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©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved.
Culture and identification of neonatal rat brain-derived neural stem cells
Qing-Zhong Zhou, Xiao-Lan Feng, Xu-Feng Jia, Nurul Huda Binti Mohd Nor, Mohd Hezery Bin Harun, Da-Xiong Feng, Wan Aliaa Wan Sulaiman
Qing-Zhong Zhou, Da-Xiong Feng, Department of Orthopedics, The Affiliated Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China
Qing-Zhong Zhou, Wan Aliaa Wan Sulaiman, Department of Neurology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia
Xiao-Lan Feng, Department of Radiology, The Affiliated Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China
Xu-Feng Jia, Department of Orthopedics, The Peoples’ Hospital of Jianyang City, Jianyang 641400, Sichuan Province, China
Nurul Huda Binti Mohd Nor, Department of Human Anatomi, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang Selangor, 43400, Malaysia
Mohd Hezery Bin Harun, Department of Orthopedics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia
Author contributions: Zhou QZ designed the study; all authors contributed to the data collect and manuscript writing; all authors have read and approve the final manuscript.
Supported by Project of Sichuan Department of Science and Technology, No. 2016PJ552; the Project of Luzhou Department of Science and Technology, No. 2016-R-70(18/24); the Project of Southwest Medical University of Science and Technology, No.15073 and 2015-YJ021; and Orthopaedic diseases (Shang Antong) special research Project of Sichuan Medical Association, No. 20220206070192.
Institutional animal care and use committee statement: This study was approved by the Institutional Animal Care and Use Committee of Southwest Medical University.
Conflict-of-interest statement: The authors declare no conflict of interest.
Data sharing statement: No additional data are available.
ARRIVE guidelines statement: The authors have read the ARRIVE Guidelines, and the manuscript was prepared and revised according to the ARRIVE Guidelines.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
https://creativecommons.org/Licenses/by-nc/4.0/ Corresponding author: Wan Aliaa Wan Sulaiman, BMedSci, MBChB BCh BAO, MRCP(UK), FRCP (Edin), Associate Professor, Department of Neurology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia.
wanaliaa@upm.edu.my
Received: March 21, 2023
Peer-review started: March 21, 2023
First decision: April 9, 2023
Revised: April 20, 2023
Accepted: April 27, 2023
Article in press: April 27, 2023
Published online: June 26, 2023
Processing time: 97 Days and 3.9 Hours
BACKGROUND
Timing of passaging, passage number, passaging approaches and methods for cell identification are critical factors influencing the quality of neural stem cells (NSCs) culture. How to effectively culture and identify NSCs is a continuous interest in NSCs study while these factors are comprehensively considered.
AIM
To establish a simplified and efficient method for culture and identification of neonatal rat brain-derived NSCs.
METHODS
First, curved tip operating scissors were used to dissect brain tissues from new born rats (2 to 3 d) and the brain tissues were cut into approximately 1 mm3 sections. Filter the single cell suspension through a nylon mesh (200-mesh) and culture the sections in suspensions. Passaging was conducted with TrypLTM Express combined with mechanical tapping and pipetting techniques. Second, identify the 5th generation of passaged NSCs as well as the revived NSCs from cryopreservation. BrdU incorporation method was used to detect self-renew and proliferation capabilities of cells. Different NSCs specific antibodies (anti-nestin, NF200, NSE and GFAP antibodies) were used to identify NSCs specific surface markers and muti-differentiation capabilities by immunofluorescence staining.
RESULTS
Brain derived cells from newborn rats (2 to 3 d) proliferate and aggregate into spherical-shaped clusters with sustained continuous and stable passaging. When BrdU was incorporated into the 5th generation of passaged cells, positive BrdU cells and nestin cells were observed by immunofluorescence staining. After induction of dissociation using 5% fetal bovine serum, positive NF200, NSE and GFAP cells were observed by immunofluorescence staining.
CONCLUSION
This is a simplified and efficient method for neonatal rat brain-derived neural stem cell culture and identification.
Core Tip: How to harvest sufficient neural stem cells (NSCs) is a basic requirement for the study and clinical application of NSCs. This study describes a simplified and efficient method for neonatal rat brain-derived NSC culture and identification comprehensively considering the influencing factors including timing of passaging, passage number, passaging approaches and methods for cell identification. It demonstrates that combination of TrypLTM Express and mechanical tapping and pipetting techniques makes a more efficient way of passaging. The optimal timing for NSC passage is on the fourth to fifth day of primary or passage NSC culture.
