TY  - GEN
KW  - Optical tissue clearing
KW  -  3D cell culture
KW  -  spheroids
KW  -  organoids
KW  -  3D microscopy
TI  - Optimizing Microscopy in Three Dimensions: Optical Clearing Techniques for the Study of hiPSC-Derived Neural Spheroids and 3D Cell Cultures
Y1  - 2024///
UR  - https://archiv.ub.uni-heidelberg.de/volltextserver/34579/
CY  - Heidelberg
AV  - public
ID  - heidok34579
N2  - 3D cell cultures are a significant advancement in cell biology, providing a more physiologically relevant environment compared to traditional 2D cultures. One notable application involves the neural differentiation of human induced pluripotent stem cells (hiPSCs) into complex 3D structures, like brain organoids, which closely resemble fetal brain development and are able to recapitulate complex processes in the human brain. While these models hold great promise for studying human brain development and diseases, they pose several challenges: a high level of expertise is necessary to generate these cultures, they are expensive to maintain, and exhibit a high variability between cell lines and experiments. In contrast, stable neural precursor cells (NPCs) derived from hiPSCs, while possessing lower differentiation potential and complexity, offer a more practical and consistent method for generating human neural cells in vitro. 
Unfortunately, the microscopic analysis of 3D-cultures presents difficulties owing to the intricate, extensively branched structure of neural cells, which span considerable distances, rendering it challenging to extract comprehensive information solely from cryosectioning techniques. One viable approach for analyzing these models involves whole mount confocal laser scanning microscopy (CLSM), which utilizes the intact sample for 3D visualization. However, optical effects, such as lateral light scattering due to refractive index (RI) mismatches prevent the generation of high-quality images beyond imaging depths of ~50 µm. Optical tissue clearing (OTC) techniques are a means to overcome these limitations by homogenizing the RI across the sample and enabling image acquisition at high resolution deep into biological specimens. Although OTC protocols have been thoroughly examined in tissue, their application was seldom extended to in vitro systems. In the limited studies conducted on this topic, a substantial variation in optical clearing efficiency is observed across different methods and cell lines, indicating the absence of a universal gold standard in this field of research.
The presented work describes the establishment of a 3D-spheroid model of hiPSC-derived NPCs, which can be generated in low attachment microwell plates, and which can be differentiated into a mixture of neurons and astrocytes. The differentiation process was monitored in cryosections over an extended time period of 50 days for marker proteins indicative of proliferation, apoptosis, as well as neuronal and astroglial differentiation. The results show a constant growth of spheroids over a period of two weeks, which correlated with KI67-expression level. Markers for young neurons were already present at the start of 3D-cultivation, whereas astrocytes emerged at a later differentiation state. While proportions of neural cells changed over time, a steady pool of SOX2+ NPCs was maintained throughout the cultivation period. 
Furthermore, a range of optical tissue clearing methods were established and evaluated for their effectiveness in clearing hiPSC-derived NPC spheroids. Specifically, ClearT2, CytoVista, ScaleS, and an 88% glycerol solution were tested to determine their capacity to maintain sample size while enhancing optical transparency. This assessment was conducted through a depth-dependent analysis of signal intensity and signal-to-noise ratio (SNR), as well as suitability for semi-automated image segmentation of nuclear signals. Although results showed the highest degree of spheroid size preservation upon ScaleS clearing, remaining analyses confirm superiority of glycerol clearing with respect to optical transparency and image quality. Furthermore, in addition to optical clearing of undifferentiated hiPSC-derived neural spheroids, glycerol clearing was applied to differentiated spheroids which were then subjected to light sheet fluorescence microscopy (LSFM). This confirmed the method's applicability to both undifferentiated and differentiated 3D samples.
In the light of high variability of optical clearing efficiency in 3D-in vitro systems reported in literature, ClearT2, CytoVista, ScaleS, and 88% glycerol were applied to additional cell culture models of varying complexity. These included four monoculture spheroid models, one complex tri-culture model of skin cancer and a chip-based co-culture model. Obtained results show a high degree of variability between the respective methods and cell culture models with respect to fluorescent dye preservation and optical transparency. Overall, optical clearing with glycerol proved to be the most reliable method across all models, although clearing efficiency was generally affected by the complexity of the cell culture model. Furthermore, application of linear z-compensation during imaging substantially improved image segmentation by stabilizing signal intensity and SNR across the sample dimensions.
A1  - Nürnberg, Elina
ER  -