Clearer Answers Ahead: Optical Clearing of 3D Spheroid Cell Cultures

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Paper Review by AxoSim Research Scientist Kevin Simpson, PhD

Three-dimensional cell cultures, such as spheroids, serve as increasingly important models in fundamental and applied research and continue to be developed for use in drug screening.  In short, spheroids as three-dimensional (3D) cell models can better mimic the characteristics of more complex cellular structures, such as networks with supporting cell types, both in the brain and peripheral nervous system.  While these models can afford more clinically relevant data for research and drug screening, imaging of three-dimensional specimens can be inherently challenging. Depending on the sample cell type(s) and spheroid size, the sample thickness commonly exceeds the depth of focus of a conventional detection system.

At the center of this problem, penetration of light into biological samples is usually limited to around 50–70 µm and light scattering considerably impairs the image quality; caused by mismatches in the refractive index (RI) between boundaries within biological tissue components, such as proteins, water, and lipids.  Additionally, the density of the spheroid can have a direct impact on the ability of imaging reagents to be uniformly applied.

To work around these limitations, researchers can run analysis of cell type or marker protein distribution in fixed frozen or paraffin-embedded biological 3D samples; typically using tissue sectioning followed by immunohistochemical staining, and confocal laser scanning microscopy (CLSM). But, these methods are destructive to the sample and can be awkward to reassemble into a meaningful 3D representation of the original spheroid system.

So, despite the widespread usage of 3D-cell culture models, there is much potential for optimization in related analytical downstream processes.  Tissue clearing is an increasingly attractive emerging option with several benefits.

Tissue clearing techniques all aim to make tissues or cell cultures more transparent to overcome their opacity, allowing for deeper penetration by visible wavelengths of light under the microscope and increased visualization of markers.  Additional potential benefits include, minimum changes to morphology, comparable sensitivity of detection with almost any fluorophore and reversible (non-destructive) procedures which keep the spheroid intact for further research if needed.

The authors of this review style publication go through a more systematic overview about the effects and applicability of optical tissue clearing on three-dimensional cell cultures.  They take the time to compare six different clearing/embedding protocols (including a selection of commercially available kits) on seven types of spheroid cell cultures (mono-, co- and tri-cultures) of approximately 300 µm in size. Quantitative analysis includes fluorescence signal intensity and signal-to-noise ratio as a function of z-depth as well as segmentation and counting of nuclei and immunopositive cells.

There is great detail in the Materials and Methods section, with a step-by-step walkthrough of the processes and full description of solution components (where available).  Gorgeously engaging figures show the visualization and depth of penetration for a selected cell type spheroid across all cell clearing techniques reviewed (with additional links to other cell type spheroid data in the Supplemental section).  Everything a lab needs to start considering different methodologies of cell clearing and begin to optimize methods to meet the needs of the cell type(s) they are working with.

Often times, while commercial vendors supply valuable information about their particular product, the practical aspects of a side-by-side comparison with other options is not present (or showcases only the strengths of the system in question).  This is a refreshingly impartial review of the methods, data, outcomes and pros/cons of different clearing systems with no spin towards a certain product or method.  And as they conclude:

“…  inherent characteristics of cell lines influence the outcome of optical clearing and that protocols should be chosen in a sample-dependent manner. Factors to consider include size, complexity and composition of 3D cultures”.

Here at AxoSim, both NerveSim® and BrainSim® utilize well established cell spheroid technologies to produce more clinically relevant data.  Current methods of fixation and immuno-histochemistry (IHC) have afforded ground breaking information from these systems.  And with growing advances in cell imaging, like cell clearing, we will continue to improve the quality of data obtained by our systems.


Read Full Research Paper:

Frontiers | Routine Optical Clearing of 3D-Cell Cultures: Simplicity Forward | Molecular Biosciences (  (