Purification and cultivation of mouse primary retinal microvascular pericytes based on pre-incubation

Authors: Liu Guanghui,  Lin Cuihong,  Yang Tianye,  Xu Chaoyang,  Zheng Yongzheng,  Zhao Li,  Meng Chun,  Pan Mingdong

DOI: 10.3760/cma.j.issn.2095-0160.2019.10.002
Published 2019-10-10
Cite as Chin J Exp Ophthalmol, 2019,37(10): 774-778.

Abstract

Objective

To establish a simple method for isolation, purification and cultivation of primary retinal microvascular pericytes (RMPs) from mice.

Methods

Retinas were isolated from mice following with mechanical morcel, enzymatic digestion and filtration.The retinal fragments were incubated with low glucose DMEM with 20% fetal bovine serum after 24 hours pre-incubation.Differential digestion was used for purification of primary RMPs.Morphological examination of cells was performed by phase contrast microscopy, and further characterization was analyzed by immunocytochemistry.Functional assay was evaluated by the pericytes-endothelial cells (ECs) co-culture system.The treatment and use of experimental animals followed the regulations on the administration of experimental animals promulgated by the state science and technology commission.

Results

Cells migrated out of fragments after 24 hours of incubation, and developed into small or large colonies gradually.The cells and their subpassages presented typical pericyte morphology with large irregular triangular cell bodies and multiple long processes.No contact inhibition was observed.Most cells uniformly expressed the cellular markers α-smooth muscle actin (α-SMA) and platelet-derived growth factor receptor-β (PDGFR-β), a few cells expressed the cellular markers glial fibrillary acidic protein (GFAP), but no cell expressed von Willebrand factor (vWF). The purity rate of RMPs was up to 97%.In the co-culture system, RMPs directly contacted with ECs to form the capillary-like cords in vitro.

Conclusions

A simple method for the isolation, purification cultivation of mouse RMPs is established, and active RMPs can be readily obtained by this method.

Key words:

Retinal microvascular pericyte; Pre-incubation; Isolation; Purification; Cultivation

Contributor Information

Liu Guanghui
Affiliated People’s Hospital, Fujian University of Traditional Chinese Medicine, Fuzhou 350004, China
Lin Cuihong
College of Biological Science and Biotechnology, Fuzhou University, Fuzhou 350108, China
Yang Tianye
College of Biological Science and Biotechnology, Fuzhou University, Fuzhou 350108, China
Xu Chaoyang
Affiliated People’s Hospital, Fujian University of Traditional Chinese Medicine, Fuzhou 350004, China
Zheng Yongzheng
Affiliated People’s Hospital, Fujian University of Traditional Chinese Medicine, Fuzhou 350004, China
Zhao Li
Affiliated People’s Hospital, Fujian University of Traditional Chinese Medicine, Fuzhou 350004, China
Meng Chun
College of Biological Science and Biotechnology, Fuzhou University, Fuzhou 350108, China
Pan Mingdong
Affiliated People’s Hospital, Fujian University of Traditional Chinese Medicine, Fuzhou 350004, China
(Read 31 times, 1 visits today)
Updated: November 20, 2019 — 8:48 am