Tissue clearing techniques in non-clinical studies

Tissue clearing techniques used in non-clinical studies include CUBIC and iDISCO. This article summarizes the background and key methods, features, and specific application examples of these techniques required in non-clinical studies.

Background for "Tissue Clearing Technology" in Non-clinical Studies

In non-clinical studies, tissue clearing technologies such as CUBIC are required. This is because traditional pathological evaluation using thin slices makes it difficult to grasp the "big picture," including complex neural networks, vascular trajectories, and micro-cancer metastases, posing a risk of missing important information.

By using tissue clearing technology to make tissues transparent and then imaging them with techniques like light-sheet microscopy, we can visualize and quantify their three-dimensional structure without damaging the organs.

Key methods of organizational transparency technologies and their respective features

CUBIC

CUBIC is an advanced clearing technology developed in Japan. It allows observation of all cells within an entire organ or the whole body by delipidating and refractive index matching the tissue, thereby achieving transparency. This enables deep tissue visualization and is characterized by high reproducibility in whole-brain and whole-body analyses.

CLARITY・iDISCO

CLARITY is a technique that makes tissues transparent using acrylamide-based hydrogels. While it takes time to achieve transparency due to hydrogel fixation, it allows for lipid removal while preserving intrinsic fluorescence and fine structures, making it suitable for multiple staining. Furthermore, in the case of iDISCO, organic solvents can be used to rapidly clear a wide range of tissues, offering excellent counter-staining properties.

However, since there are cases where changes in the sample occur during the clearing process, it is recommended to select according to the target you wish to observe, etc.

Imaging technology

After tissue clearing, imaging techniques visualize the internal structure of thick samples in 3D without sectioning. In particular, light-sheet fluorescence microscopy (LSFM) is effective for rapidly imaging entire cleared large samples.

Specific Use Cases and Results in Non-clinical Studies

Neuroscience

The CUBIC technology developed by RIKEN has become a foundational technique for acquiring three-dimensional data on gene expression and network structures across the entire brain, enabling quantitative comparisons between samples. This technology has successfully achieved the transparency of adult mouse and small monkey brains, allowing for observation at a single-cell resolution.

We tested whether CUBIC could be applied to compare brain activity states under different conditions, and succeeded in comprehensively identifying light-activated brain regions at the whole-brain level. Furthermore, we demonstrated its potential for 3D immunostaining and observing the fine structures of nerve cells.

Cancer research

In this study, 3D lung organoids created from human lung epithelial cells were monitored with transmitted light, then stained and imaged through Matrigel using automated confocal microscopy. Advanced image analysis enabled complex analysis of organoid 3D reconstruction, cell morphology, viability, and differentiation markers, characterizing multiple quantitative metrics that can be used to study disease phenotypes and compound effects. Additionally, the concentration-dependent effects of several drugs known to cause lung toxicity were measured.

Organ-specific analysis

This introduces a method for making entire organs transparent using CUBIC technology and imaging them in three dimensions. It explains how this technique visualizes entire organ information, which was difficult with traditional 2D slices, and how it can be utilized for drug pharmacokinetics and toxicity evaluation.

Process and Points to Note for Introducing Organizational Transparency Analysis into Practice

Workflow

When performing tissue transparency analysis, we first fix the sample to preserve its morphology, then reduce light scattering through delipidation and decolorization. Next, we achieve transparency using reagents, and if necessary, perform antibody staining or other methods to visualize targets. Finally, we perform imaging using techniques like light-sheet microscopy to acquire 3D images and conduct image analysis.

The Importance of Data Management

3D images of transparent samples are very large in capacity, sometimes reaching terabytes per file. Therefore, not only storage capacity becomes an issue, but also an IT infrastructure that includes transfer speed, backup, access permissions, and GPU environments for analysis becomes important. If the data management environment is insufficient, analysis after imaging may be delayed, significantly reducing efficiency.

Utilizing external resources

When using organizational transparency technologies, significant capital investment and specialized knowledge are required. Since handling everything in-house can be a heavy burden, utilizing specialized CROs (Contract Research Organizations) with advanced technical expertise makes it easier to proceed with high-quality analysis in a short period while suppressing costs, including those related to equipment. As a result, improvements in development and research speed can also be expected.

What's next for organizational transparency technology?

Organizational transparency technologies are being utilized in integration with AI analysis. This leads to the increased efficiency of cell counting and three-dimensional structure extraction using machine learning and deep learning. Furthermore, by connecting detailed 3D analysis data obtained from animal models to improved predictability of clinical trials, it is thought to contribute to translational research.