overcome. CLARITY is one of the tissue preparation techniques that makes it possible to obtain opaque biological objects transparent while maintaining the integrity of their internal structures. This method is based on a special sample preparation, during which lipids are removed from cells and replaced with hydrogel compounds such as acrylamide, while proteins and nucleic acids remain intact. CLARITY provides researchers with a unique opportunity to study three-dimensional biological structures while preserving their internal organization, including whole animals or embryos, individual organs and artificially grown organoids, in particular cerebral organoids. This protocol summarizes an optimization of CLARITY conditions for human brain organoids and the preparation of Light-Sheet microscopy samples.


Introduction
Biological tissues and organs present a complex three-dimensional structure. Due to their opacity and high level of autofluorescence, three-dimensional reconstruction of such objects is an extremely laborious, but necessary process. 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. But on the other hand, some of the protocols involve using toxic and corrosive chemicals that require special objectives to avoid damage to the microscope or require other special equipment. Most of these protocols have been developed to clarify entire organs or their big fragments.
Recently, a new method of artificial mini-organ or organoids generation from induced pluripotent stem cells (iPSC) was developed (Lancaster et al., 2013) and now many different types of organoids have already been produced (brain, lung, liver, intestine, pancreas, kidney and others). Organoids are widely used both to recreate the three-dimensional architecture and functional activity of the original organs during normal embryonic development and at various disorders and to test the biological activity of various drugs, chemical and biological agents. Usually, organoids are opaque, which makes investigating them rather difficult. For this purpose, it is advisable to use the combination of tissue clearing and 3D imaging technologies. However, it is important to select the clarifying technology that would match organoids size and fragility as much as possible and would produce sufficient resolution for investigation of tiny structures.
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). Others, such as RapiClear, Fructoseglycerol and FUnGI, also using hydrophilic components, are designed and optimized for handling small and fragile, predominantly hollow organoid structures such as intestinal organoids. 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. However, most of the organic components used in these protocols are quite toxic (for example, a mixture of benzyl alcohol and benzyl benzoate in BABB method (Renner H. et al., 2020)).
The use of hydrogel-tissue chemistry sometimes provides more opportunities for preserving the structure of organoids and increasing the optical resolution of tiny objects. 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).
Thus, it is quite important to choose the most optimal and effective tissue clearing technique for samples, especially for such complex objects as cerebral organoids.
One of the most convenient and lab-friendly techniques is CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/In situ hybridization-compatible Tissue-hYdrogel). CLARITY was developed in 2013 for obtaining high-resolution information from complex 3D structures, such as the whole mouse brain (Chung, Deisseroth, 2013). Application of this technique enabled to obtain intact-tissue imaging of long-range projections, local circuit wiring, cellular relationships, subcellular structures, protein complexes, and neurotransmitters. CLARITY protocol includes replacing lipids with hydrophilic polymers (acrylamide and bis-acrylamide), which help to stabilize tissue but make it optically transparent and permeable. It is very important that molecules like nucleic acids and proteins stuck in the hydrogel keep their structures and locations. Thus, CLARITY allows combining tissue clearing techniques with immunostaining and in situ hybridization and explores the internal structure of large three-dimensional objects without damaging their integrity. There is only one article in which CLARITY technique was used for cerebral organoid clarifying (Sakaguchi et al., 2019), but without a detailed description. Thus, the aim of our work was optimization of CLARITY protocol in application to cerebral organoids and detailed description of samples preparation for Light-Sheet microscopy.

Sample preparation for Light-Sheet microscopy
Organoid sizes can vary greatly. Therefore, we recommend using a different fixation method for Light-Sheet microscopy depending on the size.

Agarose embedding samples (for smaller samples)
Note: Use the agarose with a low melting point temperature only. Note: The percentage of agarose solutions depends on the size of the organoid. For larger organoids, use 1.5 % agarose solution.
1. Prepare the 1-ml syringe by cutting off the top (Fig. 1, A). 2. Weigh the required amount of agarose (at the rate of 1 g per 100 ml) and dissolve in 1X PBS or ddH 2 O. Prepare agarose solution by melting in the microwave. Usually, for a 1-ml syringe, 1.5 ml of agarose is enough. 3. Pour the hot agarose solution into a 12-well plate or any other laboratory glassware or plasticware. When agarose solution cools down to +40 °C, transfer samples and gently mix. Put the samples in agarose solution into the 1-ml syringe. 4. Assemble the 1-ml syringe with a sample holder (see Fig. 1, A). 5. Proceed to Light-Sheet microscopy. Note: Fill the microscope chamber with ddH 2 O or 1X PBS. No great differences were observed between the two solutions.

Agarose-free or hanging samples (for bigger samples)
1. Glue the sample to the bar (see Fig. 1, B, C)

Results
Human cerebral organoids were generated according to a protocol from Lancaster et al. (2013) with small modifications. 2-and 3-month-old cerebral organoids were used for tissue clearing protocol. At this stage, there are dense spheres more than 2 mm in diameter (Fig. 2, A). We noted that the time of tissue clearing depends on the size of organoids. Cerebral organoids ≤ 0.5 cm become transparent during ~2 weeks, for organoids ≥ 0.5 cm up to 3 weeks. This time may vary from sample to sample, however, continue cleaning until the samples become transparent (see Fig. 2, B, C). For immunostaining we chose two proteins with different subcellular localisation such as nuclear CTIP2 (Fig. 3, A) and cytoplasmic bTubb3 (see Fig. 3, C). We did not find a significant difference between penetration of antibodies into different  C 100 μm cellular compartments. In both cases, we observed specific staining throughout the entire thickness of the organoid (see Fig. 3, B).

Conclusions
Cerebral organoids are a unique novel technology that allows the reconstruction of early human neurogenesis. The outstanding feature of this in vitro system is the reproduction of the three-dimensional organization of the human embryonic brain. Standard histological methods of analysis do not allow reconstructing the internal structure of the cerebral organoids and result in information loss. The tissue clearing technique helps to overcome these limitations, allowing to recreate a three-dimensional model of cerebral organoids and explore their fine organization without internal structure destruction. This is especially important for the investigation of brain organoids since they contain a dense network of long processes of nerve cells, which is very difficult to study by serial sections (Dodt et al., 2007).
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.
Of course, a significant disadvantage of CLARITY technique is the relatively long tissue clearance procedure (approximately three weeks for 90-days cerebral organoids), but this obstacle is compensated by a quite simple protocol. To our knowledge, there is a single report in which the CLARITY technique was used for cerebral organoid clarifying (Sakaguchi et al., 2019); however, a detailed description of this technique applied to brain organoids has not been previously performed. For the first time, we make a detailed description of the human cerebral organoid samples preparation for investigation of CLARITY-treated samples for Light-Sheet microscopy.