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In vitro Maintenance of Olfactory Mucosa: with Enriched Olfactory Ensheathing Cells

Neetu Singh, Saroj C Gopal, Rajeshwar N Srivastava, Tulika Chandra, Satya P Agarwal, Sanjay K Singh, Devendra K Gupta and Anil K Balapure

Human Olfactory Mucosa (OM) regulates olfaction through axonal regeneration and myelination mediated by stem cells and Olfactory Ensheathing Cells (OECs) resident in the niche. Purified OECs/olfactory biopsies have been utilized for functional recovery in different Spinal Cord Injury (SCI) models. However, recent reports find this debatable where we propose primary culture of OM, basal cells of olfactory epithelium and olfactory ecto-mesenchymal stem cells. Our defined culture conditions improve the life span of OM with enrichment of OECs providing a strategy for employment for SCI/cochlear damage repair. Briefly, OM post-collection, was non-enzymatically sliced, cultured for 6 weeks and cells characterized morphologically, immuno-cytochemically and western blotting. By day 21, ~70% GFAP and p75NTR stained, spindle shaped astrocyte-like and flattened sheet-like OECs displayed axonal remyelination. By day 30, caspase 3, 8, 9 (gene-product and activity), phospho-p53 negative; GFAP and p75NTR positive dense, overlapping mass of cells was found. This was accompanied with degenerative changes by 6 weeks through GFAP staining. Conversely, trypsination on day 21 resulted in >95% OECs with flattened morphology, GFAP and p75NTR positivity. The human derived OECs were compared with the 2-day SD rat Olfactory Bulb Cells cultured for 2 weeks in F12 media (GFAP and p75NTR positive). Hence, cultured olfactory mucosa displaying axonal regeneration with OECs in culture provides a vehicle for SCI/cochlear damage repair studies.