Bioprocessing strategies to enhance the challenging isolation of neuro-regenerative cells from olfactory mucosa

Abstract

Olfactory ensheathing cells (OECs) are a promising potential cell therapy to aid regeneration. However, there are significant challenges in isolating and characterizing them. In the current study, we have explored methods to enhance the recovery of cells expressing OEC marker p75NTR from rat mucosa. With the addition of a 24-hour differential adhesion step, the expression of p75NTR was significantly increased to 73 ± 5% and 46 ± 18% on PDL and laminin matrices respectively. Additionally, the introduction of neurotrophic factor NT-3 and the decrease in serum concentration to 2% FBS resulted in enrichment of OECs, with p75NTR at nearly 100% (100 ± 0% and 98 ± 2% on PDL and laminin respectively), and candidate fibroblast marker Thy1.1 decreased to zero. Culturing OECs at physiologically relevant oxygen tension (2–8%) had a negative impact on p75NTR expression and overall cell survival. Regarding cell potency, co-culture of OECs with NG108-15 neurons resulted in more neuronal growth and potential migration at atmospheric oxygen. Moreover, OECs behaved similarly to a Schwann cell line positive control. In conclusion, this work identified key bioprocessing fundamentals that will underpin future development of OEC-based cell therapies for potential use in spinal cord injury repair. However, there is still much work to do to create optimized isolation methods.

Impact of Dual Cell Co-culture and Cell-conditioned Media on Yield and Function of a Human Olfactory Cell Line for Regenerative Medicine

“…Another difference between the rat and human OECs is that the human OECs do not appear to benefit from the presence of NT-3. Although the study was not carried out as part of this work, previous studies have shown that NT-3 significantly increased the expression of glial markers in rat cells [27]. For our human cell line, the presence of NT-3 seemed to increase the level of expression slightly, but not significantly.…”

“…Fibroblasts have a faster adhesion time than OECs. After 24 h, most fibroblasts will adhere to the tissue culture plastic, whereas OECs will still be in suspension [ 27 ]. After 24 h, the suspension was replated onto laminin-coated (20 μg/mL, Sigma-Aldrich, UK) wells.…”

“…Mucosae were isolated following the optimum protocol previously identified [27]. The mucosae were dissected from three adult female Sprague-Dawley 200-250g rats, which were euthanized by carbon dioxide asphyxiation (Schedule 1 method [28]) according to the U.K.…”

“…After five days, cells were fixed and stained for S100β and fibronectin. Neurotrophic factor 3 (NT-3) was added to some media conditions at 50 ng/mL as the literature indicated that NT-3 encourages the neurological repair phenotype in OECs [27].…”

“…According to the literature, five days of co-culture are optimal for observing enhanced neurite outgrowth compared with three days of co-culture [27]. After five days of co-culture, cells were labelled with antibodies against βIII-tubulin.…”