Studying Pathologic Brain Developing Using 3D Brain Organoids
Human induced pluripotent stem cell (hiPSC)-derived cerebral organoids can serve as in vitro models for the study of pathologic human brain development
Neurological disorders carry complex etiologies characterized by variable genetic and environmental factors that differ from one individual to another. This makes it all the more challenging to understand and gain insights into the pathophysiology of these disorders. Disease modeling has been an integral approach in contemporary biomedical research to tackling this challenge and has contributed to our knowledge of the development, function, and pathology of diseases.
Disease models usually share specific physiological and behavioral features with human beings and enable experiments that may not be possible in direct human studies. Classical cell lines and animal model systems have been in use since the late twentieth and early twenty-first centuries. Animal models have been quite successful in predicting effective treatment strategies and guiding the design of therapeutics for various diseases. However, these models are also riddled with translational failures due to inherent disparities between them and human biology. Current animal models available to neuroscience researchers fail to fully capture the characteristics and functionalities of the human brain, making it extremely difficult to translate said assays for clinical trials. Similarly, in-vitro models based on simplified cell culture systems, while easy to scale up, do not adequately reflect the structural and organizational complexity of the human brain.
Human in vitro 3D cell culture using stem cells from different organs of the human body, otherwise known as organoids, has emerged as a promising solution for overcoming these limitations.
3D Brain Organoids – A StemoniX story
Organoids are not a novelty and are the product of decades of research going back to the beginning of the 20th century. Organoids generally refer to 3D cultures and specifically to multi-cellular microtissues derived from stem cells or organ progenitors grown in 3D gels that closely mimic the structure and function of human organs. Naturally, organoids owe a better representation of in vivo cell responses and interactions of a given organ compared to traditional 2D cell cultures, and subsequently, provide greater insights into disease pathology and host-pathogen interactions. Patient-derived organoids thus provide an alternative but highly effective avenue for drug screening and toxicity evaluations as well as advances in personalized medicine.
Human induced pluripotent stem cell (hiPSC)-derived brain organoids have become a promising tool for neuroscience modeling. When cultured, hiPSCs differentiate into neural cells that mature to resemble structures of various regions of the human brain. A rapidly developing technology, brain organoids present great potential for understanding human brain development, neuronal diseases, and enable testing for genetic mutations, pharmaceutical drugs and their effects, and other functional genomic applications. Yet, despite their promising potential, hiPSC-derived brain organoids continue to display high variability and lack functional assays for phenotype assessment.
Recently, scientists at StemoniX have bridged this gap by developing the microBrain 3D platform, setting the stage to capture the unique features of human brain development and function in vitro. This was achieved using hiPSC-derived neural progenitor cells to create functionally active 3D neural cortical spheroids. Neurospheroids generate spontaneous oscillations of activity, that can be visualized by using calcium sensors, and measured as oscillations of fluorescence. In a platform composed of mature and active neurons and astrocytes, the resultant cortical spheroids more closely mimicked the human cortical brain, responding to neuromodulators much like primary neural cultures. These pre-plated neural spheroids thus enable researchers the ability to construct accurate, and predictive in-vitro models that can be used for drug discovery, disease modeling and toxicology screening surrounding neurological disorders, a feature that has been lacking in the field. The development of these complex organoids requires the support of sophisticated 3D imaging and analysis instruments to assist in accurately characterizing the relevant biological assays. StemoniX’s microBrain 3D Platform is similarly facilitated by Molecular Devices’ automated confocal imaging systems, multi-mode microplate readers, and high-throughput cellular screening systems.
Molecular Devices – Setting the stage
A life-science technology innovator, Molecular Devices represents an extensive platform of diverse integrated hardware and software solutions that enable automated workflows in cell line development, 3D biology, and drug screening. With a goal to maximize throughput and reproducibility, Molecular Devices’ solutions have helped researchers streamline their workflow and optimize experimental results. StemoniX’s design of the microBrain 3D platform, in 96 and 384-well formats, was achieved in conjunction with the use of high-content screening instruments from Molecular Devices including the FLIPR Tetra High-Throughput Cellular Screening System, the ImageXpress Micro Confocal High-Content Imaging System, and the SpectraMax i3x Multi-Mode microplate reader.
The FLIPR Tetra High-Throughput Cellular Screening System is essentially an automated solution for identifying early leads in the drug discovery process as well as evaluating drug efficacy and toxicity. In what is a fully integrated solution, the FLIPR screening system enables record fast kinetic cell responses using calcium-sensitive dyes including calcium oscillations that can measure neuronal activity. This helps researchers transition seamlessly from assay development for fast kinetic cellular assays to lead optimization. The ImageXpress Micro Confocal High-Content Imaging System helps capture high-quality images of relevant samples at great speed, sensitivity, and flexibility. It also provides sophisticated (comprehensive) image analysis, serving as a multi-dimensional, high-throughput screening solution. Lastly, the SpectraMax i3x Multi-Mode microplate reader allows researchers to measure spectral-based absorbance, fluorescence, and luminescence and sheds light on cellular pathways and protein activation and expression in a given system.
The ImageXpress Micro Confocal system was utilized for brightfield capture and automated size measurement thus facilitating the observation of individual plates with highly homogeneous neurospheroids (size variations less than 4%). Compositional assessment of the 3D spheroids was accomplished using immunofluorescence analysis on the ImageXpress system and helped capture the presence of cortical neurons and astrocytes. Functional activities in organoids are distinguished by Ca2+ oscillations using calcium imaging and recorded by FLIPR instrument. A combination of the ImageXpress and FLIPR systems were utilized in the case of StemoniX’s brain organoids. The FLIPR system was also used to correlate modulations of neural activity with known mechanisms of action, further confirming the functional maturity of the microBrain 3D platform and the presence of functional glutamatergic and GABAergic circuits in the spheroids.
Beyond neurological disorders, neural spheroids can also be used to screen neurotoxic effects of various chemicals. Using microBrain 3D neurospheres from Stemonix as well as the ImageXpress Micro Confocal and FLIPR Tetra systems from Molecular Devices, the researchers then successfully screened the cytotoxicity effects of anticancer and other drugs using Ca2+ oscillations in neurospheroids. Molecular Devices’ high-content screening instruments have also played focal roles as part of a toxicological case study to investigate cell toxicity of a targeted library of compounds with differing potencies against the Zika Virus. The SpectraMax microplate reader was used to translate this investigation to the microBrain 3D platform. Cellular toxicity analysis was achieved using the FLIPR system and high-resolution videos of calcium oscillations following each treatment were captured using the ImageXpress confocal microscope. The ensuing results solidified the StemoniX microBrain 3D platform’s ability for efficient in vitro investigations of neural phenotypes, toxicological profiles, and drug screening.
Conclusion
Human in vitro 3D cell culture systems have made it possible to recreate the structure and physiology of human organs with remarkable detail. Human organoids provide researchers with a unique opportunity to study human disease and complement animal models while simultaneously overcoming their corresponding limitations.
Through the genetic engineering of human stem cells, human organoids have been used to study infectious diseases as well as genetic and neurological disorders. hiPSC-derived brain organoids have been recognized as an effective tool for neuroscience modeling. StemoniX’s microBrain 3D platform provides the means to obtain 3D brain organoids to better recapitulate brain development and function in vitro. Combined with Molecular Devices’ array of integrated solutions for high-quality imaging and characterization of organoids, researchers have been able to definitively demonstrate that hiPSC-derived cerebral organoids can serve as in vitro models for the study of pathologic human brain development.
To learn more, visit moleculardevices.com.
