Conditional immortalization of olfactory ensheathing cells for intelligently regulating their proliferation state

Abstract

Olfactory ensheathing cells (OECs) are among the most promising cell types for the treatment of spinal cord injury (SCI) and other neural traumas. However, primary OECs cultured in vitro are constrained by their inherent “Hayflick limit,” resulting in poor proliferative and passaging capacities, which restricts their large-scale application in both basic and clinical research. Conditional immortalization technology provides an excellent solution for rapid, large-scale, and controllable proliferation of primary OECs. However, few studies have applied this approach to the controlled passaging of OECs. Here, we used the tetracycline-controlled system (Tet-On system) to introduce the HBLV-TetOn-SV40Tag-PURO constructed into the primary OECs via lentiviral transfection, establishing a conditionally immortalized OEC line. We comprehensively investigated its functional characteristics with or without tetracycline conditions by using immunofluorescence, qPCR, CCK-8, Scratch assay, and Transwell migration experiment. Our results demonstrated that this cell line could rapidly proliferate under tetracycline and quickly recover to proliferation and migration levels similar to primary OECs after tetracycline removal following multiple passages. This approach ensures obtaining a suitable quantity of OECs with specific functional characteristics while avoiding the malignant proliferation risks associated with traditional cell line establishment, thus offering higher safety. Moreover, the method is simple to operate, cost-effective, possessing excellent potential for basic and clinical translational applications.