Research Scientist Molecular Devices, LLC San Jose, CA, United States
Abstract: Organotypic three-dimensional (3D) cultures resemble native 3D tissue architecture and are believed to be more representative of real tissues than their 2D (monolayer) counterparts, thus providing higher in vivo relevance. Brain organoids attract a lot of interest due to their physiological relevance for the study of neurological development and disease. However, while they show great promise, brain organoids tend to be difficult to grow, and demonstrate variable shape, size, neural network density, and light transmissibility, thus rendering many unsuitable for high-throughput screening. Some of this variability can be attributed to extended maturation times, and the batch-to-batch variability of commonly used extracellular matrix (ECM) mimetics such as Matrigel® or Geltrex™.
RealBrain® micro-tissues from Tessara Therapeutics combine high physiological relevance with reproducibility at scale, and provide an ideal platform for development of optimized protocols for 3D neurite outgrowth analysis. RealBrain® micro-tissues are generated by encapsulating primary or induced pluripotent stem cell (iPSC)-derived neural precursor cells in a proprietary, chemically defined hydrogel that provides the optimal micro-environment to activate endogenous programs of neurodevelopment in vitro. The developing cells proliferate, remodel their micro-environment, migrate, self-organize in 3 dimensions, and replace the engineered hydrogel with cell-secreted ECM. Furthermore, the optical clarity of the RealBrain® constructs is ideally suited for high-content imaging applications.
Developing neurons produce new projections as they grow and integrate into a neural network. As a result, assessing the extent of neurite outgrowth is an important quantification of neural network complexity. Such quantification requires a tightly orchestrated and integrated workflow that renders visualization and analysis of these complex biological samples, at scale, with the ability to reconstruct 3D images.
In this study we imaged RealBrain® micro-tissues treated with compounds that are reported to either stimulate (NGF and Pentadecanoic acid (PD acid)) or inhibit (Rotenone) neurite outgrowth and stained with fluorescent markers using the ImageXpress® Micro Confocal High-Content Imaging System. We then reconstructed these tissues in 3D and developed a method to quantify the number of neurons and their respective outgrowths using MetaXpress® High-Content Image Acquisition and Analysis Software. With this approach, we confirmed the efficacy of the compounds with the quantification of neurite outgrowth analysis and found that NGF and PD acid significantly increase while Rotenone reduces the number of neurites compared to the control. Thus, this type of analysis can be used in multiple applications, such as assessing the effects of compounds on neurons and neural networks.