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

Melanie Georgiou,Patricia Perez Esteban,David Choi,Ying Li,Rachael Wood,Victoria Roberton,Daqing Li,Chris Mason,Ivan Wall,Joana Neves Dos Reis

doi:10.1038/s41598-018-32748-w

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.

Highlights

Regeneration within the central nervous system (CNS) generally does not occur naturally
Studies have investigated the effect of serum concentration, or the addition of neurotrophic factors amongst other variables on the isolation and culture of mucosal Olfactory ensheathing cells (OECs), but a full characterization of these cells is yet to be established in the literature
We investigated the effect of different bioprocess conditions, namely cell culture substrate, serum concentration, oxygen tension, enrichment with neurotrophic factor-3 (NT-3), and differential adhesion

Summary

Introduction

Regeneration within the central nervous system (CNS) generally does not occur naturally. Olfactory ensheathing cells (OECs), the glial cells of the olfactory system, play a key role to support the regeneration and guidance of olfactory receptor neurons from the peripheral nervous system into the central nervous system by creating a permissive environment for neurite outgrowth[1,2,3,4] Due to this unique ability, OECs have been investigated extensively over the years for use in a potential spinal cord repair cell therapy, where spontaneous regeneration does not occur after injury and so surgical intervention is required[5,6,7]. Studies have investigated the effect of serum concentration, or the addition of neurotrophic factors amongst other variables on the isolation and culture of mucosal OECs, but a full characterization of these cells is yet to be established in the literature. We chose the most beneficial process conditions amongst the ones considered in this study, by which we could isolate pure OECs, and assessed their ability to support and promote neuronal growth in 2D neuron co-culture in vitro

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Conclusion