Myelinated, synapsing cultures of murine spinal cord – validation as an in vitro model of the central nervous system

Abstract

Research in central nervous system (CNS) biology and pathology requires in vitro models, which, to recapitulate the CNS in vivo, must have extensive myelin and synapse formation under serum-free (defined) conditions. However, finding such a model has proven difficult. The technique described here produces dense cultures of myelinated axons, with abundant synapses and nodes of Ranvier, that are suitable for both morphological and biochemical analysis. Cellular and molecular events were easily visualised using conventional microscopy. Ultrastructurally, myelin sheaths were of the appropriate thickness relative to axonal diameter (G-ratio). Production of myelinated axons in these cultures was consistent and repeatable, as shown by statistical analysis of multiple experimental repeats. Myelinated axons were so abundant that from one litter of embryonic mice, myelin was produced in amounts sufficient for bulk biochemical analysis. This culture method was assessed for its ability to generate an in vitro model of the CNS that could be used for both neurobiological and neuropathological research. Myelin protein kinetics were investigated using a myelin fraction isolated from the cultures. This fraction was found to be superior, quantitatively and qualitatively, to the fraction recovered from standard cultures of dissociated oligodendrocytes, or from brain slices. The model was also used to investigate the roles of specific molecules in the pathogenesis of inflammatory CNS diseases. Using the defined conditions offered by this culture system, dose-specific, inhibitory effects of inflammatory cytokines on myelin formation were demonstrated, unequivocally. The method is technically quick, easy and reliable, and should have wide application to CNS research.

The Development of a ɛ-Polycaprolactone Scaffold for Central Nervous System Repair

“…In general, the maximum percentage of myelination in the cultures ranges from 2%-10% after 28 DIV as many of the fibers generated in culture are interneurons. Previously, we have demonstrated that myelin is compacted 25 and formed correctly with the correct location of nodal proteins in these cultures. 24 Comparisons were made to a positive control of cultures grown on a PLL-coated glass coverslip (to assure us that the cultures are differentiating correctly), and the following PLL-coated test substrates: PCL (low (L) and high (H) Mw), PC, PMMA, PS, PLLA, and PDMS (Fig.…”

“…Myelin ensheathment observed in these cultures is similar to myelination and sheath formation in vivo. [23][24][25][26] One important feature of the myelinating culture is ensuring that the neurite density is comparable between , markers of increasing oligodendroglial maturity at both 7 DIV and 14 DIV, with the percentage of A2B5 + cells and NG2 + cells calculated from a total cell number as indicated by DAPI (blue) staining. The total coverage of O4 + processes was also calculated and divided by the total DAPI count to give a ratio of O4 + coverage to total cell number.…”

“…Myelinating CNS cultures were set up as previously outlined, 24,25 with a couple of minor modifications. They comprise a monolayer of astrocytes on top of which are plated mixed rat spinal cord cells.…”

“…Since astrocytes derived from neurospheres or the cortex support myelination in these cultures, we used neurosphere-derived astrocytes as they more rapidly generate many purified cells. 24,25 Differentiating astrocytes in a flask before seeding enabled the calculation of defined cell numbers sufficient to form a confluent monolayer after seeding onto a glass coverslip or polymer substrate. This compensated for any difficulties assessing if the monolayer was confluent due to the opacity of the polymers.…”

“…These cells interact to mimic the complex cellular interactions of CNS cells in vivo. [24][25][26] Although many studies have primarily investigated biocompatibility, cytotoxicity, and the immune response following implantation, 27 we have focused on how the host CNS cells, including astrocytes, neurons, microglia, and their myelinating glia respond to the scaffold design with emphasis on differentiation as a read out, which indicates the potential effectiveness of the scaffold to support repair in the CNS.…”