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Method Article

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

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DOI:

10.3791/68129

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June 17th, 2025

 ,  ,  ,  ,  , 
1Eye School of Chengdu University of TCM, Chengdu University of Traditional Chinese Medicine, 2Key Laboratory of Sichuan Province Ophthalmopathy Prevention & Cure and Visual Function Protection with TCM Laboratory, Eye School of Chengdu University of TCM, Chengdu University of Traditional Chinese Medicine, 3Retinal Image Technology and Chronic Vascular Disease Prevention & Control and Collaborative Innovation Center, Eye School of Chengdu University of TCM, Chengdu University of Traditional Chinese Medicine, 4Department of Ophthalmology, Ziyang Hospital of Traditional Chinese Medicine, 5Department of Ophthalmology, Ineye Hospital of Chengdu University of TCM, Chengdu University of Traditional Chinese Medicine

In This Article

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 unit1,2. 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 organization4,5.

RMCs are enriched with ion channels, ligands, receptors, transmembrane transporters, and enzymes6, and they participate in retinal glucose metabolism via glycolysis7. Potassium ion channels (Kir2.1, Kir4.1) and water channel proteins (AQP4, AQP9, AQP11) collaboratively regulate ion and water homeostasis across the cell membrane, maintaining retinal physiological balance7. Additionally, RMCs absorb and clear neurotransmitters released by neurons, including glutamate, γ-aminobutyric acid (GABA), and glycine7,8.

Pathological conditions such as diabetic retinopathy7, glaucoma9, and retinitis pigmentosa10 can damage RMCs, disrupt the blood-retinal barrier, trigger inflammation, increase vascular permeability, and impair retinal function. RMCs are also recognized as latent intrinsic sources of retinal regenerative cells11,12. In fish and certain amphibians, RMCs can dedifferentiate into retinal progenitor cells that replace neurons lost to injury13. In adult mammalian retinas, RMCs express stem cell-related marker proteins14,15, granting them the potential to differentiate into retinal neurons and replace damaged cells in degenerative diseases such as age-related macular degeneration, glaucoma, and diabetic retinopathy16. Primary RMCs closely mimic their in vivo state and are of great value in studying and treating retinal degenerative diseases. However, the literature contains few established methods for isolating and culturing primary RMCs, particularly from neonatal Sprague-Dawley (SD) rats.

This protocol utilizes 3-5-day-old neonatal SD rats, without gender preference. Under sterile conditions, the eyeballs are enucleated, and retinal tissue is digested using trypsin. Primary RMCs are then isolated via centrifugation and purification for subsequent culture and passaging. Morphological analysis using inverted optical microscopy combined with hematoxylin and eosin (H&E) staining revealed that the isolated cells exhibited characteristic RMC features. Immunofluorescence staining confirmed the expression of RMC-specific protein markers, including glutamine synthetase (GS), cellular retinaldehyde-binding protein (CRALBP), Vimentin, aquaporin-4 (AQP4), and inward rectifier potassium channel 4.1 (Kir4.1). Flow cytometric analysis after labeling with GS and CRALBP antibodies demonstrated a cell purity of ≥90%, which meets the established criteria for RMC-based biological experiments and ensures suitability for subsequent functional studies. During the experimental process, cells at the fourth passage showed reduced adherence to the culture flask, morphological changes, and signs of senescence, ultimately leading to cell death. Therefore, only cells from the first three passages were used in subsequent experiments.

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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 study are provided in the Table of Materials.

1. Isolation and culture of primary RMCs

  1. Preparation of experimental items
    1. Sterilize the following items under ultraviolet light for at least 30 min on the clean bench before use: T25 culture flasks, centrifuge tubes and racks, pipettes and stands, containers for liquid waste, cigarette lighters, spirit alcohol lamps, and markers.
    2. Sterilize the following items by autoclaving before use: 1 mL pipette tips, bending plate, glass Petri dishes with lids, curved tweezers, 45 µm nylon mesh (300 mesh), toothed tweezers, toothless tweezers, and micro corneal scissors.
  2. Environmental preparation: Disinfect the environment and operating table for eyeball dissection with ultraviolet light for more than 30 min. Spray alcohol on the experimenter's hands every time before entering and after leaving the clean bench. Set the incubator to 37 °C and 5% CO2. Spray the experimenter's hands with alcohol before and after opening the incubator.
  3. Solution preparation: Prepare D-Hank's solution, phosphate-buffered saline (PBS), Dulbecco's Modified Eagle Medium (DMEM high-glucose), fetal bovine serum (FBS, heat-inactivated), penicillin/streptomycin solution (100x), and 0.25% trypsin-EDTA solution.
    NOTE: Warm DMEM and FBS in a 37 °C water bath before use.
    1. Prepare D-Hank's and PBS solutions containing 1% penicillin/streptomycin by adding 100 µL of penicillin/streptomycin solution to 10 mL of D-Hank's or PBS solution, respectively.
    2. Prepare the complete culture medium by adding 100 µL of penicillin/streptomycin solution and 2 mL of FBS to 10 mL of high-glucose DMEM.
      NOTE: Prepare solutions on a laboratory clean bench and use them immediately when possible. Store the complete culture medium at 4 °C and warm to 37 °C before each use. Discard if stored for more than one week.
  4. Eye removal
    1. Euthanize neonatal SD rats by cervical dislocation (without CO2 asphyxiation) on a sterilized workbench following institutionally approved protocols. Immerse the bodies in 75% alcohol for 5 min for disinfection, then transfer to a sterile curved dish.
    2. Pour D-Hank's solution into two 10 cm glass culture dishes.
    3. Use toothed tweezers to tear open the eyelid along the palpebral fissure to expose the eyeball.
    4. Use toothless tweezers held open and parallel to the palpebral fissure to press down the orbital. Once the optic nerve is reached and the eyeball is exposed, close the tweezers to lift and extract the eyeball.
    5. Place the eyeball in a glass culture dish with D-Hank's solution, rinse it, and transfer it to another dish with fresh D-Hank's solution.
      NOTE: Ensure the eyeball remains intact. Ideally, a portion of the optic nerve should remain attached to facilitate retinal tissue separation.
  5. Retinal dissection
    1. Prepare a sterilized, lidded empty culture dish and adjust the microscope.
    2. Place the glass culture dish from step 1.4.5 under the microscope. Use curved ophthalmic micro forceps to gently fix the region between the cornea and optic nerve to expose the cornea.
    3. Pierce the corneoscleral junction using micro corneal scissors. Cut along the limbus in a circular fashion, then make symmetrical scleral incisions approximately 2 mm in length. Release the forceps and re-clamp at the junction of the optic nerve and sclera.
    4. Use a second forceps to gently press near the optic nerve root, directing pressure toward the corneal-optic nerve interface. When the lens tissue appears, remove it carefully, and continue pressing until the retinal tissue emerges.
      NOTE: Remove the lens carefully to avoid inadvertently extracting retinal tissue.
    5. Transfer the separated retinal tissue to another sterile culture dish using forceps. Cover the dish, spray it with alcohol, and place it on the clean bench.
  6. Extraction of primary RMCs
    1. Turn on the clean bench ventilation switch.
    2. Open the culture dish lid. Use a pipette with a 1 mL tip to pipette the retinal tissue up and down about 15 times to break it into small pieces. Add 1 mL of 0.25% trypsin and incubate at 37 °C for 5 min.
    3. Remove the culture dish from the incubator and place it on the clean bench. Add 2 mL of complete medium and pipette gently to stop the digestion.
      NOTE: Use twice the volume of trypsin to terminate digestion. For example, if 1 mL of trypsin was used, terminate with 2 mL of complete medium.
    4. Filter the cell suspension through a 300-mesh nylon screen into a 15 mL centrifuge tube. Wash the dish with prepared PBS and collect the remaining suspension.
    5. Centrifuge the tube at 878 x g for 5 min at room temperature (RT). Aspirate and discard the supernatant. Resuspend the pellet in 2 mL of complete medium and centrifuge again at 878 x g for 5 min to purify the cells.
    6. After centrifugation, discard the supernatant. Resuspend the cells in 2 mL of complete medium. Add 3 mL of complete medium to each T25 flask beforehand, then add 1 mL of the cell suspension. Shake the flask in a cross pattern and place it in the incubator.
    7. Perform the first medium change after 48 h of incubation. Remove the flask from the incubator and place it on the clean bench. Discard the spent medium and wash the cell-adhering surface three times with 1 mL of PBS.
      1. Add 5 mL of fresh complete medium. Continue changing the medium every other day until cell confluency exceeds 90%, then proceed with subculturing.

2. Passaging of RMCs

NOTE: Passage the cells when confluence exceeds 90%. In this protocol, primary cultures typically require passaging at 5-6 days post-isolation. The timing of the first passage may vary depending on the plating density. Adjust the cell density to 4 x 103 cells/mL and seed the cells into T25 culture flasks. When confluence reaches >90% after 3-4 days, proceed with passaging.

  1. Remove the T25 culture flask from the incubator. Discard the culture medium and wash the cells three times with 1 mL of PBS solution containing 1% penicillin/streptomycin.
  2. Add 1 mL of 0.25% trypsin-EDTA solution to the flask and incubate for 1 min and 30 s.
  3. Observe the flask under an inverted microscope. When the cells appear round, detached, and begin to float, remove the flask from the incubator. Transfer it to the clean bench and add 2 mL of complete culture medium to terminate digestion.
  4. Use a pipette to aspirate the cell suspension. Gently pipette against the flask wall to dislodge any remaining adherent cells.
  5. Transfer the entire cell suspension to a 15 mL centrifuge tube. Rinse the flask wall with 2 mL of PBS containing 1% penicillin/streptomycin and add it to the same tube. Centrifuge the tube at 878 x g for 5 min at RT.
  6. Discard the supernatant. Resuspend the cell pellet in an appropriate volume of complete medium. Passage the cells at a ratio of 1:2 or 1:3 as required.
    NOTE: Use cells at passage 2 (P2) for downstream experiments (Figure 1).

3. Hematoxylin and Eosin (H&E) staining

  1. Seed passage 2 (P2) cells in a 6-well plate containing pre-placed sterile coverslips at a density of 2 x 105 cells per well. Add 2 mL of complete culture medium to each well. When the cells reach approximately 80% confluence, discard the culture medium and gently rinse each well twice with 1 mL of PBS.
  2. Add 1 mL of 4% paraformaldehyde fixative to each well and incubate for 30 min to fix the cells. Discard the fixative and wash the cells twice with 1 mL of PBS.
  3. Add 1 mL of hematoxylin stain to each well and incubate for 15 min. Discard the staining solution and rinse the wells 2-3 times with 1 mL of PBS.
  4. Observe the cells under a microscope. If the hematoxylin stain appears too dark, use 1% hydrochloric acid alcohol to differentiate and remove excess cytoplasmic stain. Wash 2-3 times with 1 mL of PBS.
  5. Add 1 mL of eosin staining solution to each well. Observe the staining under the microscope for approximately 30 s or until the desired color intensity is achieved. Rinse the wells 2-3 times with 1 mL of PBS. Mount the coverslips on slides using neutral resin.

4. Immunofluorescence staining

  1. Seed P2 cells in a 6-well plate containing pre-placed sterile coverslips at a density of 2 x 105 cells per well. Add 2 mL of complete culture medium to each well.
    1. When the cells reach approximately 80% confluence, discard the culture medium. Remove the coverslip and place it in a staining jar. Wash the coverslip three times with 1 mL of PBS, 5 min each time.
  2. Add approximately 50 µL of permeabilization solution (Triton X-100: PBS = 1: 200) to cover the cells. Incubate at room temperature for 5 min, then wash three times with 1 mL of PBS, 5 min each time. Add approximately 50 µL of 3% BSA blocking solution and incubate at room temperature for 20 min.
  3. Gently discard the blocking solution. Add approximately 200 µL of primary antibody solution prepared in PBS to cover the slide: AQP4 (1:100), Kir4.1 (1:200), GS (1:200), CRALBP (1:50), or Vimentin (1:200). Place the coverslip in a humidified chamber and incubate overnight at 4 °C.
  4. Wash the coverslip three times with 1 mL of PBS, 5 min each time.
  5. Add approximately 200 µL of FITC-labeled goat anti-rabbit IgG secondary antibody17. Incubate at room temperature in the dark for 50 min. Wash the coverslip three times with 1 mL of PBS, 5 min each time.
  6. Stain the nuclei with DAPI for 10 min. Wash three times with 1 mL of PBS, 5 min each time.
  7. Add one drop of anti-fade mounting medium to the slide. Mount the coverslip cell-side down. Seal and observe under a fluorescence microscope.

5. Flow Cytometry

  1. Adjust the cell concentration to 1.0 x 107 cells/mL using flow cytometry staining buffer.
  2. Aliquot 100 µL of the single-cell suspension into two separate sample tubes per condition. Designate one tube as the negative (unstained) control.
  3. Add 100 µL of permeabilization medium A (from the commercially obtained kit, see Table of Materials) to each tube. Gently mix and incubate for 15 min at room temperature in the dark.
  4. Add 3 mL of pre-cooled flow cytometry staining buffer to each tube. Centrifuge at 300 x g for 5 min at RT and discard the supernatant.
  5. Add 100 µL of permeabilization medium B (from the commercially obtained kit) to each tube and vortex to mix. Add 0.1 µL of CRALBP antibody to the designated sample tubes, and 0.8 µL of GS antibody to the corresponding tubes. Do not add any antibody to the negative control tube. Incubate at 4 °C in the dark for 30-60 min.
  6. Add 3 mL of flow cytometry staining buffer to each tube. Centrifuge at 300 x g for 5 min at RT and discard the supernatant.
  7. Resuspend each tube in 100 µL of flow cytometry staining buffer. Add 1 µL of FITC-conjugated goat anti-rabbit IgG (H&L) to each CRALBP-stained tube, and 1 µL of PE-conjugated goat anti-rabbit IgG (H&L) to each GS-stained tube. Do not add any antibody to the negative control tube. Incubate at 4 °C in the dark for 30-60 min.
  8. Add 3 mL of flow cytometry staining buffer to each tube. Centrifuge at 300 x g for 5 min and discard the supernatant.
  9. Resuspend each tube in 300 µL of flow cytometry staining buffer. Proceed with flow cytometry analysis immediately; alternatively, resuspend in 500 µL of 1%-4% paraformaldehyde, store at 2-8 °C in the dark, and analyze within 24 h.
  10. Analyze negative and positive samples using the flow cytometry software.

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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 ...

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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 ...

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Disclosures

All authors do not have a conflict of interest related to the content of this article.

Acknowledgements

This work was supported by the Sichuan Provincial Nature Foundation Fund (2023NSFSC0690).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
0.1% Triton X-100SolarbioT8200
0.25% Trypsin-EDTAHyclone SH30042.01B
1% hydrochloric acid alcoholBIOSYSTEMS3803651E
4% ParaformaldehydeSolarbioP1110
45μm nylon mesh (300 mesh)SolarbioYA0926
6 well plateServicebioIMC202302001
6 cm Glass Petri DishNormax5058541
75% AlcoholKESHIGEA004
Aquaporin-4 (AQP4)Proteintech Group, Inc16473-1-AP
BSAServicebioGC305010
Cell culture incubatorThermoFORMA311
cellular retinaldehyde-binding protein (CRALBP)HUABOIHA721330
CentrifugeHunan XiangyiTDZ5-WS
Clean benchZHICHENGZHJH-C12
Corneal scissorsJinzhongBXGJ
DAPISolarbioG1012
D-Hank solutionSolarbioH1045
DMEM  high glucose mediumGibcoC11995500BT
Fetal Bovine Serum (FBS)Sbjbio life SciencesBC-SE-FBS01
FIX&PERM Medium AMultiSciencesGAS006/2
FIX&PERM Medium BMultiSciencesGAS006/2
flow cytometry staining bufferMultiSciencesS1001
Fluoromount-G Fluorescent mounting mediaSourthernBiotech0100-01
Glutamine Synthetase Polyclonal AntibodyProteintech Group, IncPTG-11037-2-AP-50ul
Goat 324 Anti-Rabbit IgG H&L PEBiossbs-0295G-PE
Goat Anti- 323 Rabbit IgG H&L FITCBiossbs-0295G-FITC
Hematoxylin-eosin (HE) staining kitSolarbioG1120
high pressure disinfection machinezealway(xiamen) instrument IncGI80DS
Inverted fluorescence microscopeNikon SMZ1500
Inverted phase contrast microscopeLeica DMIL
inward rectifier potassium channel 4.1 (Kir4.1)Proteintech Group, Inc12503-1-AP
Neutral resinSolarbioG8590-100
Penicillin-Streptomycin Solution (100×)HycloneSV30010
Phosphate Buffer saline (PBS) (pH7.2- 7.4)SolarbioP1020-500ml
Secondary antibody (FITC-labeled goat anti-rabbit)ServicebioGB22303
Sprague-Dawley rats Chengdu Dashuo Experimental Animal Co., Ltd.Production License No.: SCXK [Sichuan] 2020-0030
T25 Culture flaskCornning 430639
Tornado Tubes: 15mLWHB WHB-15-1
Tornado Tubes: 50mLWHB WHB-50-1
ultra violet disinfection sterilizerGEMEISIxd06
VimentinAbcamab92547

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Primary Cell CultureRetinal DissectionTrypsin DigestionSprague Dawley RatsImmunofluorescence StainingFlow CytometryHematoxylin Eosin StainingGlutamine SynthetaseRetinal Stem Cells
Isolation and Culture of Primary Retinal Müller Cells from Sprague-Dawley (SD) Rats
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