Automation and High-Throughput Technologies

Alzheimer’s Disease Modeling and High-throughput Drug Screening using Homogeneous Arrays of Human Neurospheroids

• MJ

  Mehdi Jorfi, PhD

  Research Fellow
  Massachusetts General Hospital and Harvard Medical School

Neurospheroids serve as a widely accepted in vitro platform for disease modeling and drug screening. However, current approaches to recreate neurodegenerative diseases in a dish using neurospheroids rely on mixtures of spheroids that are heterogeneous in size, which limit their applications in basic mechanistic studies and drug screening. Here, we show the in vitro culture of uniformly-sized stem-cell-derived human neurospheroids in large arrays, where they can be monitored for months, and closely recapitulate key hallmarks of familial Alzheimer’s disease including pathogenic accumulation of amyloid-β (Aβ) and phosphorylated tau. The three-dimensional (3D) microarray system generates uniform-sized neurospheroids, with less than 1% variability in diameter in a 96-well array with 1,536 microwells. This performance is key to measuring with unprecedented precision the efficacy and side-effects of Aβ modulating drugs in large scale arrays. We also observed accumulation of amyloid-β and pathogenic phosphorylated tau species after 7-8 week-differentiation in our 3D neurospheroid model of Alzheimer’s disease, not in the control 3D spheroids. This accumulation of amyloid-β was blocked by β-secretase inhibitor treatment. To further extend the capability of our array platform and accelerate drug screening of the human neurospheroids for drug discovery, we leveraged microfabrication and 3D printing techniques to develop a 96-well array with 1,536 microwells. The 96-well array is comprised of five main components: (i) a 3D designed and printed 96-well frame, (ii) a high-quality glass substrate with high transmittance of over 92% and high optical clarity for fluorescence wavelengths, (iii) a microfabricated microarray with 1,536 microwells, (iv) a self-adhesive 96-well silicon superstructure that adheres to the microarray, and (v) a lid. Using this array, we generated uniformly-sized neurospheroids and treated with various compounds including gamma–secretase inhibitor, β–secretase inhibitor, gamma–secretase modulator, Imidazenil, and Methotrexate for different concentrations. We have also confirmed our microarrays can be used for differentiating and modeling disease phenotype using human iPSC-derived neurospheroids. The advantages associated with this microarray platform include, but not limited to facile microarray construction, ease of culture, high-throughput sampling, low amount of reagents required to establish the neurospheroids, cost effective, and compatible with wide range of commercially available automated handling machines. This robust in vitro platform could serve as a valuable next-generation tool for sophisticated 3D models of complex neurodegenerative diseases such as Alzheimer’s disease and expediting the central nervous system drug discovery.