Towards an improved serum-free, chemically defined medium for long-term culturing of cerebral cortex tissue

Abstract

The present study describes a series of experiments which have led to a substantially improved serum-free, chemically defined medium (CDM) for long-term culturing of reaggregated fetal rat cerebral cortex tissue. A reduction of the original medium concentrations of the hormones insuline, T3 and corticosterone, on the one hand, and an enrichment of the medium with the vitamins A, C and E, the unsaturated fatty acids linoleic and linolenic acid, and biotin, L-carnitine, D(+)-galactose, glutathione (reduced) and ethanolamine, on the other hand, formed the most important chemical adjustments of the medium. With the aid of this CDM (encoded R12), the light- and electron microscopic architecture of the tissue could be kept in a good condition (superior to that seen earlier in serum-supplemented medium) up to 23 days in vitro. From that time on, the neuronal network lying between the reaggregates degenerated for the largest part, while a portion of the large neurons (probably pyramidal cells) plus some of the neuronal network within the reaggregates degenerated too. This degeneration process continued during the following weeks, but the reaggregates nevertheless retained most of their mass, so that both small and large neuronal cell bodies (visible in transparent regions at the edge of the reaggregates) remained in good condition up to at least 103 DIV. Stout, thick nerve bundles interconnecting the reaggregates, also survived up to this point. Electron microscopic evaluation of suc…