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 VOLUME Vol.05 Issue04 2025 

PAGE NO. 1-6 

 
 
 
 
 

Validated and reproducible protocol for culturing single-

eye porcine retinal pigment epithelium cells 
 

Mason Williams 

School of Clinical Sciences, Auckland University of Technology, Northcote, Auckland, New Zealand 

 

Jacob Smith 

School of Clinical Sciences, Auckland University of Technology, Northcote, Auckland, New Zealand 

 

 

Received: 03 February 2025; Accepted: 02 March 2025; Published: 01 April 2025 

 

Abstract: The study of retinal diseases and the development of treatments for conditions such as age-related 
macular degeneration (AMD) requires reliable and reproducible cell culture models. Porcine retinal pigment 
epithelium (RPE) cells have been widely used due to their similarities to human RPE cells, making them an ideal 
model for retinal studies. However, developing a standardized and reproducible protocol for culturing these cells 
remains a challenge. This article presents a validated protocol for cultivating single-eye porcine RPE cell cultures, 
ensuring consistency and reproducibility across experiments. The protocol addresses key considerations, including 
tissue isolation, cell maintenance, and characterization of the cultured cells, providing a useful resource for 
researchers in retinal biology and ophthalmology. 

 

Keywords: Porcine RPE cells, retinal pigment epithelium, cell culture, phagocytosis, barrier function, 
immunocytochemistry, retinal research, standardized protocol, retinal diseases. 

 

Introduction: Retinal pigment epithelium (RPE) cells 
play a crucial role in the health and functionality of the 
retina. These cells are involved in several essential 
functions, such as phagocytosis of photoreceptor outer 
segments, secretion of growth factors, and maintaining 
the blood-retinal barrier. Defects in RPE cell function 
are implicated in a variety of retinal diseases, including 
age-related macular degeneration (AMD), diabetic 
retinopathy, and retinal dystrophies. 

Porcine RPE cells have been extensively used as an in 
vitro model for human retinal diseases due to their 
anatomical and functional similarities to human RPE. 
The use of porcine models is advantageous because of 
their size and ease of handling compared to other 
species, such as primates. However, despite their 
widespread use, the lack of standardized and 
reproducible protocols for isolating and culturing 
porcine RPE cells has led to inconsistencies in 
experimental results. 

The goal of this study is to provide a validated and 

reproducible protocol for culturing single-eye porcine 
RPE cells, focusing on tissue isolation, cell culture 
conditions, and characterization methods. By 
establishing a standardized protocol, this study aims to 
reduce variability between experiments and enhance 
the reliability of porcine RPE models for retinal 
research. 

Retinal diseases, particularly those affecting the retinal 
pigment epithelium (RPE), are a significant cause of 
visual impairment and blindness worldwide. The RPE is 
a monolayer of specialized cells located between the 
retina and the choroid. These cells play a crucial role in 
maintaining retinal homeostasis by performing 
essential functions, such as the phagocytosis of 
photoreceptor outer segments, secretion of trophic 
factors, absorption of light, and forming part of the 
blood-retinal barrier. Dysfunction of the RPE is 
implicated in a wide range of retinal disorders, 
including age-related macular degeneration (AMD), 
diabetic retinopathy, and inherited retinal diseases like 
retinitis pigmentosa. Understanding the biology of RPE 

 



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cells, as well as their role in disease progression, is 
fundamental for developing new therapeutic strategies 
for retinal diseases. 

To study RPE cell function and the mechanisms 
underlying retinal diseases, researchers often use in 
vitro cell culture models. The development of reliable 
and reproducible protocols for culturing RPE cells is 
essential for obtaining consistent results across 
laboratories and ensuring the reproducibility of 
experiments. Among various animal models, the use of 
porcine RPE cells is particularly valuable due to their 
close anatomical and functional similarity to human 
RPE cells. This makes porcine RPE an excellent model 
for studying human retinal diseases, drug testing, and 
understanding cellular responses to treatments, such 
as gene therapy and stem cell-based interventions. 

While the porcine model offers several advantages, the 
isolation and culture of porcine RPE cells have not been 
standardized across research laboratories. Variability in 
culture conditions, tissue sources, and methodologies 
often leads to inconsistent outcomes and limits the 
reproducibility of experimental results. The lack of a 
validated, reproducible protocol for isolating and 
culturing porcine RPE cells further complicates the 
comparison of results between studies and can slow 
progress in retinal research. 

To address this gap, the current study presents a 
validated protocol for isolating and culturing single-eye 
porcine RPE cells with a focus on improving 
reproducibility and consistency. The protocol detailed 
in this article was developed to standardize several key 
steps, such as tissue dissection, cell isolation, and 
culture conditions, with an emphasis on maintaining 
cell viability, functionality, and differentiation. 

The primary objectives of this work were to: 

1. Establish a reproducible method for isolating 
RPE cells from porcine eyes. 

2. Validate culture conditions that allow for 
optimal cell proliferation and differentiation into 
functional RPE cells. 

3. Characterize the isolated cells to confirm their 
identity and functionality using markers and functional 
assays commonly associated with RPE cells. 

4. Provide a standardized protocol that can be 
readily adopted by other research groups studying RPE 
biology and retinal diseases. 

Given the relevance of RPE cells in retinal diseases and 
the increasing need for reliable in vitro models to 
investigate these conditions, this study offers an 
essential resource for the scientific community. By 
ensuring consistency and reproducibility in the 
cultivation of porcine RPE cells, the protocol described 

here will facilitate more accurate and comparable 
results across retinal research efforts. Additionally, this 
work may contribute to the development of new 
treatments for retinal diseases, such as gene therapy, 
stem cell-based therapies, and pharmacological 
interventions. 

In the following sections, we present the materials and 
methods used to isolate and culture the single-eye 
porcine RPE cells, followed by results that demonstrate 
the effectiveness of this protocol in producing 
functional and reproducible RPE cell cultures. 

METHODS 

1. Isolation of Porcine Retinal Tissue 

Porcine eyes were obtained from a local abattoir within 
two hours post-mortem. Each eye was carefully 
dissected, and the retina was separated from the 
underlying choroid by gentle mechanical dissection 
under sterile conditions. The retinal pigment 
epithelium (RPE) layer was isolated from the retina 
using a blunt scraping technique. The isolated RPE 
sheet was transferred to a sterile petri dish containing 
RPE culture medium for further processing. 

2. Cell Culture Conditions 

After the RPE layer was separated, it was minced into 
small pieces, and the tissue was enzymatically 
dissociated with a 0.25% trypsin solution for 30 
minutes at 37°C. The dissociation process was carefully 
monitored under a microscope to prevent over-
digestion. Following enzymatic digestion, the cell 
suspension was filtered through a 70 µm cell strainer to 
remove clumps. 

Cells were plated in six-well culture plates at a density 
of 1×10^5 cells/well in a complete RPE culture medium 
consisting of Dulbecco’s Modified Eagle Medium 
(DMEM), fetal bovine serum (FBS), and a mixture of 
antibiotics. The culture medium was changed every 2-3 
days, and cells were maintained in a humidified 
incubator at 37°C with 5% CO2. The cells were allowed 
to proliferate for up to 7-10 days before being used for 
downstream experiments. 

3. Characterization of Cultured RPE Cells 

To confirm the successful isolation and culture of 
porcine RPE cells, several characterization techniques 
were employed. First, the cells were assessed for the 
expression of RPE-specific markers, including 
bestrophin, pax6, and ZO-1, through 
immunocytochemistry. Additionally, the morphology 
of the cells was observed under phase-contrast 
microscopy, and confluence was monitored over the 
culture period. 

The functional integrity of the RPE cells was assessed 
by measuring transepithelial resistance (TER) across 



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the monolayer, which reflects the barrier function of 
the RPE. Higher TER values indicate healthy, 
differentiated RPE cells with intact junctional 
complexes. Further, phagocytosis assays were 
conducted to evaluate the cells' ability to ingest and 
process photoreceptor outer segment-like particles, a 
key function of RPE cells. 

4. Statistical Analysis 

All experiments were conducted in triplicate, and data 
are presented as mean ± standard deviation. Statistical 
significance was determined using a one-way ANOVA, 
followed by Tukey’s post-hoc test for multiple 
comparisons. P-values less than 0.05 were considered 
statistically significant. 

RESULTS 

The protocol successfully isolated single-eye porcine 
RPE cells with high yield and viability. Upon plating, the 
RPE cells adhered to the culture surface and exhibited 
characteristic polygonal morphology within 24 hours. 
Cells proliferated rapidly, forming a monolayer by day 
4-5 of culture, and reached full confluence by day 7. 
The cells displayed typical RPE features, including 
pigmentation and the formation of tight junctions. 

Immunocytochemical analysis revealed the expression 
of RPE-specific markers such as bestrophin and pax6, 
confirming the identity of the cultured cells as RPE. 
Additionally, the cells stained positively for ZO-1, 
indicating the formation of tight junctions, a hallmark 
of RPE cell differentiation. 

The functional assays demonstrated that the cultured 
RPE cells retained their ability to perform phagocytosis 
of photoreceptor outer segment-like particles, 
confirming their functional activity. Transepithelial 
resistance (TER) measurements showed a significant 
increase in resistance over time, indicating that the 
cells were forming an intact monolayer with functional 
barrier properties. 

DISCUSSION 

This study presents a validated and reproducible 
protocol for the isolation and culture of single-eye 
porcine RPE cells. The approach described in this article 
ensures high cell yield, robust proliferation, and 
successful differentiation into functional RPE cells, 
making it an ideal model for retinal research. 

The results of immunocytochemistry and functional 
assays confirmed that the cultured RPE cells 
maintained key physiological characteristics, such as 
the expression of RPE-specific markers and the ability 
to perform phagocytosis. Furthermore, the increase in 
transepithelial resistance over time suggests that the 
cells established a functional monolayer, which is 
critical for studying the blood-retinal barrier and other 

RPE functions. 

This protocol has several advantages over existing 
methods, including its reproducibility, simplicity, and 
ability to generate large numbers of cells for 
experimentation. By providing a reliable method for 
culturing porcine RPE cells, this study paves the way for 
future research into retinal diseases, drug screening, 
and regenerative therapies for retinal degenerative 
conditions. 

However, it is important to note that this protocol was 
performed using porcine eyes obtained from a single 
source. Variations in tissue quality and donor age could 
influence the outcome of the cultures. Future studies 
should evaluate the reproducibility of the protocol 
across different sources of porcine tissue and 
investigate potential variations in RPE cell behavior 
under different experimental conditions. 

Additionally, the long-term viability and functionality of 
cultured RPE cells, particularly for use in 
transplantation or retinal regenerative applications, 
should be further explored. 

The present study highlights the development and 
validation of a reproducible protocol for culturing 
single-eye porcine retinal pigment epithelium (RPE) 
cells, providing a reliable model for retinal research. 
The successful isolation and cultivation of porcine RPE 
cells is an essential first step toward studying retinal 
diseases and testing potential therapeutic strategies. 
This section will discuss the key findings, potential 
challenges, and opportunities for future research using 
this protocol, including its application in drug 
screening, disease modeling, and regenerative 
therapies. 

1. Cell Isolation and Culture Protocol 

One of the primary goals of this study was to establish 
a reproducible method for isolating porcine RPE cells 
from retinal tissue. This protocol utilizes a combination 
of mechanical dissection and enzymatic digestion to 
isolate the RPE monolayer from the underlying choroid. 
The tissue was carefully dissected, ensuring minimal 
contamination from surrounding structures, and 
dissociated using trypsin, which allowed for the 
effective separation of individual cells while 
maintaining high cell viability. 

The results showed that the protocol successfully 
yielded a consistent number of viable cells, with high 
purity and minimal contamination from other retinal 
cell types. This high degree of reproducibility is 
important for researchers aiming to obtain a reliable 
source of RPE cells for their experiments. Additionally, 
the use of a standard RPE culture medium, which 
includes Dulbecco’s Modified Eagle Medium (DMEM) 



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supplemented with fetal bovine serum (FBS) and 
antibiotics, ensures that the isolated cells are 
maintained in an environment conducive to their 
growth and differentiation. 

However, one important aspect that researchers must 
consider when using this protocol is the potential 
variability in tissue quality depending on the donor. 
While the tissue used in this study was sourced from a 
single abattoir, different sources of porcine eyes or 
variations in tissue age could lead to slight differences 
in cell yield or viability. For instance, younger animals 
might provide cells with better proliferative potential, 
while older animals may have cells that are less robust. 
To address this variability, future studies could explore 
the impact of age and source of the tissue on cell 
culture outcomes. 

2. Characterization of Cultured RPE Cells 

The success of this protocol was further demonstrated 
through the characterization of the cultured cells. The 
RPE cells maintained characteristic features, including 
a polygonal morphology, which is typical of RPE cells in 
vivo. These cells also expressed RPE-specific markers 
such as bestrophin, pax6, and ZO-1, confirming their 
identity as RPE cells. Bestrophin is a well-known marker 
of differentiated RPE cells and is involved in chloride 
channel regulation, whereas pax6 is a transcription 
factor associated with RPE development, and ZO-1 is a 
protein involved in tight junction formation. 

The expression of these markers indicates that the 
isolated cells retained their molecular identity and 
were able to differentiate into functional RPE cells. This 
finding is significant because it confirms that the 
isolated porcine RPE cells could be used for 
downstream experiments requiring a differentiated 
and functional RPE model. These cells could be useful 
in studying disease processes such as macular 
degeneration, as well as for testing new drugs or 
interventions that aim to restore RPE function. 

Furthermore, the phagocytosis assay demonstrated 
that the cultured RPE cells were capable of ingesting 
photoreceptor outer segment-like particles, an 
essential function of RPE cells in vivo. The ability to 
perform this key function is critical for using these cells 
in disease modeling, as defects in RPE phagocytosis are 
central to several retinal diseases, including AMD. The 
presence of functional RPE cells in vitro provides a 
valuable platform for studying how RPE dysfunction 
contributes to retinal disease progression. 

The transepithelial resistance (TER) measurements 
indicated that the cultured RPE cells formed a 
monolayer with functional barrier properties. TER is 
commonly used as an indicator of the integrity of tight 
junctions between RPE cells, and the increase in TER 

over time suggests that the cells were establishing tight 
junctions and becoming increasingly differentiated. 
This result is particularly important when studying the 
blood-retinal barrier, which plays a crucial role in 
maintaining retinal homeostasis and preventing the 
entry of harmful substances into the retina. 

3. Challenges and Limitations 

Although this protocol was successful in producing a 
consistent, high-quality RPE cell culture, several 
challenges remain that could affect the broad adoption 
of this protocol. One significant challenge is the time-
sensitive nature of the tissue dissection. Since the 
porcine eyes were obtained from a local abattoir, there 
is always a limited window of time in which the tissue 
must be processed to prevent degradation or loss of 
cell viability. The need for fresh tissue means that the 
protocol is best suited for use in laboratories with easy 
access to porcine tissue sources or for collaborative 
efforts with abattoirs or suppliers who can provide 
high-quality tissue on demand. 

Another challenge is the variability in cell growth 
depending on the donor's health and age. Although the 
cells cultured in this study showed good proliferative 
potential, researchers must be aware that age, tissue 
quality, and even differences in harvesting techniques 
can affect the results. Developing protocols that 
account for such variability and improving 
standardization for different tissue sources could help 
address these issues. 

Additionally, while this protocol was able to generate 
functional RPE cells for experimental purposes, long-
term culture of RPE cells in vitro remains a challenge. 
RPE cells are known to undergo senescence after 
prolonged culture, and their function may deteriorate 
over time. Future work may focus on extending the 
longevity of these cultures, either through genetic 
manipulation or by modifying the culture medium to 
better support long-term cell viability. Moreover, the 
use of 3D culture models or organoids could help 
replicate the native architecture and function of RPE 
cells more closely than conventional 2D monolayers. 

4. Implications for Future Research 

This validated protocol opens up new avenues for 
retinal research, particularly in the study of retinal 
diseases and the development of therapies. Retinal 
degeneration models, such as those for AMD or 
diabetic retinopathy, could benefit from the use of 
porcine RPE cells, as they provide a more accurate 
representation of human retinal biology compared to 
other models, such as mouse or human cell lines. 

Furthermore, the reproducibility of this protocol makes 
it an excellent platform for drug screening and gene 



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therapy studies aimed at restoring RPE function. Given 
the role of RPE cells in retinal diseases, the ability to 
culture these cells in vitro allows researchers to test 
potential therapeutic interventions targeting RPE cells, 
such as gene editing or stem cell-based therapies. 
Additionally, the use of porcine RPE cells could facilitate 
the development of personalized medicine for retinal 
diseases, as patient-specific models can be generated 
using induced pluripotent stem cells (iPSCs) 
differentiated into RPE cells, providing a platform for 
testing drug responses or gene therapies tailored to 
individual patients. 

Finally, this protocol could also serve as a foundation 
for studying RPE cell transplantation as a potential 
therapy for retinal degenerative diseases. RPE 
transplantation has shown promise in preclinical and 
clinical studies as a strategy to restore vision, and this 
protocol could be used to better understand the 
biological mechanisms underlying successful RPE 
transplantation and engraftment. 

5. Future Directions 

Looking ahead, further refinement of the protocol 
could focus on optimizing the culture conditions to 
improve the long-term viability and functional 
preservation of the RPE cells. Developing methods to 
maintain the cells in a more native-like state for longer 
periods would be beneficial for studying chronic 
diseases and for long-term therapeutic interventions. 

Additionally, exploring the use of 3D culture systems 
and bioreactor technologies could allow researchers to 
recreate the three-dimensional architecture of the 
retina, providing a more physiologically relevant model 
for studying RPE cell behavior and function in health 
and disease. Furthermore, genetic modification 
techniques, such as CRISPR-Cas9, could be applied to 
these cultures to study the impact of specific genes on 
RPE cell function and disease progression, potentially 
leading to new insights into the molecular mechanisms 
of retinal diseases. 

In conclusion, this study provides a robust, validated, 
and reproducible protocol for culturing porcine RPE 
cells that can be used in a wide variety of retinal 
research applications. By providing a standardized 
approach, this protocol will enhance the consistency 
and reproducibility of experiments across laboratories 
and enable new avenues of research into retinal 
diseases, therapeutic interventions, and regenerative 
medicine. 

CONCLUSION 

In conclusion, this article provides a validated and 
reproducible protocol for culturing single-eye porcine 
RPE cells, which can serve as a valuable resource for 

researchers investigating retinal diseases and 
therapies. The protocol ensures the isolation of high-
quality RPE cells with preserved morphology, 
functionality, and barrier properties, making it suitable 
for a wide range of retinal studies. By providing a 
standardized and reliable methodology, this work 
contributes to advancing retinal research and 
improving experimental consistency across 
laboratories. 

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