Primary cell culture of meningothelial cells--a new model to study the arachnoid in glaucomatous optic neuropathy.

BACKGROUND

In a previous report, we found that the occurrence and amount of meningothelial cell nests in the subarachnoid space are significantly increased in glaucomatous optic nerves compared to normals. In order to allow research into the role of meningothelial cells during diseases of the optic nerve, an in vitro model is necessary. For this purpose, we developed a culture method for porcine meningothelial cells from the arachnoid layer covering the optic nerve.

METHODS

Meningothelial cells were scraped from the arachnoid layer of porcine optic nerves and cultured for 2-3 weeks until the cells formed a monolayer. To eliminate contaminating fibroblasts from the culture, cells were negatively selected using magnetic anti-fibroblast beads after the first passage. Cells were detached using 0.05% Trypsin-EDTA, incubated with anti-fibroblast beads, separated using a magnetic column and the flow-through was collected. The purified primary meningothelial cells were characterized by electron microscopy and immunocytochemistry using anti-glial fibrillary acidic protein (GFAP) and anti-keratan sulfate antibodies.

RESULTS

Primary cells grew out after dissection and formed a monolayer within 2-3 weeks, which was composed of two morphologically different cell types, flattened cells with round nuclei and fibroblast-like cells with long processes. The fibroblast-like cells in the culture could be labelled and selected using anti-fibroblast microbeads. The second cell type did not bind to the anti-fibroblast beads, and upon immunocytochemistry showed a marked expression of both GFAP and keratan sulphate. In addition, examination of these cells by electron microscopy revealed morphological characteristics of meningothelial cells, including hemidesmosomes and cytoplasmatic filaments.

CONCLUSIONS

The technique described in this paper for the primary culture of meningothelial cells from the subarachnoid space of the optic nerve and using magnetic beads for the removal of fibroblasts is effective in obtaining a highly enriched meningothelial cell culture.