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Into the depths: Techniques for in vitro three-dimensional microtissue visualization.

Pranita K Kabadi¹, Marguerite M Vantangoli¹, April L Rodd¹

¹Department of Pathology and Laboratory Medicine, Brown University, Providence, RI.

|November 12, 2015

Summary

This summary is machine-generated.

Optimized histology and microscopy techniques improve 3-D microtissue imaging. Refined methods enhance visualization for disease and toxicity studies using three-dimensional (3-D) cell cultures.

Keywords:

clearing histology microtissues spheroids three-dimensional

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Area of Science:

Biomedical Engineering
Cell Biology
Toxicology

Background:

Three-dimensional (3-D) in vitro models offer superior physiological relevance compared to 2-D cultures or animal models.
3-D cell culture systems are valuable for disease modeling, drug discovery, and toxicity testing.
Current limitations in imaging hinder the full potential of 3-D microtissues.

Purpose of the Study:

To optimize histology and microscopy techniques for enhanced imaging of 3-D microtissues.
To address the challenges associated with visualizing and analyzing 3-D cell culture models.
To improve the utility of 3-D in vitro systems in pharmacological and toxicological research.

Main Methods:

Developed a two-step glycol methacrylate embedding protocol for improved morphological assessment of 3-D microtissues.
Adapted immunohistochemistry, immunofluorescence, and in situ immunostaining for 3-D microtissues.
Implemented the Clear(T2) optical clearing protocol to enhance imaging depth and clarity.

Main Results:

The optimized embedding protocol improved resolution of nuclear and cellular histopathology, revealing cell death and proliferation.
Optical clearing with Clear(T2) significantly increased fluorescence signal intensity and imaging depth.
Enhanced imaging allowed for more complete confocal fluorescence microscopy of 3-D microtissues.

Conclusions:

Refined histology and microscopy techniques overcome key 3-D imaging challenges.
These methods provide new avenues for evaluating disease pathogenesis and toxicity pathways.
The improved imaging versatility enhances the application of 3-D in vitro systems in drug development and safety assessment.