Strategies for repair of white matter: influence of osmolarity and microglia on proliferation and apoptosis of oligodendrocyte precursor cells in different basal culture media

Abstract

The aim of the present study has been to obtain high yields of oligodendrocyte precursor cells (OPCs) in culture. This is a first step in facilitation of myelin repair. We show that, in addition to factors, known to promote proliferation, such as basic fibroblast growth factor (FGF-2) and platelet derived growth factor (PDGF) the choice of the basal medium exerts a significant influence on the yield of OPCs in cultures from newborn rats. During a culture period of up to 9 days we observed larger numbers of surviving cells in Dulbecco's Modified Eagle Medium (DMEM), and Roswell Park Memorial Institute Medium (RPMI) compared with Neurobasal Medium (NB). A larger number of A2B5-positive OPCs was found after 6 days in RPMI based media compared with NB. The percentage of bromodeoxyuridine (BrdU)-positive cells was largest in cultures maintained in DMEM and RPMI. The percentage of caspase-3 positive cells was largest in NB, suggesting that this medium inhibits OPC proliferation and favors apoptosis. A difference between NB and DMEM as well as RPMI is the reduced Na + -content. The addition of equiosmolar supplements of mannitol or NaCl to NB medium rescued the BrdU-incorporation rate. This suggested that the osmolarity influences the proliferation of OPCs. Plating density as well as residual microglia influence OPC survival, BrdU incorporation, and caspase-3 expression. We found, that high density cultures secrete factors that inhibit BrdU incorporation whereas the presence of additional microglia induces an increase in caspase-3 positive cells, indicative of enhanced apoptosis. An enhanced number of microglia could thus also explain the stronger inhibition of OPC differentiation observed in high density cultures in response to treatment with the cytokines TNF-α and IFN-γ. We conclude that a maximal yield of OPCs is obtained in a medium of an osmolarity higher than 280 mOsm plated at a relatively low density in the presence of as little microglia as technically achievable.

Primary Spinal OPC Culture System from Adult Zebrafish to Study Oligodendrocyte Differentiation In Vitro

“…The mixed cultures can be enriched for OPCs by different methods, the most popular and simple being the shake-off technique that is based on differential adhesive properties of glial populations, reviewed in ( Barateiro and Fernandes, 2014 ). The enrichment and survival of rat OPCs is largely dependent on the presence of growth factors, like basic fibroblast growth factor (FGF-2) and platelet derived growth factor (PDGF), the cell seeding density and also the composition of the basal medium used ( Kleinsimlinghaus et al, 2013 ). The establishment of mouse OPC cultures is much more difficult than from rat, mostly because of the lack of surface markers and the tendency of mouse OPCs to spontaneously differentiate into other lineages ( Chen et al, 2007 ; Zhu et al, 2014 ).…”

Regulation of neuronal excitability by release of proteins from glial cells

“…Microglial cells were obtained from whole brain mixed glial cultures by a shaking procedure following the procedures published by McCarthy & de Vellis [38], modified as described in [39]. In brief, mixed glial cultures were obtained from whole brains of postnatal day 0-3 Wistar Hannover rat pups rostral of the cerebellum, dissociated by passing through 125 mm and 36 mm nylon meshes.…”

“…Pipette solutions contained, in mM: CaCl 2 0.1, EGTA 1.1, MgCl 2 5, CsF 100, NaCl 5, HEPES 10; bath solutions contained, in mM: 4-aminopyridine 4, TEA-Cl 10, CaCl 2 1, CdCl 2 0.5, MgCl 2 1, glucose 10, HEPES 10, NaCl 100. Osmolarities of pipette and bath solution were adjusted to the low osmolarity of the NB medium which inhibits the proliferation of oligodendrocyte precursors [39].…”

Optimized culture of retinal ganglion cells and amacrine cells from adult mice

“…Optimization of our culture conditions revealed inverse differences in base media on their effects on cell survival versus the percentage of cells with neurite processes in RGCs and CD15 + neurons. The different base media differ by osmolarity ranking in osmotic concentration as N < NA < DN = DW < DNA [59,60]. RGCs and CD15 + cells exposed to lower osmolarity (N) increased in cell viability, contrary to adult hippocampal neuronal cultures.…”