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OBJECTIVES/GOALS: Our goal is to develop a silk fibroin scaffold-based neural tissue construct and characterize it in a rat model of cortical injury. We aim to optimize the construct for transplantation, test pharmacologic interventions that may enhance its survival, and evaluate its integration with the host brain. METHODS/STUDY POPULATION: To optimize cell density and health, silk fibroin scaffolds varying in porosity and stiffness were seeded with E18 GFP+ rat cortical neurons and imaged at DIV 5. Different seeding methods and loads were similarly tested. Constructs, loaded with an inhibitor of apoptosis (ROCK inhibitor Y-27632) or necroptosis (necrostatin-1) in a fibrin hydrogel, were transplanted into aspiration lesions created in the primary motor cortex of Sprague-Dawley rats, and graft survival was compared to negative control at 2 weeks. Lastly, constructs were transplanted and evaluated via immunohistochemistry at 1, 2, and 4-month time points for survival, differentiation, inflammation, and anatomic integration. RESULTS/ANTICIPATED RESULTS: Scaffolds with smaller pore sizes retained more cells after seeding. Softer scaffolds, which enhance hemostasis at transplantation, did not compromise cell health on live/dead assay. We anticipate that seeding concentrated cell suspensions onto multiple surfaces of the construct will produce the most evenly seeded and cell-dense constructs. Based on a prior pilot study, we anticipate that necrostatin-1 will significantly improve intermediate-term construct survival. We have observed up to 15% cell survival at 1 month with retained neuronal identity and abundant axonal projections into the brain despite evidence of persistent inflammation; we anticipate similar outcomes at later time points. DISCUSSION/SIGNIFICANCE OF IMPACT: Our construct, due to its exceptional longevity in vitro, manipulability, and modularity, is an attractive platform for neural tissue engineering. In the present work, we optimize and validate this technology for transplantation with the goal of addressing the morbidity burden of cortical injury.

ABSTRACT IMPACT: This study will provide the essential characterization of intrinsic neural activity in human brain organoids, both at the single cell and network levels, to harness for translational purposes. OBJECTIVES/GOALS: Brain organoids are 3D, stem cell-derived neural tissues that recapitulate neurodevelopment. However, to levy their full translational potential, a deeper understanding of their intrinsic neural activity is essential. Here, we present our preliminary analysis of maturing neural activity in human forebrain organoids. METHODS/STUDY POPULATION: Forebrain organoids were generated from human iPSC lines derived from healthy volunteers. Linear microelectrode probes were employed to record spontaneous electrical activity from day 77, 100, and 130 organoids. Single unit recordings were collected during hour-long recordings, involving baseline recordings followed by glutamatergic blockade. Subsequently, tetrodotoxin, was used to abolish action potential firing. Single units were identified via spike sorting, and the spatiotemporal evolution of baseline neural properties and network dynamics was characterized. RESULTS/ANTICIPATED RESULTS: Nine organoids were recorded successfully (n=3 per timepoint). A significant difference in number of units was seen across age groups (F (2,6) = 6.4178, p = 0.0323). Post hoc comparisons by the Tukey HSD test showed significantly more units in day 130 (51.67 ±14.15) than day 77 (16.33 ±14.98) organoids. Mean firing rates were significantly different in organoids based on age, with drug condition also trending toward significance (F (6,12) = 9.97; p = 0.0028 and p = 0.08 respectively). Post hoc comparisons showed a higher baseline firing rate in day 130 (0.99Hz ±0.30) organoids than their day 77 counterparts at baseline (0.31Hz ±0.066) and glutamate blockade (0.31Hz ±0.045). Preliminary network analysis showed no modularity or small-world features; however, these features are expected to emerge as organoids mature. DISCUSSION/SIGNIFICANCE OF FINDINGS: Initial analysis of brain organoid activity demonstrates changes in single unit properties as they mature. Additional work in this area, as well as further network analyses, will confer better sense of how to rationally utilize brain organoids for translational purposes.

Neuronal degeneration and the deterioration of neuronal communication lie at the origin of many neuronal disorders, and there have been major efforts to develop cell replacement therapies for treating such diseases. One challenge, however, is that differentiated cells are challenging to transplant due to their sensitivity both to being uprooted from their cell culture growth support and to shear forces inherent in the implantation process. Here, we describe an approach to address these problems. We demonstrate that rat hippocampal neurons can be grown on colloidal particles or beads, matured and even transfected in vitro, and subsequently transplanted while adhered to the beads into the young adult rat hippocampus. The transplanted cells have a 76% cell survival rate one week post-surgery. At this time, most transplanted neurons have left their beads and elaborated long processes, similar to the host neurons. Additionally, the transplanted cells distribute uniformly across the host hippocampus. Expression of a fluorescent protein and the light-gated glutamate receptor in the transplanted neurons enabled them to be driven to fire by remote optical control. At 1-2 weeks after transplantation, calcium imaging of host brain slice shows that optical excitation of the transplanted neurons elicits activity in nearby host neurons, indicating the formation of functional transplant-host synaptic connections. After 6 months, the transplanted cell survival and overall cell distribution remained unchanged, suggesting that cells are functionally integrated. This approach, which could be extended to other cell classes such as neural stem cells and other regions of the brain, offers promising prospects for neuronal circuit repair via transplantation of in vitro differentiated, genetically engineered neurons.