High-throughput Organoid Screening Platform for Investigating Novel Traumatic Brain Injury Therapeutics

Traumatic brain injury (TBI) remains one of the leading causes of morbidity and mortality among civilians and military personnel globally.  In the US there are > 220K hospitalizations and >70K deaths annually from TBI.  Despite advances in our knowledge of the complex pathophysiology of TBI, the underlying mechanisms are not yet fully understood and more effective therapeutics are required for both acute and chronic disease.  While many therapeutic approaches show promise in preclinical studies and Phase I/II trials, the majority of them turn out to be unsuccessful in Phase III clinical trials.  This project will address three critical areas for drug discovery and lead to an improved high-throughput screening (HTS) method for discovery and developing new therapeutics for TBI.  First, improved in vitro brain models are needed that are not only physiologically relevant but have the cost structure and reproducibility required for HTS.  We will use the existing capabilities of UF Scripps to create large quantities of human-derived brain organoids or neurospheres. Second, TBI events must be simulated on organoids in a device and manner suitable for HTS.  We will use precision high pressure pulses (up to 45 psi, <100 ms) delivered to organoids in a microfluidic flowchip to cause TBI.  The organoids can then be assayed for acute and chronic disease phenotypes and screened against panels of therapeutics.  The microfluidic connections offer potential for organoid-to-organoid communication or introduction of cell therapies to expand the range of possible assays.  Third, high throughput analysis of 3D neurosphere cell structures provides significant challenges from both an instrumentation and data analysis perspective.  We will use AI-trained models to efficiently extract phenotypic information (e.g. organoid viability, neurogenesis) from widefield fluorescent and transmitted light images.  This will be coupled with measurements of neuronal firing response to provide a multiparametric characterization of how organoids react to and recover from TBI events.   Ultimately, this platform will provide scientists an unprecedented ability to evaluate therapeutics for multiple phases of TBI: Initial impact and short term (< 1 hr) recovery including neuroprotective therapies, mid-term (1-7 days) effects of cell death, intracranial pressures (ICP), and other damage, and longer-term recovery from chronic cerebral disease including neurogenesis and cell therapies.  The results from these studies have the potential to save millions of lives and improve recovery of our military personnel from blunt impact and explosive blasts.