Alu RNA-transfected Primary Mouse Retinal Pigment Epithelium: A Pathologically Relevant In Vitro Model for Age-related Macular Degeneration

Summary

In this protocol, we describe the full process of establishing a pathology-relevant model for age-related macular degeneration (AMD) in primary mouse retinal pigment epithelial (RPE) cells using Alu RNA transfection.

Abstract

Age-related macular degeneration (AMD), particularly non-exudative AMD, requires experimental models that better replicate human pathology. Current in vivo models remain technically demanding and time-intensive, whereas conventional in vitro systems fail to recapitulate disease-specific pathological triggers. Here, we present a method to establish a retinal pigment epithelial (RPE) degeneration model using primary mouse RPE cells transfected with Alu RNA, a retrotransposon directly implicated in geographic atrophy pathology.

This protocol details the enzymatic isolation of primary mouse RPE cells, followed by Alu RNA transfection to induce RPE degeneration. Validation integrates morphological (hexagonal architecture), functional (polarity loss and mouse protein ZO-1 disruption), and molecular analysis (quantitative PCR). As a result, we observed multifactorial changes triggered by Alu RNA transfection: inflammatory cytokine secretion (mouse genes Ifn-β, Il-6, Tnf-α; p < 0.05) and cellular senescence (mouse genes p21 and p53 upregulation; p < 0.05). Compared to traditional acute stress models, this system recapitulates chronic inflammatory-degenerative cascades of AMD through standardized techniques, ensuring reproducibility. By combining aspects of simplified in vitro assays and complex in vivo models, this approach could serve as a preliminary platform for exploring retrotransposon-driven mechanisms and screening potential therapeutic candidates.

Introduction

Age-related macular degeneration (AMD) is a predominant cause of global visual impairment. However, effective treatments for non-exudative AMD remain limited. Thus, there is an urgent need for reproducible and efficient research models to uncover disease mechanisms and identify therapeutic targets^1,2. Central to this effort is the retinal pigment epithelium (RPE), a monolayer situated between Bruch's membrane (choroid) and photoreceptors (retina), which plays a pivotal role in sustaining retinal homeostasis by supporting photoreceptor function, regulating the blood-retinal barrier, and facilitating waste clearance^3,4.

Dysfunction or loss of RPE cells is a salient feature of many retinal degenerative diseases, especially in AMD, where RPE dysfunction represents an early and critical step in its progression^2,4,5. Notably, in patients manifesting geographic atrophy (GA), the advanced phase of AMD, reduced levels of the microRNA-processing enzyme Dicer 1 ribonuclease III (DICER1) result in toxic accumulation of Alu RNA, a retrotransposon transcript directly linked to RPE degeneration and implicated in human pathology⁶.

Traditional in vivo models, such as the sodium iodate (NaIO₃) model and amyloid-β (Aβ) model, retain systemic disease complexity but face critical limitations. The NaIO₃ model rapidly induces retinal oxidative stress and RPE damage, however, its acute toxicity disrupts both RPE and photoreceptors, failing to mimic the chronic pathology-related progression of AMD and obscuring therapeutic evaluation of subtle RPE dysfunction⁷. Similarly, the Aβ model introduces disease-specific stressors but demands extensive technical expertise and prolonged timelines compared to in vitro approaches^8,9. Conventional in vitro RPE models, like H₂O₂ and hydroquinone exposure models, on the other hand, lack pathological relevance, failing to recapitulate disease-specific stressors such as toxic protein aggregates or inflammatory triggers¹.

Here, we provide a detailed protocol for establishing an RPE degeneration model in primary mouse RPE cells using Alu RNA, a retrotransposon-derived non-coding RNA directly implicated in GA pathogenesis⁶. Alu RNA activates inflammatory pathways and compromises RPE barrier integrity^10,11, thereby mimicking the pathological features associated with non-exudative AMD. This method employs the enzymatic isolation and culture of RPE cells, followed by the induction of RPE degeneration using Alu RNA. Additionally, we provide a comprehensive validation system, combining morphological (hexagonal architecture), functional (barrier integrity and polarity loss), and molecular (inflammatory and senescence markers) readouts to assess this degeneration model.

Protocol

This study was approved by the Animal Ethics Committee of Shanghai General Hospital affiliated to Shanghai Jiao Tong University School of Medicine. This protocol strictly follows the Regulations for the Administration of Affairs Concerning Experimental Animals (issued by the State Science and Technology Commission) and the guidelines for the ethical use of animals in ophthalmic and vision research established by the Association for Research in Vision and Ophthalmology (ARVO). See the Table of Materials for more details related to the materials used in this protocol.

1. Isolation of primary mouse RPE cells

1. Preparation of coated plates
   1. Dilute Matrigel at a ratio of 150 µL of Matrigel to 13 mL of DMEM/F12 (final concentration approximately 1.14%).
   2. Evenly coat the bottom of the plate with the diluted Matrigel (approximately 150 µL for a 48-well plate). Incubate overnight at 37 °C to form a gel.
2. Solution preparation
   1. Prepare the complete RPE culture medium using Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) complemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin.
   2. Prepare Papain Mix by adding 4.5 mL of Earle's Balanced Salt Solution (EBSS) to a papain vial (100 Units/vial).
   3. Prepare DNase I Mix by adding 500 µL of EBSS to a DNase I vial (1,000 Units/vial).
   4. Prepare papain digestion solution by adding 250 µL of DNase I Mix to 4.5 mL of Papain Mix (adjust the protocol provided by the supplier to achieve a final papain concentration of 22 IU/mL), making the final volume 4.75 mL.
3. Euthanasia and tissue collection
   1. Euthanize mice 2-3 weeks old via intraperitoneal injection of 5% sodium pentobarbital at 150-200 mg/kg body weight. Confirm deep anesthesia and death by verifying cessation of heartbeat and respiration.
   2. Thoroughly spray the ocular region with 70% ethanol prior to enucleation to eliminate surface contaminants^12,13,14. Gently press the periorbital rim to protrude the globe. Then, use forceps to extract the eyeball, preserving the optic nerve for easier dissection.
      NOTE: All tools (e.g., forceps, scissors) are autoclaved, and procedures are performed under a laminar flow hood to maintain sterility.
   3. Immerse the eyeballs immediately in sterile complete RPE culture medium prewarmed to 37 °C.
4. Enzymatic digestion
   NOTE:All steps, including enzymatic digestion and subsequent procedures, must be performed under a laminar flow hood to maintain sterility.
   1. Transfer the eyeball to a dish and use forceps to eliminate the adhering connective tissue and residual extraocular muscles under a stereomicroscope.
   2. Gently rinse the eyes 2x with precooled Dulbecco's Phosphate Buffered Saline (D-PBS; no Ca²⁺/Mg²⁺; at 4 °C).
      NOTE: Residual complete RPE culture medium may compromise papain enzymatic activity.
   3. Fully immerse the eyes in 1 mL of papain digestion solution and incubate at 37 °C for 45 min.
5. Separation of RPE cells
   1. After digestion, neutralize the enzymatic activity with complete RPE culture medium.
   2. Transfer the eyeball to a dish, and under a stereomicroscope, excise the cornea, iris, and lens, and preserve the posterior eyecup.
      NOTE: RPE cells were isolated and maintained as intact sheets (rather than dissociating into single cells) during the entire process.
   3. Separate the retina-RPE complex from the choroid, then peel the RPE layer away from the retina as continuous sheets in complete RPE culture medium. Collect the detached RPE layers uniformly into fresh complete RPE culture medium as a mixture and subsequently culture them on Matrigel-coated surfaces.
6. Cell culture
   1. Seed the mixture in a Matrigel-coated 24-well plate (8-10 eyeballs per well on coverslip, for immunostaining), a Matrigel-coated 48-well plate (6-8 eyeballs per well, for qPCR), or a Matrigel-coated 96-well plate (5-6 eyeballs per well).
   2. Incubate at 37 °C with 5% CO₂. Perform the first medium change after 48 h using complete RPE culture medium. Change the medium every 48 h after the first change.
      NOTE: To maintain the polarity of RPE cells as much as possible, avoid passaging them whenever possible, as repeated subculturing can lead to loss of differentiation and functional integrity.
7. Cell morphology monitoring
   1. Allow cells to form a monolayer with characteristic hexagonal morphology. Monitor for morphology under an inverted cell culture microscope.

2. Induction of the RPE degeneration model

1. Alu RNA synthesis and verification
   1. Synthesize Alu RNA using an in vitro transcription system.
      NOTE: The synthesis of Alu RNA used in our study was based on the protocol described by the Ambati Lab⁶.
   2. Purify the RNA and confirm its quality using a microspectrophotometer.
2. Alu RNA transfection
   NOTE: We designated the day of primary RPE cell seeding as day 0. On day 5, the adherent primary RPE cells exhibited a uniform morphology and reached more than 50% cellular confluence, at which point Alu RNA transfection was carried out using a cationic lipid-based transfection reagent, according to the manufacturer's guidelines.
   Two control groups were included to validate the results: i) a negative control (NC), in which neither Alu RNA nor the transfection reagent was added and low-serum medium was used as a substitute; ii) liposome control (LC), in which Alu RNA was omitted but the same amount of transfection reagent was mixed with low-serum medium and applied to cells, to control for lipofection-associated effects. The following steps outline the Alu RNA transfection procedure.
   1. Replace the existing medium with 300 µL of complete RPE culture medium per well (48-well plate) before transfection procedures.
   2. Prepare transfection complexes with Alu RNA and cationic lipid-based reagent in low-serum medium.
      1. Prepare Mix 1 with 1.2 µL of cationic lipid-based reagent and 18.8 µL of the low-serum medium. Gently mix (do not vortex) and incubate it at room temperature for 5 min.
      2. Prepare Mix 2 with 6 µL of Alu RNA (100 µg/mL) and 14 µL of low-serum medium.
      3. Prepare transfection complexes by adding 20 µL of Mix 2 dropwise into 20 µL of Mix 1.
         NOTE: Alu RNA was transfected at a final concentration of 0.6 µg per well using 1.2 µL of cationic lipid-based reagent per well (1.2 µL of reagent: 0.6 µg RNA ratio).
      4. Gently flick to mix. Then, leave it at room temperature for 20 min.
   3. Add 40 µL/well of the transfection complexes to each well of a 24-well plate. Place the plate in a 37 °C/5% CO₂ incubator for 48 h.
   4. Perform qPCR, immunostaining, and other experimental procedures 48 h post transfection.

3. Validation of the degeneration model

NOTE: Comprehensive model validation experiments were performed on day 7.

1. Immunofluorescence staining for polarity and barrier integrity evaluation
   1. Fix cultured RPE cells with 4% methanol-free formaldehyde for 20 min at room temperature. Wash 3 x 10 min with 1x PBS.
   2. Block nonspecific binding with 5% bovine serum albumin (BSA) in Phosphate-buffered saline (PBS) for 1 h.
   3. Incubate with primary antibodies anti-ZO1 (1:1,000 dilution in 3% BSA) and Phalloidin 488 (1:1,000 dilution in 3% BSA) overnight at 4 °C, avoiding light.
   4. Wash the cells 3 x 10 min gently with PBS.
   5. Incubate with secondary antibodies (1:1,000 dilution in 3% BSA) for 1 h at room temperature.
   6. Wash the samples 3 x 10 min with 1x PBS.
   7. Counterstain with DAPI (1:1,000 dilution in PBS) for 10 min.
   8. Wash the samples for 3 x 10 min with 1x PBS.
   9. Carefully remove the coverslip from the 24-well plate and place it onto a glass slide preloaded with a drop of mounting medium. Capture confocal images using a 40x/0.95 objective.
2. Quantitative PCR (qPCR) for senescence and inflammatory markers
   NOTE: Glyceraldehyde-3-phosphate dehydrogenase (mouse gene Gapdh) was employed as the internal reference gene for normalization of threshold cycle (Ct) values. All quantitative PCR (qPCR) reactions included three biological replicates per experimental group. Primer sequences are provided in Supplemental Table S1.
   1. RNA extraction
      1. Add lysis buffer (Total RNA Kit) to the culture plate (1 mL per 10 cm² surface area) and incubate at room temperature for 5 min.
      2. Centrifuge at 13,400 × g for 5 min at 4 °C. Transfer the supernatant to a new RNase-free tube.
         NOTE: Skip this step if samples are free of debris (e.g., clear cell lysate).
      3. Add 200 µL of chloroform to the supernatant, vortex for 15 s, and incubate at room temperature for 3 min.
      4. Centrifuge at 13,400 × g for 10 min at 4 °C. Transfer the upper aqueous phase (~500 µL) to a new tube.
      5. Add 0.5 volumes of ethanol to the aqueous phase. Mix gently by pipetting.
      6. Load the mixture onto a silica membrane column (included in Total RNA Kit). Centrifuge at 13,400 × g for 30 s. Discard the flowthrough.
      7. Add 500 µL of protein removal buffer (Total RNA Kit)to the column. Centrifuge at 13,400 × g for 30 s. Discard the flowthrough.
      8. Add 500 µL of wash buffer (Total RNA Kit) to the column. Centrifuge at 13,400 × g for 30 s. Discard the flowthrough.
      9. Repeat the previous step 3.2.
      10. Centrifuge the empty column at 13,400 × g for 2 min to dry the membrane.
      11. Elute RNA by adding 30-100 µL of RNase-free water to the membrane. Incubate at room temperature for 2 min. Centrifuge at 13,400 × g for 2 min.
   2. Complementary DNA (cDNA) synthesis
      1. Measure RNA concentration using a spectrophotometer at 260 nm and 280 nm.
         NOTE: An A₂₆₀/A₂₈₀ ratio between 1.8 and 2.0 is considered to be associated with acceptable RNA purity.
      2. Prepare the reaction mix in an RNase-free tube: RNA template (500-1,000 ng): X µL (related to its concentration), 5x reverse transcription buffer: 4 µL, RNase-free water: to 20 µL.
      3. Incubate the reaction mix in a thermal cycler using the following program: 37 °C for 15 min, 85 °C for 5 s, and an indefinite hold at 4 °C.
   3. qPCR
      1. Prepare qPCR master mix for each reaction (10 µL total): TB Green (included in Premix Taq) : 5 µL, ROX dye (included in Premix Taq): 0.2 µL, Forward primer (1 µM): 0.2 µL, Reverse primer (1 µM): 0.2 µL, RNase-free water: 3.4 µL, cDNA template: 1 µL.
      2. Add 10 µL of the master mix into each well of a 96-well plate.
      3. Seal the plate with an adhesive optical cover. Centrifuge at 300 × g for 1 min.
      4. Run the following program on a real-time PCR system: Stage 1: 95 °C for 30 s; Stage 2: 40 cycles of: 95 °C for 5 s, 60 °C for 30 s; Stage 3: Melting curve analysis.
      5. Calculate the relative expression levels of target mRNAs using the 2^−ΔΔCt method¹⁵.

Results

Primary mouse RPE cells before Alu RNA transfection
On Day 0 (Figure 1), primary mouse RPE cells were isolated and exhibited the characteristic hexagonal morphology, which is typical of healthy RPE cells. These cells maintained a well-organized monolayer. The cells were cultured to allow for initial structural organization and formation of tight junctions, as evidenced by the hexagonal shape and well-defined boundaries. This morphology served as the baseline for the subs...

Discussion

Our goal was to establish a reproducible and accessible RPE injury model that enables high-resolution imaging and mechanistic analysis with minimal resource constraints. This approach provided clear visualization of Alu RNA-induced structural disruption comparing with control group, including apical tight junction fragmentation and basal cytoskeletal disorganization through confocal Z-stack imaging (0.5 µm step size). Two critical steps ensure the system's reliability: enzymatic digestion and Alu RNA transfectio...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1300 Series Class II, Type A2 Biological Safety Cabinet	Thermo Scientific
37 °C/5% CO2 incubator	Thermo Scientific
4% Paraformaldehyde (PFA)	Servicebio	G1101-500ML
human Alu RNA			Gifted by L.Z.
Anti-rabbit secondary antibody,Alexa Fluor 568(goat)	Invitrogen Thermo Scientific	A11011
Applied Biosystems MicroAmp adhesive optical cover	Thermo Fisher Scientific
Applied Biosystems MicroAmp Optical 96-Well Reaction Plates	Thermo Fisher Scientific
Applied Biosystems ViiA 7 Real-Time PCR System	Thermo Fisher Scientific		Real-time PCR system for quantitative gene expression analysis
Bovine Serum Albumin(BSA, Fraction V)	Beyotine	ST023-50g
Carl Zeiss Primovert inverted cell culture microscope	Zeiss		For cell morphology monitoring
Centrifuge 5424 R	eppendorf
Chloroform	Sigma		Phase separation in RNA extraction.
Confocal laser scanning microscope(LSM900+Cell discover 7)	Zeiss		High-resolution imaging of immunofluorescence markers (e.g., ZO-1 and F-actin).
Costar 24/48/96 well plate	Corning
coverslip	Thermo Fisher Scientific		Used on 24-well plate for immunofluorescence imaging.
DAPI	Thermo	62248
Digital dry bath	Mulab
DMEM/F12	gibco		Media to grow RPE cells
Dulbecco's Phosphate-Buffered(D-PBS)	ShareBio	SB-CR017
Earle’s Balanced Salt Solution (EBSS)	gibco		Balanced salt solution used for papain and DNase I dilution during RPE isolation.
Ethanol (Absolute)	Sigma		RNA precipitation and column washing.
Fetal Bovine Serum (FBS)	Sigma		For complete RPE cell culture media
forceps			For dissection
Lipofectamine 3000 (Cationic Lipid-Based Transfection Reagent)	Thermo Fisher	L300015	To transfect Alu RNA into RPE cells
matrigel	Corning	356234	Matrix (1.14% dilution) for coating plates to support RPE adhesion and polarization.
Methanol-Free Formaldehyde	Thermo Scientific		Alternative fixative (4%) for preserving epitopes in immunofluorescence
Micropipette	eppendorf
Nanodrop 2000 spectrophotometer	Thermo Scientific		Measures RNA concentration and purity
Nikon ECLIPSE Ti-S fluorescent Inverted microscope	Nikon		Fluorescence imaging of stained samples
Olympus SZX16 stereomicroscope	Olympus		For dissection of mouse eyeballs and RPE isolation
Opti-MEM	gibco		low-serum medium for transfection complex preparation
Papain dissociation system	Worthington	LK003150	For working enzyme
Penicillin-Streptomycin	gibco		For complete RPE cell culture media
Pentobarbital sodium salt	Sigma		Euthanasia agent
Phalloidin 488	Abcam	176753	For polarity assessing
PrimeScript RT reagent Kit	TaKaRa	RR037A	Reverse transcription kit for cDNA synthesis (qPCR template preparation)
RNAsimple Total RNA Kit	TIANGEN	DP419	For extracting RNA
TB Green Premix Ex Taq (Tli RNaseH Plus)	TaKaRa	RR420A	For qPCR
ZO-1 Rabbit Polyclonal Antibody	Proteintech	21773	For morphological evaluation