Isolation and Culture of Primary Retinal Müller Cells from Sprague-Dawley (SD) Rats

Summary

This article presents a detailed protocol for isolating primary retinal Müller cells from neonatal Sprague-Dawley (SD) rats. The procedure includes enucleation of the eyeballs, dissection of retinal tissue, extraction and identification of cells, and key considerations for subsequent cell culture.

Abstract

Retinal Müller cells (RMCs) play a crucial role in providing structural support and regulating various functions within the retina. As a core component of the retinal microenvironment, RMCs perform several vital functions. Through their abundant ion channels, ligands, receptors, transmembrane transporters, and enzyme systems, these cells contribute to neurotransmitter and trophic factor secretion, regulate retinal metabolism, and maintain water-ion homeostasis. Notably, RMCs have recently been identified as a significant source of endogenous retinal regenerative stem cells, offering novel therapeutic targets for the treatment of retinal degenerative diseases. Therefore, studying RMCs is essential for understanding the pathological mechanisms underlying retinal disorders. This study systematically establishes a standardized experimental protocol that includes trypsin digestion and purification of primary RMCs, morphological observation using inverted optical microscopy and hematoxylin and eosin (HE) staining, specific protein identification through immunofluorescence staining, and cell purity analysis via flow cytometry. This protocol serves as a valuable reference for both basic research and clinical applications related to RMCs, supporting the exploration of their mechanisms in retinal diseases and advancing the development of therapeutic strategies.

Introduction

Retinal Müller glial cells (RMCs), the predominant macroglia in the retina, form the core of the retinal neurovascular unit^1,2. Spanning nearly the entire retinal thickness, RMCs ensheathe almost all retinal neurons and microvasculature. Their processes extend upward to the inner limiting membrane (ILM), forming apical endfeet, and downward to the outer limiting membrane (OLM), where they develop specialized microvilli³. This unique architecture enables RMCs to function as the primary structural scaffold for retinal organization^4,

Protocol

All animal experiments were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Animal Ethics Committee of Chengdu University of Traditional Chinese Medicine (Ethics Number: 2024069). Fifty specific pathogen-free (SPF) Sprague-Dawley rats (3-5 days old, sex unspecified, body weight 7-10 g) were used in the study. The animals were commercially obtained and housed at the Experimental Animal Center of Chengdu University of Traditional Chinese Medicine (Facility Use License No.: SYXK [Sichuan] 2024-049) under standard laboratory conditions. Details of the reagents and equipment used in this stud....

Results

After primary cells are seeded into the culture flask, subsequent medium changes and passaging help eliminate poorly adherent RMCs and remove cellular debris generated during passaging, thereby enriching the RMC population in the culture. RMCs were identified based on their morphology using an inverted optical microscope (Figure 1) and hematoxylin and eosin (H&E) staining (Figure 2). Immunofluorescence staining was performed .......

Discussion

This article provides a detailed protocol for isolating primary RMCs from neonatal SD rats. The protocol covers all stages, including pre-experimental preparations, eyeball enucleation, retinal tissue isolation, and step-by-step procedures for RMC extraction, culture, passaging, and identification. It also emphasizes key considerations that should be observed throughout the entire cell culture process, offering standardized technical guidance for related research.

For RMC extraction, neonatal .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.1% Triton X-100	Solarbio	T8200
0.25% Trypsin-EDTA	Hyclone	SH30042.01B
1% hydrochloric acid alcohol	BIOSYSTEMS	3803651E
4% Paraformaldehyde	Solarbio	P1110
45μm nylon mesh (300 mesh)	Solarbio	YA0926
6 well plate	Servicebio	IMC202302001
6 cm Glass Petri Dish	Normax	5058541
75% Alcohol	KESHI	GEA004
Aquaporin-4 (AQP4)	Proteintech Group, Inc	16473-1-AP
BSA	Servicebio	GC305010
Cell culture incubator	Thermo	FORMA311
cellular retinaldehyde-binding protein (CRALBP)	HUABOI	HA721330
Centrifuge	Hunan Xiangyi	TDZ5-WS
Clean bench	ZHICHENG	ZHJH-C12
Corneal scissors	Jinzhong	BXGJ
DAPI	Solarbio	G1012
D-Hank solution	Solarbio	H1045
DMEM  high glucose medium	Gibco	C11995500BT
Fetal Bovine Serum (FBS)	Sbjbio life Sciences	BC-SE-FBS01
FIX&PERM Medium A	MultiSciences	GAS006/2
FIX&PERM Medium B	MultiSciences	GAS006/2
flow cytometry staining buffer	MultiSciences	S1001
Fluoromount-G Fluorescent mounting media	SourthernBiotech	0100-01
Glutamine Synthetase Polyclonal Antibody	Proteintech Group, Inc	PTG-11037-2-AP-50ul
Goat 324 Anti-Rabbit IgG H&L PE	Bioss	bs-0295G-PE
Goat Anti- 323 Rabbit IgG H&L FITC	Bioss	bs-0295G-FITC
Hematoxylin-eosin (HE) staining kit	Solarbio	G1120
high pressure disinfection machine	zealway(xiamen) instrument Inc	GI80DS
Inverted fluorescence microscope	Nikon	SMZ1500
Inverted phase contrast microscope	Leica	DMIL
inward rectifier potassium channel 4.1 (Kir4.1)	Proteintech Group, Inc	12503-1-AP
Neutral resin	Solarbio	G8590-100
Penicillin-Streptomycin Solution (100×)	Hyclone	SV30010
Phosphate Buffer saline (PBS) (pH7.2- 7.4)	Solarbio	P1020-500ml
Secondary antibody (FITC-labeled goat anti-rabbit)	Servicebio	GB22303
Sprague-Dawley rats	Chengdu Dashuo Experimental Animal Co., Ltd.	Production License No.: SCXK [Sichuan] 2020-0030
T25 Culture flask	Cornning	430639
Tornado Tubes: 15mL	WHB	WHB-15-1
Tornado Tubes: 50mL	WHB	WHB-50-1
ultra violet disinfection sterilizer	GEMEISI	xd06
Vimentin	Abcam	ab92547