In case you missed them - below are our posters and abstracts from SOT 2018 in San Antonio:
3D Histological Characterization of Precision-Cut Lung Slices for Inhalation Studies
Inhalation studies for allergens and pathogens typically rely upon flow cytometry which provides quantitative analysis of cell proteins associated with immune cells. However, flow cytometry does not provide information on the migration of these cells within the lung and is highly limited in describing the complexities of the lung. Due to these limitations, researchers have begun adopting precision cut lung slices (PCLS) as an in vitro tool as they are able to provide significantly improved in vivo relevancy. However, one of the current limitations in using PCLS models is that they are approx. 250 µm in thickness, and are too thick to image in their entirety using confocal microscopy. Therefore, it is challenging to capture data from these tissues past a few cell layers due to optical attenuation. Through this work, a rapid tissue clearing technique was applied to PCLS models in a high-throughput/well-plate-compatible format to enable whole mount 3D imaging of the entire PCLS model with confocal microscopy. It was shown that this tissue clearing approach was compatible with several commonly used labels (e.g. DAPI, CD68, tomato lectin, PDGFR, alpha-smooth muscle actin) and that uniform labeling as well as complete tissue characterization could be accomplished.
Optical Projection Tomography with a Tissue-Clearing Agent for Developmental and Reproductive Toxicology Studies
Developmental and reproductive toxicology (DART) testing is one of the most expensive and time-consuming stages in determining the toxicological profile of new chemical entities. Within DART studies, gross morphological evaluation of fetal animal skeletons for developmental abnormalities presents a major bottleneck. Current processing techniques involve digestion of soft tissue using a strong base, followed by qualitative assessment of the remaining skeletal tissue by a pathologist. Micro computed tomography (microCT) has been proposed as a non-destructive image-based alternative. Such methods eliminate the need for extensive tissue processing and can be paired with quantitative analysis algorithms. However, due to the significant capital and operational expenses required for microCT imaging, this approach has yet to gain widespread traction. Here, we propose a cost effective optical imaging alternative. A novel tissue clearing agent was used in 1-week old rats to temporarily render soft tissue optically transparent. Alizarin red was used to stain skeletal tissues. High dynamic range (HDR) optical trans-illumination images were then acquired with a low-cost optical-CT imaging system and rendered as 3D images of skeletal structure. HDR-based optical-CT imaging of chemically cleared tissues can rapidly generate high resolution (50–250 um) reconstructions of whole skeletons. This study demonstrates that the combination of tissue clearing, optical imaging, and novel reconstruction algorithms may present a new paradigm for low-cost, high-throughput evaluation of tissues in DART testing.
Improved Characterization of Compound Toxicity through the Application of a Tissue-Clearing Technique to 3D In Vitro Model Screening
The use of in vitro three-dimensional (3D) cell cultures has increased dramatically for drug screening as 3D culture models more accurately mimic the in vivo environment compared to traditional monolayer cultures. However, unlike for traditional cell culture, imaging analysis of 3D cultures is limited due to the thickness of 3D cell cultures (typically >100 μm) which causes light scattering, limiting imaging to the surface-layer cells of the 3D culture. This limitation prevents the complete characterization of the cell population within whole-mount 3D cultures. Furthermore, this technical limitation introduces an unavoidable sampling bias in imaging analysis, since only the exterior cells can be imaged where concentrations of nutrients, oxygen, and drug compound are greatest. It was sought to solve the problem of 3D cell culture opacity by employing an optical clearing agent designed specifically for these tissues. Here, we combine HepG2 Spheroids and the Visikol® HISTO-M™ clearing agent to illustrate the effect of tissue clearing on high content confocal screening of steatosis and cytotoxicity induced by several known hepatotoxic agents. It was demonstrated that the addition of tissue clearing allowed for a 3-fold increase in cells detected, the ability to characterize dose response as a function of tissue depth and an increase in dose response sensitivity.