Ontogenetic stage affects rat neural cell spheroids formation and properties

Abstract

Aim. To compare the formation efficiency and morphometric, mechanical, and morphofunctional properties of neural spheroids derived from brain cells of rat embryos (embryonic day 15, E15) and neonatal rats (postnatal day 0, P0). Methods. Neural cells were isolated by mechanical-enzymatic dissociation, and spheroids were formed by the hanging drop method. Spheroid formation efficiency, diameter, sphericity, mechanical stability, and cell behavior after spheroid transfer to an adhesive substrate were evaluated. Results. Embryonic brain-derived cells demonstrated higher spheroid formation efficiency, producing larger, more spherical structures with less size variability and greater mechanical sta- bility than neonatal cells. After attachment, E15 spheroids rapidly disintegrated, accompanied by intense radial cell migration and the formation of a confluent layer exhibiting signs of neuronal and glial differentiation. In contrast, neonatal P0 spheroids maintained a compact 3D structure and formed stable neuronal networks. Conclusions. The ontogenetic stage of neural cells significantly affects spheroid self-organisation, mechanical stability, and morphofunctional properties, highlighting its importance in selecting cell sources for 3D neural tissue models.