Culturing Olfactory Ensheathing Glia from the Mouse Olfactory Epithelium

The majority of studies centered on understanding the in vitro properties of olfactory ensheathing glia (OEG) have utilized OEG prepared from the nerve fiber layer of the embryonic or neonatal olfactory bulb ( 1 – 3 ), summarized in chapter 4. In fact, a significant fraction of the OEG population is found within the lamina propria of the olfactory epithelium as they ensheath olfactory receptor axons en route to the olfactory bulb Fig. 1 ). Generating olfactory ensheathing glia from the lamina propria presents a unique set of obstaclesfreeing the lamina propria tissue from the neuronal epithelium, the cartilaginous turbinates, and the extracellular matrix (ECM) in which the cells are embedded, in a way that will avoid fibroblast contamination. Thus, the method used to culture epithelium-derived OEGs is quite different from the method for bulb-derived glia, and encompasses steps to minimize cartilage contamination (careful dissection), digest away ECM (using a defined enzyme treatment), and remove fibroblasts, which certainly will find their way into the culture (cytotoxic lysis of contaminating fibroblasts). This method generates a culture of proliferating olfactory ensheathing glia that express, thus far, the same antigenic markers as bulb-derived olfactory ensheathing cells (OECs) ( 4 ). The procedure outlined in this chapter has been shown to eliminate 99% of nonglial cells from the glial culture by passage 3, and to produce a glial culture that is 98-100% positive for the glial markers (glial fibrillary acidic protein) (GFAP), S100Beta and p75, at 4 wk in vitro.

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