Optimizing cell encapsulation condition in ECM-Collagen I hydrogels to support 3D neuronal cultures

The emergence of three-dimensional (3D) cell culture in neural tissue engineering has significantly elevated the complexity and relevance of in vitro systems. This is due in large part to the incorporation of biomaterials to impart structural dimensionality on the neuronal cultures. However, a comprehensive understanding of how key seeding parameters affect changes in cell distribution and viability remain unreported.

In this study, we systematically evaluated permutations in seeding conditions (i.e., cell concentration and atmospheric CO₂ levels) to understand how these affect key parameters in 3D culture characterization (i.e., cell health and distribution). Primary rat cortical neurons (i.e., 2 × 10⁶, 4 × 10⁶, and 1 × 10⁷ cells/mL) were entrapped in collagen blended with ECM proteins (ECM-Collagen) and exposed to atmospheric CO₂ (i.e., 0 vs 5% CO₂) during fibrillogenesis.

At 14 days in vitro (DIV), cell distribution within the hydrogel was dependent on cell concentration and atmospheric CO₂ during fibrillogenesis. A uniform distribution of cells was observed in cultures with 2 × 10⁶ and 4 × 10⁶ cells/mL in the presence of 5% CO₂, while a heterogeneous distribution was observed in cultures with 1 × 10⁷ cells/mL or in the absence of CO₂. Furthermore, increased cell concentration was proportional to the rise in cell death at 14 DIV, although cells remain viable >30 DIV.

ECM-Collagen gels have been shown to increase cell viability of neurons long-term.

In using ECM-collagen gels, we highlight the importance of optimizing seeding parameters and thorough 3D culture characterization to understand the neurophysiological responses of these 3D systems.