Purity determining of cultured OECs from olfactory mucosa of rats\' pups

Abstract

Introduction: Cell transplantation is one of the main strategies for spinal cord injury repair. As OECs of the olfactory mucosa can be obtained by simple biopsy in all individuals without affecting their smell sensation, OECs considered as a promising candidate for autologous transplantation in the nervous system injury, especially for spinal cord repair. Thereby in the current study OECs were cultured from olfactory mucosa of 7 days old rats' pups and their purity was examined by flow-cytometry after simultaneous double staining for p75 and GFAP markers. Materials and methods: 7 days old Wistar rats' pups were deeply anesthetized by ketamine / xylazine (60/6mg/Kg). Then the nasal cavity was opened sagittally and the olfactory mucosa was separated from posterior part of nasal septum and at last OECs were obtained from lamina properia of olfactory mucosa and were cultured. The cultured cells were simultaneously immunolabeled for p75 and GFAP markers and finally purity of cultured cells assessed by flow-cytometry. Results: cultured OECs demonstrated two different morphologies, a spindle shape Schwannlike and an astrocyte-like OECs with flat sheet-like morphology. In addition, simultaneous immunolabeling for p75 and GFAP markers of OECs exhibited OECs were positive for both markers at the same time. The flow-cytometry results displayed that 87.9±2.4% of cells were p75/ GFAP double positive cells,1.05±0.4 only p75 positive and 5.8±1.5% were single positive for GFAP. Conclusion: Purity of cultured OECs in our study is probably more than 87.9% by flowowing to p75+/S100+ and GFAP+/S100+ olfactory ensheathing cells were not counted. Thus the culture procedure of this study seems to be a good protocol for OECs purifying and cell therapy in CNS damages.

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“…The cells begin to stick to the bottom of the flask with two different appearances: spindle-shaped Schwann-like cells and astrocyte-like cells with a flat appearance with multiple redundancies. After 10–12 days of culture, when the bottom of flask was completely filled with proliferated cells, fourth Cell Passage was performed 41. The cultured cells were exposed to NG (glucose 5.5 mM), HG (glucose 30 mM), or mannitol (30 mM) at 24, 48 and 72 hrs and 48 hrs was selected.…”