Cell-based platform for studying trigeminal satellite glial cells under normal and inflammatory conditions.

Aims Satellite glial cells (SGCs) in sensory ganglia contribute to the pathogenesis of chronic pain. In vitro, providing enough fresh primary SGCs poses some practical limitations; hence, frozen stocks of primary cells for culture could be an attractive alternative for cell-based studies or drug screening. This study was designed to investigate the morphology and marker expression of frozen and freshly isolated trigeminal SGCs under normal and inflammatory conditions. Methods SGCs from trigeminal ganglia of three male Sprague-Dawley rats and three frozen (sub cultured and passaged) batches of stored primary SGCs were cultured. Their morphology was observed by phase microscopy and the phenotype was characterized by immunocytochemistry of glutamine synthetase (GS) and glial fibrillary acidic protein (GFAP). Lipopolysaccharide (LPS) was used to simulate a state of neurogenic inflammation in vivo. A pilot test was performed to determine the optimal concentration of LPS to activate SGCs based on GFAP expression. A long-term activation of the SGCs with 50 ng/mL LPS was chosen for further characterization. Results The fresh and frozen primary SGCs elicited similar phenotypes based on GS marker expression. However, frozen primary SGCs differed in terms of size and morphology. GFAP was constantly expressed in frozen primary SGCs regardless of LPS stimulation. Activation of primary fresh SGCs with LPS spread the GFAP expression from around the cell body throughout the longer processes and activation was only seen in the LPS treatment. Conclusions The phenotypic marker, GS was independent of culture conditions. There was no difference in upregulation of GFAP in thawed SGCs regardless of LPS stimulation. This indicates that freeze-thawing might activate SGCs and therefore frozen and passaged cells cannot be suitable for use in cell-based models for inflammation. Fresh primary cells are therefore optimal for studying SGCs under normal and inflammatory conditions.