Evaluating Various Whole-Mount Organoid Tissue Clearing Techniques for High Throughput Experimentation with Microfluidics
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Originating from human pluripotent stem cells, organoids are three-dimensional structures that resemble the developing human brain. These multicellular structures are capable of recapitulating distinct features of organ structure and function. As a result, they provide novel opportunities for studying human brain development. Due to the thickness & density of brain organoid tissue, these complex structures are not transparent, making them difficult to analyze using microscopy. Whole organoids can be imaged using confocal or multiphoton microscopy, but their imaging quality decreases with depth focus. In contrast, tissue clearing allows for high resolution imaging of thicker tissue sections by increasing tissue transparency in order to observe development deep within organoids. Using whole-mount tissue clearing allows for analysis of protein and gene expression within deep tissue without compromising the complexity of their internal structures. As a result, this enables understanding of cellular composition, cell-to-cell interactions including sensitivity and specificity. Various tissue clearing techniques exist that enable high-resolution whole-mount imaging of 3-D samples. For example, organic solvent-based techniques (BABB, iDISCO, 3DISCO, etc.) utilize methanol dehydration prior to clearing with their respective clearing agents. There are also hyperhydration techniques (CUBIC, SCALE, CLARITY) which utilize delipidation with amino alcohols and urea prior to clearing. However, these protocols are time-consuming and labor intensive, requiring several sequential experimental steps. Microfluidics can help address some of these limitations by shortening the time of experiments conducted using perfusion for clearing, enabling in situ and real-time visualization of the clearing process, while using high-resolution detection for understanding neurological disease states. This project assesses various clearing techniques using metrics such as antibody penetration depth and localization for characterizing brain organoid development. Following the assessment, the successful protocols were modified and adapted for use with microfluidics. The findings from this work provide key considerations for developing a high throughput pipeline for whole-mount tissue clearing and immunostaining to better understand the underlying pathology of neurological diseases.