Journal of Shanghai Jiao Tong University (Medical Science) >

2025 , Vol. 45 >Issue 11: 1515 - 1526

DOI: https://doi.org/10.3969/j.issn.1674-8115.2025.11.011

Techniques and methods

An efficient isolation method for pericytes of the cochlear stria vascularis

  • ZHU Yue ,
  • LI Xiaoqin ,
  • WANG Wenxiao ,
  • ZHOU Lingyun ,
  • WU Hao ,
  • TAO Yong ,
  • DU Tingting
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  • 1.Department of Otolaryngology, Head and Neck Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China
    2.Ear Institute, Shanghai Jiao Tong University School of Medicine, Shanghai 200125, China
    3.Shanghai Key Laboratory of Translation Medicine on Ear and Nose Disease, Shanghai 200125, China
TAO Yong, E-mail: taoyent@sjtu.edu.cn
DU Tingting, E-mail: tingtingdu@shsmu.edu.cn. #Co-correspongding authors.

Received date: 2025-02-13

  Accepted date: 2025-04-25

  Online published: 2025-12-03

Supported by

Young Scientists Fund of National Natural Science Foundation of China(82000975);Shanghai Key Laboratory of Translational Medicine on Ear and Nose Diseases(14DZ2260300)

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Abstract

Objective ·To develop a stable and efficient method for isolating and obtaining pericytes from the cochlear stria vascularis while preserving their original physicochemical properties as much as possible. Methods ·Stria vascularis tissues were dissected from 6-day-old wild-type C57BL/6J mice under a microscope. Experimental conditions such as enzyme type, digestion time, and mechanical dissociation duration were optimized by evaluating single-cell yield and viability via flow cytometry (FCM). The single-cell samples were incubated with CD140b (i.e., platelet-derived growth factor receptor-β, PDGFR-β) and CD31 antibodies, and CD140b+CD31- and CD140b- cells were obtained by fluorescence-activated cell sorting (FACS). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed to assess the expression of pericyte markers [Pdgfrb and desmin (Desm)], endothelial cell markers [von Willebrand factor (Vwf)and glucose transporter 1 (Glut1)], and melanin-like macrophage markers [glutathione S-transferase α4 (Gsta4) and adhesion G protein-coupled receptor E1 (Adgre1)] to validate cell identity and the effectiveness of the isolation method. Results ·Flow cytometric analysis revealed that papain digestion of stria vascularis tissue caused significant cell damage and a high debris rate, cold-active protease showed low dissociation efficiency, and thermolysin combined with Accutase yielded unstable digestion efficiency and a relatively high debris rate. In contrast, Accutase alone consistently produced single-cell suspensions with relatively high yield and viability, and was therefore selected as the optimal enzyme. High-purity pericytes were successfully obtained by digesting stria vascularis tissue from 6-day-old mice with Accutase followed by sorting with CD140b and CD31 antibodies. RT-qPCR showed that CD140b+CD31- cells specifically exhibited high expression levels of pericyte markers Pdgfrb and Desm, and low expression levels of endothelial cell markers Vwf and Glut1, as well as melanin-like macrophage markers Gsta4 and Adgre1. Conclusion ·A stable and effective flow cytometry-based method for isolating pericytes from the stria vascularis is successfully established.

Key words： cochlea; stria vascularis; pericyte; cell isolation; flow cytometry

Cite this article

ZHU Yue , LI Xiaoqin , WANG Wenxiao , ZHOU Lingyun , WU Hao , TAO Yong , DU Tingting . An efficient isolation method for pericytes of the cochlear stria vascularis[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2025 , 45(11) : 1515 -1526 . DOI: 10.3969/j.issn.1674-8115.2025.11.011

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