Three-dimensional cultures of fetal mouse cerebral cortex in a collagen matrix

Abstract

A 3-dimensional tissue culture system for fetal mouse cerebral cortex was devised in order to study neuronal migration in vitro. The dorsolateral cerebral mantle of E16 mouse was dissected into slices of 250–350 μm. Explants were embedded in a matrix formed of thin layers of collagen gel above and below the tissue. The optimal conditions for maintaining explant survival within the matrix included (1) placing the explants on membrane inserts with large (70 μm) pores which permits access of nutrient to the cultures from below as well as from above, and (2) intermittent exposure of the explants to ambient oxygen (20% O 2 with 5% CO 2) by partially covering the upper surface with medium and placing the cultures on a rocking platform. Thickness measurements of serially sectioned intact cultures at 6 days in vitro ranged from 128 to 210 μm (average: 172 μm). In intact explants, maintained for up to 14 days in vitro, the preservation of neurones, neuronal subtypes and glia was confirmed with immunofluorescence staining for neurone-specific nucleoprotein (NeuN), microtubule-associated protein (MAP-2), a glutamate receptor subunit (GluR1), GABA and glial fibrillary acidic protein (GFAP) respectively. Some cortical differentiation was obtained in vitro, at 6 days, with the development of upper and lower cortical layers, similar to that seen in an P1 animal.