Zhang Chengyi, Hao Wanzhu, Yang Yubing, Li Jiaxi, Zhang Qiongchi, Chang Su'e, Yang Kun, He Xijing
Department of Orthopaedics, the Second Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an, China.
Department of Neurorehabilitation, Xi'an International Medical Center Hospital, Xi'an, China.
Sci Rep. 2025 Aug 19;15(1):30398. doi: 10.1038/s41598-025-15144-z.
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.
嗅鞘细胞（OECs）是治疗脊髓损伤（SCI）和其他神经创伤最有前景的细胞类型之一。然而，体外培养的原代嗅鞘细胞受其固有的“海弗利克极限”限制，增殖和传代能力较差，这限制了它们在基础研究和临床研究中的大规模应用。条件永生化技术为原代嗅鞘细胞的快速、大规模和可控增殖提供了一个很好的解决方案。然而，很少有研究将这种方法应用于嗅鞘细胞的可控传代。在这里，我们使用四环素调控系统（Tet-On系统），通过慢病毒转染将构建好的HBLV-TetOn-SV40Tag-PURO导入原代嗅鞘细胞，建立了一种条件永生化嗅鞘细胞系。我们通过免疫荧光、qPCR、CCK-8、划痕试验和Transwell迁移实验，全面研究了其在有无四环素条件下的功能特性。我们的结果表明，该细胞系在四环素存在下能快速增殖，在多次传代后去除四环素后能迅速恢复到与原代嗅鞘细胞相似的增殖和迁移水平。这种方法确保获得适量具有特定功能特性的嗅鞘细胞，同时避免了与传统细胞系建立相关的恶性增殖风险，从而提供了更高的安全性。此外，该方法操作简单、成本效益高，在基础研究和临床转化应用方面具有极好的潜力。
