Kim Samhwan, Kim So Yeun, Choi Seong-Kyun, Bae Jisub, Jeon Won Bae, Jang Jae Eun, Moon Cheil
Department of Brain and Cognitive Sciences, Daegu Gyeongbuk Institute of Science and Technology, Daegu 42988, Korea.
Convergence Research Advanced Centre for Olfaction, Daegu Gyeongbuk Institute of Science and Technology, Daegu 42988, Korea.
Exp Neurobiol. 2018 Dec;27(6):574-592. doi: 10.5607/en.2018.27.6.574. Epub 2018 Dec 28.
The olfactory system can detect many odorants with high sensitivity and selectivity based on the expression of nearly a thousand types of olfactory receptors (ORs) in olfactory receptor neurons (ORNs). These ORs have a dynamic odorant detection range and contribute to signal encoding processes in the olfactory bulb (OB). To harness the capabilities of the olfactory system and develop a biomimetic sensor, stable culture and maintenance of ORNs are required. However, monolayer culture models have several key limitations: i) short available period of cultured neurons, ii) low cultural efficiency, and iii) long-term storage challenges. This study aims to develop a technique: i) to support the spheroid culture of primary ORN precursors facilitating stable maintenance and long-term storage, and ii) to demonstrate the viability of ORN spheroid culture in developing an olfactory system mimetic bioelectronic nose. Recombinant protein (REP; TGPG[VGRGD(VGVPG)]WPC) was used to form the ORN spheroids. Spheroid formation enabled preservation of primary cultured ORNs without a significant decrease in viability or the expression of stemness markers for ten days. Physiological characteristics of the ORNs were verified by monitoring intracellular calcium concentration upon odorant mixture stimulation; response upon odorant stimulation were observed at least for ten days in these cultivated ORNs differentiated from spheroids. Coupling ORNs with multi electrode array (MEA) enabled the detection and discrimination of odorants by analyzing the electrical signal patterns generated following odorant stimulation. Taken together, the ORN spheroid culture process is a promising technique for the development of a bioelectronic nose and high-throughput odorant screening device.
嗅觉系统能够基于嗅觉受体神经元（ORN）中近千种嗅觉受体（OR）的表达，以高灵敏度和选择性检测多种气味分子。这些OR具有动态的气味分子检测范围，并有助于嗅球（OB）中的信号编码过程。为了利用嗅觉系统的能力并开发仿生传感器，需要对ORN进行稳定的培养和维持。然而，单层培养模型存在几个关键局限性：i）培养神经元的可用时间短；ii）培养效率低；iii）长期储存存在挑战。本研究旨在开发一种技术：i）支持原代ORN前体细胞的球状体培养，以促进稳定维持和长期储存；ii）证明ORN球状体培养在开发模拟嗅觉系统的生物电子鼻中具有可行性。重组蛋白（REP；TGPG[VGRGD(VGVPG)]WPC）用于形成ORN球状体。球状体的形成能够在十天内保存原代培养的ORN，而其活力或干性标记物的表达没有显著下降。通过监测气味混合物刺激后细胞内钙浓度来验证ORN的生理特征；在这些从球状体分化而来的培养ORN中，至少在十天内观察到了对气味刺激的反应。将ORN与多电极阵列（MEA）耦合，能够通过分析气味刺激后产生的电信号模式来检测和区分气味分子。综上所述，ORN球状体培养过程是开发生物电子鼻和高通量气味分子筛选装置的一种有前途的技术。
