Single-Eye Porcine Retinal Pigment Epithelium Cell Cultures—A Validated and Reproducible Protocol

Abstract

Background: Age-related macular degeneration (AMD) is the leading cause of severe vision loss in industrialized nations. AMD models based on standardized optimized cell culture models are warranted. The aim of this study is to implement a standard operation protocol for the preparation of porcine retinal pigment epithelium (RPE) from pig eyes with cells from one eye designated for one culture, generating a genetic homology within one culture and genetic heterogeneity between cultures, thereby implementing a relevant in vitro model for AMD investigations. In addition, the use of pigs’ eyes from slaughterhouse waste material is an active measure to reduce animal experimentation in ophthalmological research. Methods: Primary porcine RPE cells were prepared, and cells from one eye were seeded into one well of a twelve-well polystyrene plate. They were cultured for 7, 14, and 28 days. Different post-mortem times (2, 4, and 6 h), coatings (collagen I, IV, Poly-d-Lysine, fibronectin, and laminin), and serum content of media (1%, 5%, and 10%) were tested. The success rate for confluence and survival was determined. At the different time points, cell number (trypan blue exclusion assay), confluence and morphology (microscope imaging), VEGF content of supernatants, and gene and protein expression, as well as tight junctions (fluorescence imaging), were examined. In addition, a baseline for RPE morphometry using CellProfiler software was established. Results: A post-mortem time of 4 to 5 h is most suitable. A coating with Poly-d-Lysine is recommended due to high success rates, the fastest confluence, the highest expression of RPE65, and suitable morphologic properties. The results for confluence, protein expression, and morphology showed that a reduction of 10% to 5% FBS is acceptable. Conclusion: A basic best practice protocol for the preparation of porcine single-eye cultures with optimized parameters was established and is provided.

1. Introduction

Reliable ocular cell models for the research of ophthalmologic diseases are highly warranted. One of these diseases is age-related macular degeneration (AMD), the main cause of severe central vision loss in the elderly and Western nations, with increasing patient numbers expected to go up to 288 million by 2040 [1]. Early forms of AMD are without symptoms, whereas the two late forms lead to severe vision disturbance. In the late dry form, atrophy develops in the retinal pigment epithelium (RPE) and photoreceptors, while in the exudative (wet) form of late AMD, VEGF is oversecreted and blood vessels grow from the choroid under and into the retina [2]. Molecular pathomechanisms in the development of AMD are oxidative stress, inflammation, lipid dysregulation, and proangiogenic factors [3]. These are primarily targeting RPE within the photoreceptor/RPE/Bruch’s membrane/choriocapillaris complex [4]. The RPE maintains visual functions by supplying nutrients, taking up waste, forming the outer blood–retinal barrier, contributing to the immune privilege, phagocyting shed photoreceptor outer segments, releasing protective chemokines, and protecting against light and oxidative stress [5]. The RPE is supported by an extracellular structure known as Bruch’s membrane, which consists of collagen Type IV, fibronectin, and laminin [6]. These substances are also used for the coating of cell culture plates.

For a better understanding of all underlying physiological cell functions and pathways involved in the development of ocular diseases, or to develop new therapeutics, optimized and conclusive cellular model systems are highly desired. For an overview, the reader is referred to current comprehensive reviews [7,8]. Cell lines, like the human RPE cell line ARPE-19, are frequently used but differ considerably from a differentiated RPE cell in terms of morphology, barrier function, or gene expression [7,9,10,11]. There are published protocols that can induce a more RPE-like differentiation of ARPE-19 cells (e.g., [12] or [13]), but mostly, ARPE-19 cell culture protocols are implemented for fast results. Furthermore, these transformed, immortal cells have been passaged for years and have changed their characteristics [14,15]. Many researchers now rely on induced pluripotent stem cell (iPSC)-derived RPE cells from human donors. While these have the great advantage of being human, an iPSC-derived RPE cell also has its difficulties. Cultures of RPE derived from iPSC show subpopulations with different levels of maturity and gene expression with high variability, even within a single culture, including the population of stem cell-like progenitors and cells specialized for different RPE functions in the same culture or cultures from the same donor displaying either fetal or adult characteristics [16,17,18,19]. In addition, iPSC-derived cells may contain epigenetic modifications that are not found in natural RPE cells [20]. However, in spite of these challenges, iPSC-derived stem cells also have advantages compared to porcine cell models (see below), not only because of their human origin but also because of the possibility to use harvested patient material, giving the opportunity to directly investigate the impact of, e.g., genetic variants of patients on cell behavior and pathology development [21,22]. Furthermore, they can be developed for RPE transplantation [17,23]. Both are features that cannot be modeled by porcine cells. Mice are also widely used in retinal research. However, the use of mice to study diseases like AMD has severe disadvantages. Mice are nocturnal animals with very small eyes. Mice have very low visual acuity with no area of high(er) acuity vision, rely mainly on rods, express cones not found in humans (ultraviolet and double cones), and show features, such as an inverted rod chromatin structure, that are not found in humans [7]. Furthermore, their RPE is rather different from the human situation, with a high percentage of multi-nucleated RPE cells and different claudins used in their tight junctions [24,25]. In contrast, pigs are diurnal animals with eyes of similar size and anatomic features resembling the human eye; their eyes rely highly on cone vision with an area of higher visual acuity found in the retina (area centralis). Their cones resemble human blue and red/green cones (the division between red and green has only happened in primates), and they show the same degree of multi-nucleus characteristics and the same claudins in tight junctions as humans [7,24,25,26,27]. Despite the relevance of the porcine RPE as a model for human diseases, there are limitations to the use of this model. While the pig resembles the human situation much more closely than the mouse, it is still not identical to the human situation [7,27]. As pointed out above, porcine material cannot be used for transplantation. Furthermore, pigs, like all non-primate mammals, do not contain a macula, and SNPs related to AMD development are not described in the pig. Another limitation of using the pig as a model is the limited availability of analytics, such as primers or antibodies, which are readily available for mice and humans. Furthermore, creating transgenic animals, while possible in the pig [28,29], is cheaper and more easily performed in the mouse. Finally, housing conditions are much more expensive for pigs compared to mice.

However, the availability of porcine eyes as waste products makes them easily available without the need to house, breed, and kill animals purely for scientific purposes. Their use is, therefore, an active contribution to the 3R principle. Indeed, porcine cells are frequently used, and several protocols are available. In all published protocols, however, RPE cells of different eyes are usually mixed (e.g., [30,31,32,33,34]), so that cultures contain cells of different genetic origins, which implies different responses in one cell culture.

This study aims to generate a standardized and optimized operation protocol for the preparation of porcine RPE single-eye cultures. Here, the RPE cells from one pig eye are seeded into one well of a twelve-well polystyrene plate. This is in contrast to the usual protocols in which RPE cells from several eyes are mixed before seeding. The conventional protocols create genetically heterogeneous cultures where certain effects may be confounded by the heterogeneity of the cells. With single-eye cultures, each culture (or well of a plate) is genetically homogeneous, resembling an individual. At the same time, the cultures are genetically heterogeneous between the separate cultures (or wells). This setting will potentially generate scientific data more relevant to the real-life situation, as it resembles the human situation much closer. Also, with the use of porcine eyes from waste material in the food industry, animal experiments can be avoided, contributing to the 3R principle [30]. Furthermore, porcine material is far closer to the human eyes compared to the widely used mouse eye models, as described above. In addition, porcine RPE can be prepared after short post-mortem times (PMT), does not need characterization, and is extremely economical compared to human or stem-cell-derived RPE [31].

A new single-eye porcine RPE cell model with potentially higher biological relevance would, therefore, be a cost-effective and reproducible model that could be globally applied in research targeting RPE-based diseases, such as AMD. This model can be used to address specific classical pathology-related aspects, such as oxidative stress, inflammation, or proangiogenic signaling [3,8], but also lesser investigated pathways, such as RPE epithelial–mesenchymal transformation in subretinal fibrosis [32] or autophagy [33]. In addition, the homogenous genetic background of the RPE cell culture renders these cells highly suitable for proteomic or gene expression studies in pathogenic AMD pathways [34]. In addition, the data we present can be used as a basis for establishing quality control in porcine cell culture, especially considering cell morphology and morphometry.

Parameters that are important for the efficiency, differentiation, and expression of the RPE are PMT, plate coating substances, and the serum level of the medium. The outcome parameters of this study are cell number, confluency, morphology, differentiation, protein, and gene expression, as well as VEGF secretion. In addition, basic morphometric data were established for porcine RPE cells. For these parameters, a standard basic protocol for optimized preparation and cultivation was generated and is provided in this study. The overall experimental set up is depicted in Figure 1.

Figure 1. Flowchart of the experimental set up. The aim of this study is to establish a basic protocol for the preparation of porcine retinal pigment epithelial cells (RPE) as a single-eye culture model. The RPE cells of one eye are seeded into one well of a twelve-well plate. The preparation parameters post-mortem time, coating of the well plates, and serum content are tested and optimized. The results are analyzed with regard to cell numbers, confluence, morphology/morphometry, and the secretion and expression of various RPE markers.

2. Materials and Methods

2.1. Cell Culture

All RPE cultures were prepared from freshly slaughtered pigs’ eyes at 2 to 6 h PMT. All pigs were examined by veterinarians and were used for food production; therefore, they can be considered disease free. Pigs from Deutsche Landrasse race, mixed sex, and ages of 5 to 6 months were used.

The use of these eyes for experimental purposes was conducted in agreement with the animal welfare officer of the University of Kiel. According to the German Animal Welfare Act (TierSchG, https://www.gesetze-im-internet.de/tierschg (accessed on 8 February 2025). This is not considered to be animal research, but an alternative to the use of animals in research. The overall method is based on the preparation of RPE from mixed cultures [35] and was adapted to seed RPE cells from one eye into one well of a twelve-well polystyrene plate for adherent cells (Sarstedt, Nümbrecht, Germany; #83.3921) with a growth area of 3.65 cm² and a culture medium volume of 1 mL. A detailed protocol can be found in the Appendix B. All RPE cells harvested from one pig eye were seeded into one well of one twelve-well plate. The time difference between the first and the last eye of the 12-eye batch was ten minutes.

In brief, adjacent tissue was removed and the eyes were disinfected with betaisodona (Mundipharma, Germany; Frankfurt am Main, Germany; #04923204) and washed with 0.9% NaCl (Fresenius Kabi, Bad Homburg, Germany; #04801702), both on ice. The cornea, iris, ciliary body, and vitreous body were removed under aseptic conditions. The retina was detached in sterile Dulbecco’s phosphate-buffered saline without Ca²⁺ and Mg²⁺ (DPBS, Pan-Biotech, Aidenbach, Germany; #P04-53500) plus 1% penicillin/streptomycin (Pe/St, Sigma-Aldrich, St. Louis, MO, USA; #P0781). RPE cells were incubated for 10 min with 0.25% trypsin (Pan-Biotech; #P10-021100), followed by a 35 min incubation in 0.25% trypsin/0.02% ethylenediaminetetraacetic acid (EDTA, Pan-Biotech; #P10-020100). Cells from one eye were collected in a 15 mL tube (Sarstedt; #62.554.502), filled with 5 mL standard RPE media. Standard RPE media consists of Dulbecco’s Modified Eagle Medium with high glucose, L-glutamine, and phenol red (DMEM, Gibco™, Thermo Fisher Scientific, Waltham, MA, USA; #41965062), supplemented with 1% non-essential amino acids (Pan-Biotech; #P08-32100), 11 mM sodium pyruvate (Pan-Biotech; #P04-43100), 1% Pe/St, and 10% fetal bovine serum (FBS Mexican origin, Gibco™, Thermo Fisher Scientific; #10437028, Lot: 2405703RP). For serum testing assays, three days after preparation, the concentration of serum was changed from 10% to 5% or 1%, respectively. Cells were washed twice in 5 mL media (centrifugation), and the pellet was suspended in 1 mL media and seeded into one well of a twelve-well plate. The cells were incubated at 37 °C and 5% CO₂ in a humidified incubator. The protocol for the preparation process is provided (Appendix B).

To assess barrier function, single-eye RPE were seeded on twelve-well plates with uncoated transwell inserts (Sarstedt; #83.3931.041) and cultured for 28 days in media containing 10% FBS. Transepithelial electrical resistance (TEER) was measured with an EVOM3 Epithelial Volt/Ohm (TEER) Meter (World Precision Instruments Germany GmbH, Friedberg, Germany) after 7, 14, and 28 days.

As season may influence the quality of the eyes, we present the months in which the specific experiments were conducted. Tests regarding the qualities of the eyes and post-mortem times were conducted from May to November 2022, whereas actin evaluation samples were prepared from July to October. Coating tests with collagens were conducted from November to December 2022. Other coatings were tested from January to June 2023, whereas samples for tight junction assessment were prepared from March to September 2023. Serum content tests were conducted from May to September 2023, whereas samples for tight junction staining were prepared from August to September 2023.

2.2. Coating

Different coatings were applied on individual wells of twelve-well plates. Collagen Type I in monomeric (CIm) or fibrillar (CIf) form (Collagen A, 1 mg/mL, Pan Biotech, Aidenbach, Germany; #P06-20030), collagen Type IV (CIV, Advanced Biomatrix, Inc., Carlsbad, CA, USA; #5022), laminin (La, Sigma-Aldrich; #11243217001), fibronectin (Fn, Advanced Biomatrix; #5080), or Poly-d-Lysine (PDL, Sigma-Aldrich; #P7886) were used. All substances were applied as described in the manufacturers’ instructions with recommended concentrations (CIm/CIf—1 mg/mL or 100 µg/cm², CIV—100 µg/mL or 10 µg/cm², La—20 µg/mL or 2 µg/cm², Fn—50 µg/mL or 5 µg/cm², PDL—100 µg/mL or 10 µg/cm²) and a well volume of 365 µL (100 µL/cm² well).

2.3. Trypan Blue Exclusion Assay and Light Microscopy

Cells from one eye or one well, respectively, were counted before seeding (one eye) and after specific culture times (one well). For this, adherent cells were detached with trypsin/EDTA incubation; a 20 µL cell suspension was collected, mixed with 20 µL 0.4% trypan blue solution (Sigma-Aldrich; #T8154), and incubated for 3 min. The mixture was applied to a Neubauer counting chamber (Carl Roth GmbH + Co. KG, Karlsruhe, Germany; #T729.1). Cells were counted in a bright field using an Axiovert 160 (Carl Zeiss AG, Oberkochen, Germany) microscope with a 10× objective. The same microscope was used to take pictures of the cells to assess the morphology after 7, 14, and 28 days of cultivation. The photos were taken at distinct coordinates at the center, the middle ring, and the outer border of the wells using Axiovision Release 4.8.0.0 (Carl Zeiss AG). The percentage of epithelial, mesenchymal, and undivided cells, as well as gaps, were determined by a researcher. Also, confluence in percent 7 days after seeding as well as the day of 95–100% confluence was determined.

2.4. Enzyme-Linked Immunosorbent Assay

Secreted VEGF-A was determined with ELISA. For this, supernatants of single-eye culture RPE were collected for 4 h, centrifuged, and stored at −20 °C. These samples were assessed in Human VEGF DuoSet ELISA (R&D systems, Minneapolis, MN, USA; #DY293B) as described by the manufacturer.

2.5. Fluorescence Imaging—Tight Junctions and Cell Parameters

The labeling of actin filaments, cell nuclei, and tight junctions (zonula occludens 1, ZO-1) was performed on cells seeded on coverslips (Th. Geyer GmbH & Co. KG, Renningen, Germany; #CB00180RA1). Cells were fixated in 3% para-formaldehyde (Carl Roth GmbH + Co. KG; #0335.1) and permeabilized with 0.1% Triton-X (Carl Roth GmbH + Co. KG; #3051.2). Cells were blocked with 3% BSA diluted in PBS for one hour (Thermo Fisher Scientific; #37525). After blocking, cells were incubated for one hour with rabbit ZO-1 polyclonal antibody (Thermo Fisher Scientific; #61-7300, diluted 1:50), followed by a one-hour incubation with a secondary antibody mixture containing Hoechst (Sigma-Aldrich; #14533, diluted 1:500), Phalloidin-Atto488 (Sigma-Aldrich; #49409, diluted 1:50), and Alexa Fluor 555 goat anti-rabbit (Thermo Fisher Scientific; #A32732, diluted 1:1000). Coverslips were mounted with Fluoromount-G (Thermo Fisher Scientific; #00-4958-02) on microscope slides (Th. Geyer GmbH & Co. KG; #42406010). Fluorescence images were taken with a fluorescence microscope Imager.M2 (Carl Zeiss AG) with a 20× objective. At least three pictures from different positions were taken per coverslip. The quality of the actin filaments and tight junctions for each set of experiments were assessed by a single researcher, blinded to the conditions. For actin experiments, cell nuclei were evaluated using Fiji (Image J2). The ZO-1 signal as well as cell nuclei were evaluated with CellProfiler Software, version 4.2.8. (Broad Institute of MIT and Harvard). These data were used to determine cell parameters (cell and nuclei number, cell area, cell perimeter, eccentricity, form factor, and cell radius).

2.6. Quantitative Polymerase Chain Reaction

RNA was isolated after 28 days of cultivation in different conditions (PDL or no coating, respectively, combined with 10% or 5% serum, respectively) with a NucleoSpin RNA Mini Kit and digested with DNase (Macherey-Nagel, Düren, Germany; #740955), according to manufacturer’s instructions. RNA quality and concentration were determined with NanoDrop™ One (Thermo Fisher Scientific; #ND-ONE-W). cDNA was generated with the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific; #4368814) as described in the manual. Real-time qPCR was performed with TaqMan™ gene expression assays (Thermo Fisher Scientific; #4351372) and TaqMan™ Fast Advanced Master Mix (Thermo Fisher Scientific; #4444557) as described in the manual for the master mix. Also, self-designed gene arrays for RPE-relevant porcine genes were applied containing 96 different gene targets with dye label 5(6)-carboxyfluorescein-minor groove binder (FAM-MGB) [36]. Endogenous controls ACTG, GAPDH, and GUSB were used for normalization. 18s rRNA was used as manufacturing control by Thermo Fisher Scientific. The design of the gene array is depicted in Table 1. For the calculation of relative gene expression, the ΔΔCT method was used [37]. ΔCT [=CT (gene of interest) − CT (housekeeping gene)], ΔΔCT [=ΔCT (treated sample) − ΔCT (untreated sample)] and the relative fold gene expression level RQ (=2 ΔΔCT) were assessed with Thermo Fisher Connect (RQ module, relative quantification).

Table 1. Gene array design. Gene targets with well position and gene expression ID are shown, including the name of the encoded gene product. Underlined targets were used as endogenous controls.

Well	Target	Product	Assay ID
A1	18s rRNA	18s rRNA (Human)	Hs_99999901_s1
A2	ABCA4	ATP-Binding Cassette Subfamily A Member 4	Ss06884373_m1
A3	ACTG	Actin, Cytoplasmic 2	Ss03376081_u1
A4	ADRB2	Adrenoceptor Beta 2	Ss03818941_s1
A5	ANGPT2	Angiopoietin 2	Ss03392362_m1
A6	ANGPTL2	Angiopoietin-Like 2	Ss03389615_m1
A7	ANXA5	Annexin A5	Ss06880508_m1
A8	APOE	Apolipoprotein E	Ss03394681_m1
A9	BDNF	Brain-Derived Neurotrophic Factor	Ss03822335_s1
A10	BEST1	Bestrophin 1	Ss03376235_u1
A11	C2	Complement C2	Ss03389255_m1
A12	C3	Complement C3	Ss03391255_m1
B1	C5	Complement C5	Ss03391586_m1
B2	C9	Complement C9	Ss03388866_m1
B3	CASP1	Caspase 1	Ss03394224_m1
B4	CAT	Catalase	Ss04323025_m1
B5	CCL2	C-C Motif Chemokine Ligand 2	Ss03394377_m1
B6	CCL5	C-C Motif Chemokine Ligand 5	Ss03648939_m1
B7	CD46	CD46 Molecule	Ss03392461_u1
B8	CD55	CD55 Molecule (Cromer Blood Group)	Ss03392383_m1
B9	CD59	CD59 Glycoprotein (Protectin)	Ss03394252_m1
B10	CFB	Complement Factor B	Ss03389385_g1
B11	CFH	Complement Factor H	Ss03391439_m1
B12	CFI	Complement Factor I	Ss06935384_m1
C1	COL14A1	Collagen Type XIV Alpha 1 Chain	Ss06865093_m1
C2	CRP	C-Reactive Protein	Ss03390889_m1
C3	CRYAA (CRYA1)	Crystallin Alpha A	Ss06837084_m1
C4	CRYAB	Alpha-Crystallin B Chain	Ss06921086_m1
C5	CSF2	Colony-Stimulating Factor 2	Ss03394096_m1
C6	CST3	Cystatin C	Ss03388477_m1
C7	CTSD	Cathepsin D	Ss03379762_u1
C8	CX3CR1	CX3C Chemokine Receptor 1	Ss06883230_m1
C9	CXCL10	C-X-C Motif Chemokine Ligand 10	Ss03391846_m1
C10	CXCL12	C-X-C Motif Chemokine Ligand 12	Ss03391855_m1
C11	DICER1	Dicer 1, Ribonuclease III	Ss04248150_m1
C12	ELN	Elastin	Ss04955056_m1
D1	FASLG	Fas Ligand	Ss03381579_u1
D2	FLT1	Fms-Related Tyrosine Kinase 1 (VEGF-R1)	Ss03375679_u1
D3	FMO1	Flavin-Containing Dimethylaniline Monoxygenase 1	Ss03393883_u1
D4	FN1	Fibronectin 1	Ss03373673_m1
D5	FST	Follistatin	Ss03378467_u1
D6	GAPDH	Glyceraldehyde-3-Phosphate Dehydrogenase	Ss03375629_u1
D7	GFAP	Glial Fibrillary Acidic Protein	Ss03373547_m1
D8	GPX4	Glutathion Peroxidase 4	Ss03384646_u1
D9	GSS	Glutathione Synthetase	Ss04328106_m1
D10	GUSB	Glucuronidase Beta	Ss03387751_u1
D11	HIF1A	Hypoxia-Inducible Factor 1 Subunit Alpha	Ss03390447_m1
D12	HMOX1	Heme Oxygenase 1	Ss03378516_u1
E1	HTRA1	HtrA Serine Peptidase 1	Ss06876775_m1
E2	ICAM-1	Intercellular Adhesion Molecule 1	Ss03392385_m1
E3	IGF1	Insulin-Like Growth Factor 1	Ss03394499_m1
E4	IL1B	Interleukin 1 Beta	Ss03393804_m1
E5	IL1R2	Interleukin 1 Receptor Type 2	Ss04324011_m1
E6	IL6	Interleukin 6	Ss07308316_g1
E7	Il6R	Interleukin 6 Receptor	Ss03394904_g1
E8	IL8 (=CXCL8)	Interleukin-8 (CXCL-8)	Ss03392437_m1
E9	KDR	Kinase Insert Domain Receptor (VEGF-R2)	Ss03375683_u1
E10	KIT	KIT Proto-Oncogene, Receptor Tyrosine Kinase	Ss03380145_u1
E11	LEP	Leptin	Ss03392404_m1
E12	LIPC	Lipase C	Ss03820991_s1
F1	LPL	Lipoprotein Lipase	Ss03394608_m1
F2	MAPK1	Mitogen-Activated Protein Kinase 1 (ERK2)	Ss04248225_m1
F3	MAPK14	Mitogen-Activated Protein Kinase 14 (p38)	Ss06880885_m1
F4	MMP2	Matrix Metallopeptidase 2	Ss03394318_m1
F5	MMP9	Matrix Metallopeptidase 9	Ss03392100_m1
F6	MTOR	Mechanistic Target Of Rapamycin Kinase	Ss03377427_u1
F7	NFE2L2	Nuclear Factor, Erythroid 2 Like 2 (NRF2)	Ss06886076_m1
F8	NKAP	NFKB-Activating Protein	Ss04322419_m1
F9	NOS1	Nitric Oxide Synthase 1	Ss06838170_m1
F10	NOS2	Nitric Oxide Synthase 2 (iNOS)	Ss03374608_u1
F11	PLA2G2D	Phospholipase A2 Group IID	Ss04953157_m1
F12	PTGS1	Prostaglandin–Endoperoxide Synthase 1	Ss03373347_m1
G1	PTGS2	Prostaglandin–Endoperoxide Synthase 2 (COX2)	Ss03394694_m1
G2	RDH11	Retinol Dehydrogenase 11	Ss06878003_m1
G3	RLBP1	Retinaldehyde-binding protein 1	Ss06905574_m1
G4	RPE65	Retinoid Isomerohydrolase RPE65	Ss06891920_m1
G5	SCARB1	Scavenger Receptor Class B Member 1	Ss03391104_m1
G6	SERPINF1	Serpin Family F Member 1	Ss03385090_u1
G7	SERPING1	Serpin Family G Member 1	Ss03387977_u1
G8	SOD1	Superoxide Dismutase 1	Ss03375614_u1
G9	SOD2	Superoxide Dismutase 2	Ss03374828_m1
G10	SPARC	Secreted Protein Acidic And Cysteine Rich	Ss03392006_m1
G11	TF	Transferrin	Ss03374732_m1
G12	TGFB1	Transforming Growth Factor Beta 1	Ss04955543_m1
H1	TIMP1	TIMP Metallopeptidase Inhibitor 1	Ss03381944_u1
H2	TIMP-3	TIMP Metallopeptidase Inhibitor 3	Ss03375447_u1
H3	TLR2	Toll-Like Receptor 2	Ss03381278_u1
H4	TLR3	Toll-Like Receptor 3	Ss03388862_m1
H5	TLR4	Toll-Like Receptor 4	Ss04956023_s1
H6	TNF	Tumor Necrosis Factor	Ss03391318_g1
H7	TYR	Tyrosinase	Ss03379283_u1
H8	VCAM1	Vascular Cell Adhesion Molecule 1	Ss03390912_m1
H9	VEGFA	Vascular Endothelial Growth Factor A	Ss03393990_m1
H10	VIM	Vimentin	Ss04330801_gH
H11	VLDLR	Very Low-Density Lipoprotein Receptor	Ss03374049_m1
H12	VWF	Von Willebrand Factor	Ss04322692_m1

2.7. Protein Expression

Western blotting was performed to assess claudin-19 (CLDN19) and retinal pigment epithelium-specific 65 kDa protein (RPE65) expression. After 2 or 4 weeks of cultivation, cells were washed with PBS and lysed with Nonidet P40 solution (NP40) containing 1% Nonidet P40 Substitute (Sigma-Aldrich; #11332473001), 150 mM NaCl (Carl Roth GmbH + Co. KG; #3957.1), and 50 mM Tris (Sigma-Aldrich; #T1503, pH8) and supplemented with phosphatase inhibitor cocktail 1 and 2 (Sigma-Aldrich; #P2850, #P5726) as well as protease inhibitor cocktail (Sigma-Aldrich; #P8340) for 45 min. Protein content was assessed with DC Protein Assay (Bio-Rad Laboratories, Munich, Germany; #5000111), according to the manufacturer’s instructions. SDS-PAGE and Western blotting were conducted as previously described, with modifications [38]. A 12% acrylamide gel was used. PageRuler™ Plus Prestained Protein Ladder (Thermo Fisher Scientific; #26620), with a range of 10 to 250 kDa, was applied. Membranes were blocked with 4% skimmed milk (Carl Roth GmbH + Co. KG; #T145.2) in Tris-buffered saline with 0.05% Tween (TBST, Merck KGaA, Darmstadt, Germany; #8221840) for one hour. Membranes were incubated with primary antibodies overnight, rocking at 4 °C (mouse anti-RPE65, 65 kDa, 1:2000 in 2% skimmed milk, Novus Biologicals, Minneapolis, MN, USA; #NB100-355; rabbit anti-CLDN19, 23 kDa, 1:1000 2% skimmed milk, Absource Diagnostics GmbH, München, Germany; #00219; rabbit anti-β-actin, 37 kDa, 1:1000 in 2% skimmed milk, Cell Signaling Technologies, Denver, CO, USA, #4967). After several washing steps with TBST, secondary antibody conjugates were applied for one hour (horseradish peroxidase-conjugates, HRP, anti-mouse-HRP (Cell Signaling Technologies; #7076), anti-rabbit-HRP (Cell Signaling Technologies; #7074), both 1:1000 in 2% skimmed milk). For the chemiluminescence signal, Clarity Western ECL Substrate (Bio-Rad Laboratories; #170-5061) was applied for 5 min. The signal was detected with a ChemiDoc MP Imaging System (Bio-Rad Laboratories). Band volumes were normalized with β-actin expression and evaluated with Image Lab 6.1.0 build 7 software (Bio-Rad Laboratories).

2.8. Statistical Analysis

All experiments were independently conducted at least three times. Data management, diagrams, and calculations were carried out using Microsoft Excel (Excel 2010, Microsoft, Redmond, WA, USA). qPCR data were evaluated with the cloud software Thermo Fisher Connect. Statistical analysis was performed with GraphPad Prism 9 (GraphPad Software, Inc., San Diego, CA, USA, 2021) and Thermo Fisher Connect for the PCR data. Parametric distribution was assessed with the Shapiro–Wilk test. The significance of parametric data was determined with ANOVA (analysis of variance) and Student’s t-test; the significance of non-parametric data was determined with the Kruskal–Wallis and Mann–Whitney tests. The error bars of the column charts show the standard deviation. The bars of the box plots range from lowest (minimum) to highest values (maximum). For transparency regarding the range of data, an overview of parametric data with the mean, standard deviation, minimum, and maximum is listed in Table A3 (Appendix A). Also, individual data points of parametric data are shown. PCR data were evaluated with Student’s t-test calculated with Thermo Fisher Connect. Data were considered significant with p-values < 0.05. The number of experiments n represents the number of individual eyes used for the experiments.

3. Results

3.1. Quality of the Eyes and Post-Mortem Time

3.1.1. Qualities of the Eyes

In order to assess the barrier function of single-eye cultures, RPE cells were seeded on 12-transwell plates, and TEER was measured after 7, 14, and 28 days of cultivation (Figure 2). All cells were seeded on uncoated wells and fed with media containing 10% serum. Cells reached a TEER of 228 ± 110 Ω*cm², 315 ± 116 Ω*cm², and 497 ± 315 Ω*cm² after 7, 14, and 28 days, respectively. These high TEER values confirm that the single-eye cultures form a strong barrier. Further assays of this study were conducted with twelve-well plates without transwell inserts.

Figure 2. Barrier measurement of porcine retinal pigment epithelium single-eye cultures. Single-eye porcine cultures were seeded on 12-transwell plates and cultured for 28 days. Transepithelial electrical resistance (TEER) in Ω*cm² was measured after 7, 14, and 28 days. n = 24.

Freshly slaughtered pigs’ eyes can be of different qualities when they reach the laboratory. Their cornea and lens can be bleared or clear, depending on the time the pigs were scalded before the eyes were removed, which may denature the proteins of the cornea and the lens as seen in bleared eyes. Data for testing post-mortem times (PMTs) were collected and summarized for all PMTs (2, 4, and 6 h) and evaluated concerning bleared and clear eyes (Figure 3 and Figure 4). Eyes with light-reflecting effects or a blue iris were not used for preparation as they contained fewer (pigmented) RPE cells. Data were evaluated for cell number after 7, 14, and 28 days (Figure 3A), number of days to reach 95–100% confluency (Figure 3B), confluency in % on day 7 of cultivation (Figure 3C), and success rates for survival and confluence (Figure 3D). Clear eyes reached a cell number of 7.91 ± 3.18 cells × 10⁵/mL, 8.37 ± 3.83 cells × 10⁵/mL, and 8.70 ± 3.78 cells × 10⁵/mL after 7, 14, and 28 days, respectively. Bleared eyes reached a cell number of 7.96 ± 3.33 cells × 10⁵/mL, 8.58 ± 3.83 cells × 10⁵/mL, and 9.54 ± 3.02 cells × 10⁵/mL after 7, 14, and 28 days, respectively. The day of confluence for clear eyes was day 8.00 ± 3.00, and for bleared eyes, it was day 10.00 ± 3.00. Confluence on day 7 was 95.00% ± 40.00% for clear eyes and 82.50% ± 50.00% for bleared eyes. Clear eyes showed a death rate of 25.26% and a confluence rate of 60.00%, and bleared eyes showed a death rate of 24.11% and a confluence rate of 56.74%. Overall, no significant differences were found, but the values concerning confluency were better with clear eyes.

Figure 3. Influence of the qualities of porcine eyes on different parameters. Data to test post-mortem times were collected and evaluated regarding the quality of the porcine eyes divided into clear and bleared eyes. They were evaluated for cell number ((A), n = 13–165), day when they reach 95–100% confluence ((B), n = 81–113), confluence in % on day 7 ((C), n = 94–126) of cultivation, and rates for dead and confluence cultures ((D), n = 34–190). Data for (A) were parametric; mean and standard deviation are depicted. Significances were calculated between each group with analysis of variance (ANOVA) and Student’s t-test. Data for (B,C) was not parametric; median and interquartile ranges are depicted. Significances were calculated between each group with the Kruskal–Wallis test and Mann–Whitney tests. No significant differences were found. Data for (D) are descriptively shown as stacked bars with a percentage ratio. d = days.

Figure 4. Influence of the qualities of porcine eyes on different culture parameters. Single-eye retinal pigment epithelium cells were seeded for seven ((A), n = 126–129), 14 ((B), n = 94–95), and 28 days ((C), n = 50–53). A portion of epithelial, mesenchymal, undivided cells, and ungrown areas of clear or cleared eyes was determined with light microscopy after incubation. Data were not parametric; median and interquartile ranges are depicted. Significances were calculated between each group with the Kruskal–Wallis test and the Mann–Whitney test. No significances were detected.

Also, cell morphology was determined after 7 (Figure 4A), 14 (Figure 4B), and 28 days (Figure 4C) of cultivation. Light microscopy images of different areas of the well were visually analyzed for the proportion of epithelial, mesenchymal, and undivided cells, as well as ungrown areas, all as % portions of 100% well area. Data were again very similar between qualities (please refer to Table A1 in Appendix A). Regarding the entire data set, the % of undivided cells between day 7, day 14, and day 28 remained the same at 3.33% at all time points. Ungrown areas were filled up over time, decreasing from 12.50% to 0.00%. These areas were filled up both with mesenchymal cells (from 6.67% to 11.67% and 13.33%, respectively) and epithelial cells (from 63.33% to 72.50% and 73.33%, respectively).

All data were very similar and showed no significant or relevant differences. For the following assays, it was assumed that these qualities (clear, bleared) would not confound the results.

3.1.2. Post-Mortem Time

Single-eye RPE cells were prepared after a PMT of 2, 4, and 6 h and cultivated for 7, 14, and 28 days. Cell number after respective cultivation time (Figure 5A), the number of days until reaching 95–100% confluence (Figure 5B), confluence area on day 7 of cultivation (Figure 5C), and overall survival and confluence success rates were determined (Figure 5D).

Figure 5. Influence of post-mortem time on different culture parameters. Single-eye retinal pigment epithelium cells were prepared after 2, 4, and 6 h post-mortem time and seeded for 7, 14, and 28 days. After different cultivation times, cells were counted ((A), n = 11–112), the number of days until reaching 95–100% confluence was determined ((B), n = 56–91), a confluence area on day 7 of cultivation was estimated ((C), n = 66–94), and the overall survival and confluence success rates were determined ((D), n = 108–143). Data for (A) were parametric; mean and standard deviation are depicted. Significances were calculated between each group with analysis of variance (ANOVA) and Student’s t-test. Data for (B,C) were not parametric; median and interquartile ranges are depicted. Significances were calculated between each group with the Kruskal–Wallis test and Mann–Whitney tests. Data for (D) are descriptively shown as stacked bars with a percentage ratio. * p < 0.05, ** p < 0.01, h = hours, d = days.

Regarding cell numbers, data for all PMT were summarized and descriptively evaluated concerning seeding cell number and success of the culture (Table 2). A living and successful culture was defined as containing cells that were quantifiable in a trypan blue exclusion assay (showing a living cell number of at least 0.05 × 10⁵ cells/mL) over the course of 28 days of cultivation. Overall, the mean and median cell seeding numbers between successful (living) and unsuccessful (dead) cultures were not relevantly different. However, a seeding cell number of at least 1 × 10⁵ cells/mL increased the success rate, as in dead cultures, cell number was more often below 1 × 10⁵ cells/mL than in succeeding cultures (61.70% compared to 28.39% for living cultures).

Table 2. Descriptive analyses of summarized cell seeding number data. Numbers are depicted as ×10⁵ cells/mL. Min = minimum; Max = maximum.

	Total	Living	Dead
Mean	3.55	3.80	2.28
Deviation	7.16	6.91	8.25
Median	1.65	2.00	0.75
Range	72.80	71.75	56.45
Min	0.00	0.05	0.00
Max	72.80	72.80	56.45
Count total (n)	283.00	236.00	47.00
% <1.00	33.92%	28.39%	61.70%
% >1.00	66.08%	71.61%	38.30%

Concerning the influence of PMT, seeding cell number after 2 h PMT showed high variation. After 7 days of cultivation, 4 h PMT showed the highest cell number (mean: 9.85 ± 2.69 cells × 10⁵/mL), which was significantly higher than 2 h PMT (mean: 6.81 ± 3.10 cells × 10⁵/mL, p = 0.002) or 6 h PMT (mean: 7.19 ± 3.17 cells × 10⁵/mL, p = 0.003). The data after 14 days of cultivation were not significantly different. After 28 days of cultivation, both 4 h PMT (mean: 9.30 ± 2.81 cells × 10⁵/mL, p = 0.024) and 6 h PMT (mean: 10.45 ± 4.20 cells × 10⁵/mL, p = 0.014) exhibit significantly higher cell numbers than 2 h PMT (mean: 6.51 ± 2.78 cells × 10⁵/mL). Confluence data were similar between the groups, with no significant differences. Four and 2 h PMT were the fastest to reach full confluence (median: 8.00 ± 2.25 days) and median: 8.00 ± 3.00 days, respectively), while 6 h PMT needed more time (median: 10.00 ± 4.00 days). The seven-day confluence area was highest at 6 h PMT (median: 92.50% ± 36.25%) compared to 2 h PMT (median: 87.50% ± 38.75%) or 4 h PMT (median: 90.00% ± 60.00%). The rate of unsuccessful cultures was highest for 2 h PMT with 33.33% compared to 4 h PMT with 20.88% and 6 h PMT with 25.25%. Also, the confluence rate was highest at 4 h PMT with 63.19% compared to 2 h and 6 h PMT with 51.85% and 61.11%, respectively.

For morphology, cells were prepared after 2, 4, and 6 h PMT and cultured for 7 (Figure 6A), 14 (Figure 6B), and 28 days (Figure 6C). Light microscopy images were analyzed for the composition of epithelial, mesenchymal, and undivided cells as well as ungrown areas. Between day 7 and day 28, gaps were mainly filled up with mesenchymal cells. After 14 days of cultivation, ungrown areas displayed no differences (0.00% for each condition). After 7 days of cultivation, mesenchymal cells did not show significant differences between the groups. Four-hour PMT showed significantly more epithelial cells (median: 78.33% ± 37.50%) than two-hour PMT (median: 58.34% ± 33.76%, p < 0.001) or six-hour PMT (median: 63.33% ± 36.67%, p = 0.003), respectively. Six-hour PMT showed significantly more undivided cells (median: 6.67% ± 10.83%) than two-hour PMT (median: 3.33% ± 6.67%, p < 0.001) and four-hour PMT (median: 3.33% ± 6.67%, p < 0.001). Four-hour PMT showed significantly less ungrown area (median: 1.67% ± 26.67%) than two-hour PMT (median: 18.34% ± 33.34%, p < 0.001) or six-hour PMT (median: 8.33% ± 35.84%, p = 0.042). Taken together, 4 h PMT can be considered the optimal preparation starting time, as it leads to more epithelial cells and higher cell numbers after one week of cultivation.

Figure 6. Cell morphology after different post-mortem times. Single-eye retinal pigment epithelium cells were prepared after post-mortem times of 2, 4, and 6 h. The portion of epithelial, mesenchymal, undivided cells, and ungrown areas of each well was determined with light microscopy after 7 ((A), n = 68–159), 14 ((B), n = 46–83), and 28 days ((C), n = 22–40) of cultivation. Data were not parametric; median and interquartile ranges are depicted. Significances were calculated between each group with the Kruskal–Wallis test and Mann–Whitney tests. * p < 0.05, ** p < 0.01, *** p < 0.001, h = hours.

The morphology of actin filaments and cell nuclei was also determined after different PMTs. Cells were prepared after 2, 4, and 6 h PMT and seeded on monomeric collagen I-coated glass slides. Of note, this coating was used as a standard procedure before knowing the results of the coating tests that followed. After 28 days of cultivation, cell nuclei and actin filaments were stained, and images were taken with fluorescence microscopy. Visual ranking of the quality of the actin cytoskeleton was performed from 0/disturbed (stress fibers and cell gaps) to 5/physiological (proper/circumferential actin) morphology (Figure 7A) by a single observer, which was blinded to the post-mortem time of the cells. Images were evaluated by Fiji (ImageJ2) regarding the number of cell nuclei (Figure 7B), cell nuclei size (Figure 7C), and form factor of cell nuclei (Figure 7D). Example photos for physiological and disturbed actin structure are depicted in Figure 7E. Regarding cell nuclei size and form factor, no significant differences were detected. The quality of the actin cytoskeleton was significantly better after 2 h PMT (median: 4.00 ± 2.50) compared to 4 h (median: 3.50 ± 3.00, p = 0.017) and 6 h PMT (median: 3.00 ± 2.50, p = 0.016), resembling the physiological RPE situation more closely. Cell nuclei number was significantly higher after 2 h PMT (mean: 499.20 ± 161.08) compared to 4 h (mean: 333.02 ± 168.42, p < 0.001) or 6 h PMT (mean: 361.07 ± 114.30, p < 0.001). Regarding the actin cytoskeleton, a PMT of 2 h seems to be the optimal condition, which is in contrast to the findings of the former PMT assays. However, the overall ranking differences between 2 h and 4 h PMT are quite small, and only a few cultures survive from a PMT of 2 h, rendering 2 h PMT highly inefficient. Overall, a PMT of 4 h was chosen as the optimal condition regarding cell number, morphology, and success rate. In addition, it also is the most practicable time regarding delivery and lab working hours.

Figure 7. Morphology of actin cytoskeleton and cell nuclei. Single-eye retinal pigment epithelium cells were prepared after post-mortem times of 2, 4, and 6 h and seeded on monomeric collagen I-coated glass slides. After 28 days of cultivation, cell nuclei and actin filaments were stained, and images were taken by fluorescence microscopy. Visual ranking of the quality of the actin cytoskeleton was performed from 0 (disturbed) to 5 (proper) morphology ((A), n = 31–51). Images were evaluated by Fiji (ImageJ2) regarding number of cell nuclei ((B), n = 30–51), cell nuclei size ((C), n = 27–45), and form factor of cell nuclei ((D), n = 27–45). Data for (B) were parametric; mean and standard deviation are depicted. Significances were calculated between each group with analysis of variance (ANOVA) and Student’s t-test. Data for (A,C,D) were not parametric; median and interquartile ranges are depicted. Significances were calculated between each group with the Kruskal–Wallis test and the Mann–Whitney test. * p < 0.05, *** p < 0.001, h = hours. Exemplary photos are shown ((E), cell nuclei = blue, actin filaments = green, 20× objective, scale bar = 100 µm).

3.2. Coating

3.2.1. Cell Numbers on Different Coatings

Single-eye RPE cells were seeded on different coatings, and the cell numbers were determined. Differences between the cell number on the specific days to the initial seeding cell number were calculated (Figure 8).

Figure 8. Cell number on different coatings. Before seeding, cells were counted with a trypan blue exclusion assay. ((A), n = 7–17) Cells were seeded on monomeric collagen I (CIm), fibrillar collagen I (CIf), collagen IV (CIV), or uncoated wells (co). ((B), n = 8–16) Cells were seeded on CIm, Poly-d-Lysine (PDL), laminin (Lam), fibronectin (Fn), or co. Cells were counted again after 7, 14, and 28 days of cultivation, and the difference in the seeding cell number is depicted as cell number × 10⁴. The initial cell number mean before seeding is listed for collagen tests ((C), n = 7–17) and for coating tests ((D), n = 8–16). Data were parametric; mean and standard deviation are shown. Significances were calculated between each group with analysis of variance (ANOVA) and Student’s t-test. * p < 0.05, ** p < 0.01, d = days.

First, different collagens were tested (Figure 8A) with monomeric collagen I (CIm), fibrillar collagen I (CIf), collagen IV (CIV), or uncoated control (co). Numerically, the cell number was highest at all time points when no coating was used (co), followed by CIm. The co condition displayed significantly higher cell numbers than CIV on day 7 (mean: 63.69 ± 28.19 cells × 10⁴/mL compared to 32.03 ± 29.68 cells × 10⁴/mL, p = 0.006). In addition, after 14 days, co displayed significantly higher cell numbers than CIf (mean: 106.13 ± 37.36 cells × 10⁴/mL compared to 68.45 ± 31.44 cells × 10⁴/mL, p = 0.030).

The next step was to investigate the cell number with other coatings (Figure 8B): Poly-d-Lysine (PDL), laminin (Lam), fibronectin (Fn), co, and the most suitable collagen from the collagen testing (CIm). There were no significances or tendencies determined. Numerically, the highest cell number was achieved with PDL on day 7 and day 14, while on day 28, the cell number was highest with fibronectin, followed by PDL.

3.2.2. Confluence on Different Coatings

Single-eye RPE cells were seeded on different coatings and confluency (cell growth area in %) of the well after 7 days, and the day of 95–100% confluency was determined. First, different collagens (CIm, CIf, CIV) compared to the uncoated control (co) were tested. Confluency on day 7 (Figure 9A) was highest if no coating was used (mean: 85.62% ± 24.32%), and this was significantly higher than CIm (mean: 58.66% ± 25.00%, p < 0.001), CIf (mean: 39.85% ± 26.65%, p < 0.001), or CIV (mean: 48.09% ± 20.34%, p < 0.001). CIm displayed a significantly higher confluence on day 7 than CIf (p = 0.005). In addition, co reached confluency at the earliest timepoint (Figure 9B, median: day 8.00 ± 2.75), and this was significantly faster than CIm (median: day 13.00 ± 4.00, p < 0.001), CIf (median: day 21.00 ± 8.00, p = 0.004), or CIV (median: day 15.50 ± 7.70, p = 0.001), respectively.

Figure 9. Confluence on different coatings. ((A), n = 29–34; (B), n = 2–20) Single-eye retinal pigment epithelium cells were seeded on monomeric collagen I (CIm), fibrillar collagen I (CIf), collagen IV (CIV), or uncoated wells (co). ((C), n = 28–36; (D), n = 17–27) Cells were seeded on CIm, Poly-d-Lysine (PDL), laminin (Lam), fibronectin (Fn), or co. Confluence as a cell growth area in % of the well was determined with light microscopy after 7 days of cultivation (A,C), and the day of 95–100% confluency was determined (B,D). For (A), data were parametric; mean and standard deviation are shown. Significances were calculated between each group with analysis of variance (ANOVA) and Student’s t-test. For (B–D), data were not parametric; median and interquartile ranges are depicted. Significances were calculated between each group with the Kruskal–Wallis test and the Mann–Whitney test. * p < 0.05, ** p < 0.01, *** p < 0.001.

The next step was to investigate the confluence of the coatings’ PDL, Lam, and Fn, together with CIm and co. Regarding 7-day confluency (Figure 9C), PDL reached complete confluency (median: 100.00% ± 0.00%), which was significantly different to CIm (median: 85.00% ± 50.00%, p < 0.001), Lam (median: 100.00% ± 15.00%, p < 0.030), Fn (median: 92.50% ± 19.70%, p < 0.001), or co (median: 80.00% ± 60.00%, p < 0.001), respectively. Lam also exhibited full confluency on day 7, and this was significantly higher than CIm (p = 0.020) and co (p = 0.030). Concerning the day of full confluence (Figure 9D), PDL was the first to reach it (median: day 6.00 ± 3.00). This effect was significantly different compared to CIm (median: day 8.00 ± 4.00, p = 0.007), Fn (median: day 8.00 ± 3.00, p < 0.001), or co (median: day 7.00 ± 3.00, p < 0.002), respectively. The second best coating regarding the day of confluence was Lam (median: day 6.00 ± 1.25). This was significantly better than CIm (p = 0.030), Fn (p < 0.001) or co (p = 0.020), respectively. Overall, confluence on day 7 and time to reach full confluence was optimal with PDL.

3.2.3. Morphology on Different Coatings

Single-eye RPE cells were seeded on different coatings for 7, 14, and 28 days, and light microscopy images of different areas of the well were analyzed for composition of epithelial, mesenchymal, and undivided cells, as well as ungrown areas (% well area).

First, different collagens (CIm, CIf, CIV) were tested and compared to the uncoated control (co) (Figure 10A–C). After 7 days of cultivation (Figure 10A), co contained most epithelial cells (median: 80.00% ± 45%), which was significantly higher than CIf (median: 12.50% ± 58.80%, p < 0.001) or CIV (median: 40.00% ± 50.00%, p < 0.001). Also, CIm (median: 55.00% ± 60%) contained more epithelial cells than CIf (p = 0.004) or CIV (p = 0.040). Ungrown areas were the least likely to be found with co (median: 2.50% ± 32.50%). CIf (median: 65.00% ± 73.75%, p = 0.003) and CIV (median: 15.00% ± 57.50%, p = 0.030) showed significantly more gaps in the cell layer compared to co. After 14 days of cultivation (Figure 9B), again, co contained most epithelial cells (median: 92.50% ± 15.00%), which was significantly higher than CIf (median: 45.00% ± 47.50%, p < 0.001), CIV (median: 65.00% ± 65.00%, p < 0.001), or CIm (median: 80.00% ± 20.00%, p = 0.020). CIm had significantly more epithelial cells than CIf (p < 0.001). Accordingly, co had the fewest mesenchymal cells (median: 7.50% ± 18.80%), which was significantly lower than CIf (median: 47.50% ± 51.20%, p < 0.001), CIV (median: 30.00% ± 60.00%, p = 0.001), or CIm (median: 12.50% ± 23.75%, p = 0.040). CIm had significantly fewer mesenchymal cells than CIf (p < 0.001). After 28 days of cultivation (Figure 10C), all conditions reached full confluency (no ungrown areas, all medians: 0.00% ± 0.00%). Undivided cells were quite few, with CIf showing the most (CIm median: 0.00% ± 3.75%; CIf median: 5.00% ± 5.00%; CIV median: 0.00% ± 5.00%; co median: 0.00% ± 5.00%). Most epithelial and the least number of mesenchymal cells were achieved on uncoated wells (median: 90.00% ± 13.70% and 5.00% ± 13.80%, respectively), followed by CIm (median: 85.00% ± 33.80% and 15.00% ± 38.75%, respectively). Co condition showed significantly more epithelial cells than CIm (p = 0.001), CIf (median: 22.50% ± 61.30%, p < 0.001), or CIV (median: 70.00% ± 72.50%, p = 0.001). Also, CIm contained more epithelial cells than CIf (p < 0.001). Correspondingly, co contained significantly fewer mesenchymal cells than CIm (p = 0.030), CIf (median: 70.00% ± 73.70%, p < 0.001), or CIV (median: 30.00% ± 77.50%, p < 0.001). Additionally, CIm had significantly fewer mesenchymal cells than CIf (p = 0.002).

Figure 10. Cell morphology on different collagens and other coatings. Single-eye retinal pigment epithelium cells were seeded on monomeric collagen I (CIm), fibrillar collagen I (CIf), collagen IV (CIV), or uncoated wells (co) ((A), n = 21–24; (B), n = 21–24; (C), n = 21–24). After determining optimal collagen, coatings CIm, Poly-d-Lysine (PDL), laminin (Lam), fibronectin (Fn), and uncoated wells (co) were tested ((D), n = 21–33; (E), n = 21–33; (F), n = 21–33). The portion of epithelial, mesenchymal, undivided cells, and ungrown areas of each well was determined with light microscopy after 7 ((A) + (D)), 14 ((B) + (E)), and 28 days ((C) + (F)) of cultivation. Data were not parametric; median and interquartile ranges are shown. Significances were calculated between each group with the Kruskal–Wallis test and the Mann–Whitney test. * p < 0.05, ** p < 0.01, *** p < 0.001.

In addition, different coatings (PDL, Lam, Fn) were tested and compared to CIm and co (Figure 10D–F). After 7 days of cultivation (Figure 10D), CIm (median: 67.50% ± 60.00%) showed significantly fewer epithelial cells than PDL (median: 85.00% ± 35.00%, p = 0.004), Lam (median: 85.00% ± 20.00%, p < 0.001), or co (median: 80.00% ± 40.00%, p = 0.005). Fn (median: 65.00% ± 50.00%) also contained fewer epithelial cells than Lam (p = 0.009) or co (p = 0.040). CIm (median: 15.00% ± 25.00%) displayed significantly more mesenchymal cells than PDL (median: 5.00% ± 20.00%, p = 0.020), Lam (median: 5.00% ± 15.00%, p = 0.030), or co (median: 10.00% ± 20.00%, p = 0.049). Fn (median: 10.00% ± 45.00%) contained significantly more mesenchymal cells than PDL (p = 0.020), Lam (p = 0.040), or co (p = 0.049). After 14 days of cultivation (Figure 10E), PDL (median: 95.50% ± 12.50%) showed significantly more epithelial cells than CIm (median: 80.00% ± 42.50%, p < 0.001) or Fn (median: 80.00% ± 25.00%, p = 0.030). Lam (median: 90.00% ± 25.00%) contained more epithelial cells than CIm (p = 0.020). PDL (median: 0.00% ± 10.00%) had fewer mesenchymal cells than CIm (median: 15.00% ± 31.30%, p < 0.001), Fn (median: 15.00% ± 25.00%, p = 0.004), or co (median: 10.00% ± 30.00%, p = 0.007). Also, Lam (median: 0.00% ± 10.00%) had fewer mesenchymal cells than CIm (p = 0.007). After 28 days of cultivation (Figure 10F), all conditions reached full confluency. Undivided cells were almost undetectable and not significantly different for any tested condition. Most epithelial and the least number of mesenchymal cells were achieved on PDL-coated wells (median: 95.00% ± 10.00% and 0.00% ± 5.00%, respectively), followed by Lam-coated wells (median: 90.00% ± 20.00% and 0.00% ± 10.00%, respectively). PDL showed significantly more epithelial cells than CIm (median: 75.00% ± 48.80%, p < 0.001) and Fn (median: 80.00% ± 27.50%, p = 0.030). Also, Lam showed more epithelial cells than CIm (p = 0.02). The co condition was similar to Lam (mean: 90.00% ± 35.00%). In accordance, PDL displayed significantly fewer mesenchymal cells than CIm (mean: 10.00% ± 42.50%, p < 0.001), Fn (median: 10.00% ± 25.00%, p = 0.007), or co (median: 5.00% ± 35.00%, p = 0.010). Additionally, Lam had significantly fewer mesenchymal cells than CIm (p = 0.020). Overall, RPE on PDL-coated wells showed the highest proportion of epithelial cells.

3.2.4. Success Rates on Different Coatings

For RPE wells used for the experiments of collagen pretesting, survival rates and confluence rates were determined for all cultures combined. Of all 141 seeded wells, 128 were viable (please refer to Section 3.1.2 for the definition of success rates) until the day of the experiment, which equals a survival rate of 90.78%. From these 128 wells, only 27.34% reached 95–100% confluence, which is quite low and strongly indicates that collagen coating is suboptimal. These data were also assessed for the different collagen coatings (CIm, CIf, and CIV) compared to uncoated co after 7, 14, and 28 days (Table 3). In contrast to co, none of the collagen coatings reached confluence after day 7. For all time points, co showed the highest confluence rate, followed by CIm. The survival rate was the highest with the CIV coating (97.14%). Overall, co was better suited than any collagen coating tested.

Table 3. Success rates depending on the collagen type. Confluence and survival rates are listed according to the specific collagen coating with monomeric collagen I (CIm), fibrillar collagen I (CIf), collagen IV (CIV), or uncoated wells (co), as well as days of cultivation (d). The highest rates are marked. Numeric and percentage rates of success to the overall number of cultures are depicted.

Success Rates
Coatings	co	CIm	CIV	CIf
7 d confluence % (living wells)	9/29	0/32	0/34	0/33
	31.03%	0.00%	0.00%	0.00%
14 d confluence % (living wells)	20/29	5/32	1/34	0/33
	68.97%	15.63%	2.94%	0.00%
28 d confluence % (living wells)	20/29	9/32	4/34	2/33
	68.97%	28.13%	11.76%	6.06%
7 d confluence % (total wells)	9/34	0/36	0/35	0/36
	26.47%	0.00%	0.00%	0.00%
14 d confluence % (total wells)	20/34	5/36	1/35	0/36
	58.82%	13.89%	2.86%	0.00%
28 d confluence % (total wells)	20/34	9/36	4/35	2/36
	58.82%	25.00%	11.43%	5.56%
Survival (living wells/total wells)	29/34	32/36	34/35	33/36
	85.29%	88.89%	97.14%	91.67%

Also, for the follow-up experiments with coating substances CIm, PDL, Lam, Fn, and co, success rates for survival and confluence were determined for all cultures combined. Altogether, the survival rate was 96.25%, and the confluence rate was 69.48%. Stratified for the coating (Table 4), the data reveal that PDL shows the highest confluence rates, reaching 90% and more after 14 and 28 days, followed by Lam. Conversely, survival rates of CIm and Fn were at a maximum of 100% (Table 4).

Table 4. Success rates depending on the coating. Confluence and survival rates are listed according to the specific coating with monomeric collagen I (CIm), Poly-D-Lysine (PDL), laminin (Lam), fibronectin (Fn), or uncoated wells (co), as well as days of cultivation (d). The highest rates are marked. Numeric and percentage rates of success to the overall number of cultures are depicted.

Success Rates
Coatings	CIm	PDL	Lam	Fn	co
7 d confluence % (living wells)	10/36	23/28	17/31	7/30	9/29
	27.78%	82.14%	54.84%	23.33%	31.03%
14 d confluence % (living wells)	21/36	27/28	22/31	20/30	17/29
	58.33%	96.43%	70.97%	66.67%	58.62%
28 d confluence % (living wells)	21/36	27/28	22/31	20/30	17/29
	58.33%	96.43%	70.97%	66.67%	58.62%
7 d confluence % (total wells)	10/36	23/30	17/32	7/30	9/32
	27.78%	76.67%	53.13%	23.33%	28.13%
14 d confluence % (total wells)	21/36	27/30	22/32	20/30	17/32
	58.33%	90.00%	68.75%	66.67%	53.13%
28 d confluence % (total wells)	21/36	27/30	22/32	20/30	17/32
	58.33%	90.00%	68.75%	66.67%	53.13%
Survival (living wells/total wells)	36/36	28/30	31/32	30/30	29/32
	100.00%	93.33%	96.88%	100.00%	90.63%

3.2.5. Growth Factor Secretion on Different Coatings

To assess the secretion of VEGF-A of single-eye RPE cells cultured on different coatings (CIm, PDL, Lam, Fn, and co), supernatants were collected for 4 h on days 7, 14, and 28 after preparation and analysis with VEGF ELISA (Figure 11). Secreted VEGF ranged from 735.33 ± 164.03 pg/mL (PDL, 28 days) and 1109.33 ± 276.78 pg/mL (co, 28 days), which was in the normal range of the VEGF secretion of primary porcine mix culture RPE [39]. On day 7 and day 14, no significant differences between coatings were detected. On day 28 of cultivation, Lam showed significantly higher VEGF-A concentrations than PDL (mean: 1038.50 ± 248.47 pg/mL compared to 735.33 ± 164.03 pg/mL, p = 0.009), Fn displayed significantly lower concentrations than Lam (mean: 738.00 ± 161.08 pg/mL compared to 1038.50 ± 248.47 pg/mL, p = 0.020), and co showed significantly higher concentrations than Fn (mean: 1109.33 ± 276.78 pg/mL compared to 738.00 ± 161.08 pg/mL, p = 0.010) or PDL (mean: 1109.33 ± 276.78 pg/mL compared to 735.33 ± 164.03 pg/mL, p = 0.006). It is of interest that the previously determined most suitable coating, PDL, displayed the lowest VEGF secretion.

Figure 11. VEGF secretion on different coatings. Single-eye retinal pigment epithelium cells were seeded on monomeric collagen I (CIm), Poly-d-Lysine (PDL), laminin (Lam), fibronectin (Fn), or uncoated wells (co). The supernatant was collected for 24 h after 7, 14, and 28 days of cultivation and analyzed in ELISA for vascular endothelial growth factor A (VEGF-A). n = 6–17. Data were parametric; mean and standard deviation are shown. Significances were calculated between each group with analysis of variance (ANOVA) and Student’s t-test. * p < 0.05, ** p < 0.01, d = days.

3.2.6. Protein Expression on Different Coatings

RPE65 and CLDN19 are physiologically expressed RPE proteins, indicating differentiation and tight junction formation [40]. RPE cells were seeded on CIm, PDL, Lam, Fn, or on uncoated wells (co), and protein expression of RPE65 and CLDN19 was investigated with Western blotting (Figure 12 with example images) after 14 and 28 days of cultivation.

Figure 12. RPE65 and CLDN19 expression on different coatings. Single-eye retinal pigment epithelium cells were seeded on monomeric collagen I (CIm), Poly-d-Lysine (PDL), laminin (Lam), fibronectin (Fn), or uncoated wells (co). Lysates were made after 14 ((A), n = 6–7; (C), n = 6–7) and 28 days ((B), n = 5–6; (D), n = 6) of cultivation and analyzed with Western blotting for claudin-19 (CLDN19, (A,B) and retinal pigment epithelium-specific 65 kDa protein (RPE65, (C,D). Data were parametric; mean of band volumes normalized to β-actin expression and standard deviation are depicted. Significances were calculated between each group with analysis of variance (ANOVA) and Student’s t-test. * p < 0.05, ** p < 0.01. (E,F) Exemplary blots for 14 and 28 days are depicted.

Regarding CLDN19 expression on day 14 (Figure 12A), Lam showed the highest values with 1.74 ± 4.08 [arb. unit] and PDL showed the lowest values with 0.08 ± 0.05 [arb. unit]. Fn (mean: 0.79 ± 0.52 [arb. unit], p = 0.008) and co (mean: 0.42 ± 0.19 [arb. unit], p = 0.002) were significantly higher than PDL. CIm also exhibited higher CLDN19 expression than PDL (mean: 0.67 ± 0.55 [arb. unit], p = 0.025). Concerning CLDN19 expression on day 28 (Figure 12B), co showed the highest expression with 0.20 ± 0.13 [arb. unit] and Lam showed the lowest expression with 0.10 ± 0.04 [arb. unit]. Fn showed significantly higher values than Lam (mean: 0.17 ± 0.04 [arb. unit], p = 0.017).

Regarding RPE65 expression on day 14 (Figure 12C), both co and PDL showed equally high expressions with 0.37 ± 0.36 [arb. unit] and 0.37 ± 0.28 [arb. unit], respectively. Fn displayed the lowest values with 0.06 ± 0.10 [arb. unit]. PDL was significantly higher than Lam (mean: 0.08 ± 0.10 [arb. unit], p = 0.030) and Fn (p = 0.033). After 28 days of cultivation (Figure 12D), no significant differences were determined, but PDL showed the highest values with 0.29 ± 0.25 [arb. unit] and Lam showed the lowest values with 0.22 ± 0.29 [arb. unit]. Overall, PDL showed the highest RPE65 expression, indicating a higher level of differentiation. After 14 days of cultivation, the PDL coating showed little CLDN19 expression, but after 28 days, expression levels were comparable to the other coatings.

3.2.7. Cell Parameters on Different Coatings

To further investigate the differentiation and formation of single-eye RPE cells on different coatings, RPE cells were assessed for tight junction/cell border formation and other cellular parameters (see below). RPE cells were seeded on CIm, PDL, Lam, or Fn-coated glass coverslips (compared to uncoated slips, co) for 28 days. Cells were labeled for cell nuclei (blue) and tight junction protein CLDN19 (orange), and fluorescence images were taken.

First, a visual ranking of the quality of the cell borders (orange channel CLDN19) was conducted (Figure 13) by a single researcher, blinded to culture conditions. The ranking is from 0 for disturbed cell formation to 5 for differentiated and honeycomb-like RPE. The highest ranking was achieved by the Lam coating and co (median: 4.00 ± 1.50 and 4.00 ± 1.00, respectively). CIm, PDL, and Fn exhibited similar quality (median: 3.50 ± 1.00). Lam showed significantly better tight junction formation than Fn (p = 0.040) or PDL (p = 0.010). Co had a significantly higher ranking than CIm (p = 0.009), PDL (p < 0.001), or Fn (p = 0.001).

Figure 13. Formation of tight junctions. Single-eye retinal pigment epithelium cells were seeded on monomeric collagen I (CIm), Poly-d-Lysine (PDL), laminin (Lam), fibronectin (Fn), or uncoated wells (co). Tight junction protein claudin-19 was labeled and detected by fluorescence microscopy after 28 days of cultivation. n = 53–89. (A) The quality of the cell formation was visually ranked from 0 (disturbed) to 5 (honeycomb structure). Data were not parametric; median and interquartile ranges are shown. Significances were calculated between each group with the Kruskal–Wallis test and the Mann–Whitney test. * p < 0.05, ** p < 0.01, *** p < 0.001. (B) Exemplary images are shown for ranking 0 and ranking 5 (20× objective, scale bar = 100 µm). (C) Exemplary photos of PDL-coated glass coverslips and uncoated glass coverslips (co) after the staining procedure for immunofluorescence imaging (20× objective, light microscopy, scale bar = 100 µm) are shown. Single-eye retinal pigment epithelium cells were cultivated on these slips for 28 days. Cells on the PDL coating were affected by the staining procedure.

PDL seemed not to be suitable for coverslip coating. RPE cells on PDL-coated glass coverslips were affected by the staining procedure, disturbing the ranking quality of the cell border formation or even leading to cell detachment (Figure 13C).

The highest quality images of the cells (ranking 4 to 5) were used to determine morphological parameters and calculate optimal parameters for porcine primary RPE (Table 5) with CellProfiler software. Out of 341 images, 42.82% could not be evaluated by the software. For evaluation in CellProfiler, clearly visible cell borders and cell nuclei in a honeycomb structure were needed. Based on the evaluation of the analyzable images, the mean cell number of the images of a porcine single-eye RPE culture was 472.31 ± 97.61. Cells had a size of 324.25 ± 76.15 µm², a perimeter of 76.02 ± 8.04 µm², an eccentricity of 0.63 ± 0.01, a form factor of 0.68 ± 0.02 (which equals hexagonal shape), and a radius of 2.93 ± 0.32 µm. If data are differentiated based on the coating condition (Table 6), the PDL coating displayed the closest values to the standard calculation for cell area with 303.10 µm² and a perimeter of 75.28 µm. Eccentricity and form factor were nearly identical. Cell number and radius were closest to the standard values for no coating (co).

Table 5. Standard values of differentiated porcine single-eye retinal pigment epithelium cells. Single-eye retinal pigment epithelium cells were seeded on glass coverslips and stained for cell nuclei and tight junction protein claudin-19 after 28 days of cultivation. Fluorescence images were taken and evaluated with CellProfiler. n = 54.

	Cell Number	Area (µm2)	Perimeter (µm)	Eccentricity	Form Factor	Radius (µm)
Mean	472.31	324.25	76.02	0.63	0.68	2.93
Standard deviation	97.61	76.15	8.04	0.01	0.02	0.32

Table 6. Cell parameters depending on coating. Porcine single-eye retinal pigment epithelium cells were seeded on monomeric collagen I (CIm), Poly-d-Lysine (PDL), laminin (Lam), fibronectin (Fn), or uncoated glass coverslips (co) and stained for cell nuclei and tight junction protein claudin-19 after 28 days of cultivation. Fluorescence images were taken and evaluated with CellProfiler. Green marked data are closest and yellow marked data are second closest to the calculated standard values in Table 3. n = 12–43.

Coating	Cell Number	Area (µm2)	Perimeter (µm)	Eccentricity	Form Factor	Radius (µm)
CIm	444.97	357.55	80.11	0.64	0.67	3.02
PDL	499.23	303.10	75.28	0.65	0.66	2.77
Lam	517.65	295.40	74.41	0.64	0.66	2.74
Fn	416.17	366.34	80.97	0.64	0.67	3.07
co	463.64	348.50	80.40	0.65	0.66	2.95

The results of this section indicate that cells should be seeded on uncoated glass for staining assays.

3.3. Serum Content

3.3.1. Cell Numbers with Different Serum Content

Single-eye RPE cells were cultivated in different serum conditions, and cell number was determined before seeding and after cultivation periods of 7, 14, and 28 days with a trypan blue exclusion assay. Differences between cell count on the respective day of culture to the initial seeding cell number were calculated (Figure 14).

Figure 14. Cell number in different serum conditions. Before seeding single-eye retinal pigment epithelium, cell number was assessed with a trypan blue exclusion assay. Cells were seeded on Poly-d-Lysine (PDL) or uncoated wells (co) and cultivated in medium with 1%, 5%, or 10% serum. Cells were counted again after 7, 14, and 28 days of cultivation, and the differences in the seeding cell are depicted as cell number × 10⁴/mL ((A), n = 8–21). The initial cell number mean before seeding is listed ((B), n = 8–21). Data were parametric; mean and standard deviation are shown. Significances were calculated between each group with analysis of variance (ANOVA) and Student’s t-test. * p < 0.05, ** p < 0.01, *** p < 0.001.

On day 28, no significant differences were determined. On day 14, only co 1% displayed a negative cell growth of −7.71 ± 25.12 cells × 10⁴/mL. After 7 days, co 10% (59.73 ± 17.85 cells × 10⁴/mL) was significantly higher than co 1% (0.73 ± 17.35 cells × 10⁴/mL, p < 0.001), PDL 1% (9.82 ± 29.48 cells × 10⁴/mL, p = 0.010), PDL 5% (13.92 ± 21.79 cells × 10⁴/mL, p < 0.001), or PDL 10% (15.63 ± 44.25 cells × 10⁴/mL, p = 0.010). Co 5% (46.69 ± 18.17 cells × 10⁴/mL) was significantly higher than co 1% (p < 0.001), PDL 1% (p = 0.010), or PDL 5% (p = 0.005). After 14 days of cultivation, co 1% showed significantly fewer cells than co 5% (17.30 ± 27.12 cells × 10⁴/mL, p = 0.019), co 10% (42.15 ± 25.90 cells × 10⁴/mL, p < 0.001), PDL 5% (24.50 ± 34.39 cells × 10⁴/mL, p = 0.001), or PDL 10% (28.47 ± 33.50 cells × 10⁴/mL, p = 0.005), respectively. Co 10% showed a significantly higher cell number than co 5% (p = 0.018) or PDL 1% (0.80 ± 35.41 cells × 10⁴/mL, p = 0.006). Overall, in the first 2 weeks of cultivation, cells without coating and with a higher serum ratio show an increased cell number, but this difference is lost after 4 weeks of cultivation.

3.3.2. Confluence with Different Serum Content

Single-eye RPE cells were seeded on PDL or uncoated wells (co) with different serum content of medium (1%, 5%, and 10%), and confluency (cell growth area in % of the well) after 7 days as well as the day of 95–100% confluency was determined in light microscopy.

Confluency on day 7 (Figure 15A) was the highest with PDL 10% (median: 100.00% ± 15.00%), and the lowest with co 1% (median: 80.00% ± 47.50%). PDL 10% was significantly higher than co 10% (median: 90.00% ± 30.00%, p = 0.019), co 5% (median: 85.00% ± 40.00%, p = 0.004), co 1% (p = 0.002), or PDL 1% (median: 90.00% ± 45.00%, p = 0.005), respectively. PDL 5% (median: 95.00% ± 22.50%) showed higher confluence on day 7 than co 1% (p = 0.034).

Figure 15. Confluence in different serum conditions. Single-eye retinal pigment epithelium cells were seeded on Poly-d-Lysine (PDL) or uncoated wells (co) and cultivated in medium with 1%, 5%, or 10% serum. Confluence as a cell growth area in % of the well after 7 days of cultivation ((A), n = 43–51) and the day of 95–100% confluency was determined ((B), n = 16–27). Data were not parametric; median and interquartile ranges are depicted. Significances were calculated between each group with the Kruskal–Wallis test and the Mann–Whitney test. * p < 0.05, **p < 0.001.

The median of the day of full confluence (Figure 15B) was the same for all conditions (day 7.00). The only variation was in the interquartile range, which was the highest for co 5% with 3.75 days and significantly different from PDL 10% with an interquartile range of 0.00 (p = 0.038).

The data show that in cultivation periods of 7 and 14 days, cell number and confluency increase with increasing serum content. The faster division of the cells is highly likely due to the growth factor provided by the serum. After 28 days, however, cell number and confluence are no longer dependent on serum content.

3.3.3. Morphology with Different Serum Content

The morphology of cells grown in a medium with different serum content (1%, 5%, 10%) was analyzed by light microscopy after 7 (Figure 16A), 14 (Figure 16B), and 28 days (Figure 16C).

Figure 16. Cell morphology in different serum conditions. Single-eye retinal pigment epithelium cells were seeded on Poly-d-Lysine (PDL) or uncoated wells (co) and cultivated in medium with 1%, 5%, or 10% serum. The portion of epithelial, mesenchymal, undivided cells, and ungrown areas of each well was determined with light microscopy after 7 ((A), n = 126–153), 14 ((B), n = 96–120), and 28 days ((C), n = 33–51) of cultivation. Data were not parametric; median and interquartile ranges are shown. Significances were calculated between each group with the Kruskal–Wallis test and the Mann–Whitney test. * p < 0.05, ** p < 0.01, *** p < 0.001.

After 7 days of cultivation, PDL 5% (median: 90.00% ± 32.50%) displayed significantly more epithelial cells than PDL 1% (median: 80.00% ± 65.00%, p = 0.024). Also, PDL 5% (median: 10.00% ± 35.00%) displayed significantly fewer ungrown areas than co 1% (median: 20.00% ± 80.00%, p = 0.005), co 5% (median: 20.00% ± 85.00%, p = 0.045), co 10% (median: 20.00% ± 65.00%, p = 0.019), or PDL 1% (median: 20.00% ± 75.00%, p = 0.001). PDL 10% (median: 15.00% ± 75.50%) had significantly fewer ungrown areas than PDL 1% (p = 0.027). After 14 days of cultivation, co 1% (median: 32.50% ± 72.25%) exhibited significantly fewer epithelial cells than co 5% (median: 80.00% ± 90.00%, p = 0.038) or PDL 5% (median: 90.00% ± 60.00%, p < 0.001). Co 1% (median: 40.00% ± 80.00%) displayed significantly more ungrown area than PDL 5% (median: 15.00% ± 62.50%, p = 0.002). After 28 days of cultivation, co 10% (median: 80.00% ± 78.75%) exhibited significantly more epithelial cells than PDL 10% (median: 30% ± 81.25%, p < 0.001). Overall, a 5% serum content is suitable for a good RPE morphology but does not generally outperform 10% serum.

3.3.4. Success Rates with Different Serum Content

Success rates for survival and confluence for all seeded wells for serum testing were determined. Of note, the general rate of survival was reduced to 53.70%, compared to 96,25% in the previous work package. The main difference in these packages was the season and quality of the eyes, as this work package was conducted in the late spring season of 2023 (refer to Section 2.1) in which the quality of the eyes and with it the success rate of the culture was reduced by unknown reasons, despite cooling the eyes during transportation. Usually, a reduction of RPE quality during the late spring or early summer season can be observed, which was especially noticeable in the year 2023. Success rates of all single-eye RPE cultures from serum testing were determined, depending on the coating (PDL or uncoated) and the serum content of the medium (1%, 5%, or 10%). The percentage of surviving cultures was calculated. Also, the percentage of wells from the surviving cultures that reached a confluence of 95–100% was determined (Table 7). PDL 5% had the highest success rate with a survival rate of 71.43% followed by co 5% with 59.42%. The lowest survival rate was achieved by co 1% with 42.03%. Concerning the confluence rate, PDL 10% had the highest success rate with 34.29% followed by PDL 5% with 31.43%. Again, co 1% showed the lowest success rate with 18.84%. Overall, PDL coating and 5% serum seem to be a good choice for successful single-eye RPE cultures but do not outperform PDL 10%.

Table 7. Success rates of cultures in different serum conditions. PDL = Poly-d-Lysine, co = uncoated control well, x% = serum content of medium, n = 417.

	co 1%	co 5%	co 10%	PDL 1%	PDL 5%	PDL 10%
survived	42.03	59.42	50.72	44.29	71.43	55.71
confluent	18.84	23.19	21.74	22.86	31.43	34.29

3.3.5. Growth Factor Secretion in Different Serum Conditions

To determine if the physiological growth factor secretion of VEGF-A is maintained with different serum content (1%, 5%, or 10%), supernatants of single-eye RPE culture were collected for 4 h on days 7, 14, and 28 after preparation and analyzed with ELISA (Figure 17). Cells were seeded on PDL or uncoated wells (co). After 28 days of cultivation, secreted VEGF ranged from 504.27 ± 171.99 pg/mL (co 1%) to 1104.05 ± 443.52 pg/mL (co 5%), which falls within the normal range of VEGF secretion of primary porcine mix culture RPE [39]. Co 5% secreted significantly more VEGF than co 1% (p = 0.015) or PDL 5% (mean: 582 ± 246 pg/mL, p = 0.029). After 7 days of cultivation, no significant differences were found. But, regarding 14 days, PDL 10% (mean: 754.21 ± 158.56 pg/mL) was significantly higher than co 10% (mean: 550.33 ± 65.29 pg/mL, p = 0.041) or PDL 1% (mean: 460.74 ± 189.28 pg/mL, p = 0.020). Also, PDL 5% (mean: 713.06 ± 233.49 pg/mL) was significantly higher than PDL 1% (mean: 460.74 ± 189.28 pg/mL, p = 0.027) and co 10% (mean: 550.33 ± 65.29 pg/mL, p = 0.045).

Figure 17. VEGF secretion in different serum conditions. Single-eye retinal pigment epithelium cells were seeded on Poly-d-Lysine (PDL) or uncoated wells (co) and cultivated in medium with 1%, 5%, or 10% serum. Supernatants were collected for 24 h after 7, 14, and 28 days of cultivation and analyzed in ELISA for vascular endothelial growth factor A (VEGF-A). n = 3–26. Data were parametric; mean and standard deviation are shown. Significances were calculated between the groups with analysis of variance (ANOVA) and Student’s t-test. * p < 0.05.

3.3.6. Protein Expression in Different Serum Conditions

Single-eye retinal pigment epithelium cells were seeded on PDL or uncoated wells (co) and treated with media containing 1%, 5%, or 10% serum. Protein expression of RPE65 and CLDN19 was investigated after 14 and 28 days of cultivation and analyzed with Western blotting (Figure 18 with example images).

Figure 18. RPE65 and CLDN19 expression in different serum conditions. Single-eye retinal pigment epithelium cells were seeded on Poly-d-Lysine (PDL) or uncoated wells (co) and cultivated in medium with 1%, 5%, or 10% serum. Lysates were made after 14 ((A), n = 7–12; (C), n = 7–12) and 28 days ((B), n = 5–12; (D), n = 6–12) of cultivation and analyzed with Western blotting for claudin-19 (CLDN19, (A,B)) and retinal pigment epithelium-specific 65 kDa protein (RPE65, (C,D)). Data were parametric; the mean of band volumes normalized to β-actin expression and standard deviation are depicted. Significances were calculated between each group with analysis of variance (ANOVA) and Student’s t-test. * p < 0.05. (E,F) Exemplary blots for 14 and 28 days are depicted.

Regarding CLDN19 expression on day 14 (Figure 18A), no significant differences were observed. Descriptively, PDL 10% showed the highest CLDN19 expression (mean: 0.92 ± 0.73 [arb. unit]) and PDL 1% the lowest (mean: 0.38 ± 0.40 [arb. unit]). After 28 days of cultivation, CLDN19 expression (Figure 18B) was the highest with PDL 1% (mean: 0.45 ± 0.33 [arb. unit]), which was significantly higher than PDL 10% (mean: 0.10 ± 0.09 [arb. unit], p = 0.044). Independent of coating, CLDN19 expression decreased with increasing serum content.

RPE65 expression day 14 (Figure 18C) was highest in 5% serum and uncoated wells (mean: 0.48 ± 0.40 [arb. unit]), closely followed by 5% serum and PDL-coated wells (mean: 0.45 ± 0.43 [arb. unit]). PDL 1% showed the lowest RPE65 expression (mean: 0.14 ± 0.09 [arb. unit]), which was significantly lower than co 5% (p = 0.030) or PDL 5% (p = 0.032). After 28 days of cultivation (Figure 18D), PDL 1% displayed the highest expression (mean: 0.44 ± 0.26 [arb. unit]), which was significantly higher than co 5% (mean: 0.17 ± 0.17, p = 0.015).

Overall, the data indicate that after 14 days, these tight function and visual cycle proteins are more strongly expressed with higher serum content, while after long-term cultivation, lower serum content contributes to a higher protein expression.

3.3.7. Gene Expression

The gene expression pattern of single-eye RPE cultures was investigated with porcine-specific gene arrays for AMD and RPE-relevant genes [31] of our own design. The most suitable conditions determined in the former experiments were chosen (coating: PDL, serum content: 5%) and compared to the standard routine parameters (uncoated well (co), serum content 10%). RPE cells were seeded with conditions co 10%, co 5%, PDL 10%, or PDL 5% and cultivated for 28 days. All data and significances were calculated against standard condition co 10% (Rq = 1.000) and are listed in Appendix A Table A2. For a better overview, volcano plots are shown (Figure 19). Three flat gene expression changes were detected (significant values, but less than a 2-fold change). With PDL 10%, gene CRYAB was slightly up-regulated (Rq = 1.815, p = 0.040) and gene VWF was slightly down-regulated (Rq = 0.587, p = 0.010). With PDL 5%, gene ANGPTL2 was slightly up-regulated (Rq = 1.657, p = 0.039). Clearly up-regulated (2-fold changed or higher) was proinflammatory and proangiogenic factor CXCL8 with co 5% (Rq = 2.960, p = 0.032) and PDL 5% (Rq = 4.084, p = 0.016) conditions. Significances between other groups were not detected. Also, genes with more than a 2-fold expression change that did not reach significance are also highlighted in the volcano plots. Additionally, some genes were not expressed under specific conditions. Co 5% did not express CRP and PLA2G2D, and co 10% as a standard did not express CRP, CX3CR1 PLA2G2D, and LEP. PDL 5% and PDL 10% showed no expression of CRP, CX3CR1, or PLA2G2D, with the former also lacking an expression of NOS1 and the latter lacking FASLG. Overall, the gene expression for all conditions is similar, only an up-regulation of CXCL8 with 5% serum can be found. However, a general inflammatory response is not seen, as other inflammation-relevant genes were not influenced.

Figure 19. Gene expression in different serum conditions (volcano plots). Single-eye retinal pigment epithelium (RPE) cells were seeded on Poly-d-Lysine (PDL) or uncoated wells (co) and cultivated in a medium with 5% or 10% serum. RNA was isolated after 28 days of cultivation and analyzed with qPCR for different RPE-relevant genes. Relative quantification quotient (Rq) and significances were calculated between each group with analysis of variance (ANOVA) and Student’s t-test by the cloud software Thermo Fisher Connect and are logarithmically shown. ((A), n = 3) Co 5% vs. co 10%, ((B), n = 3) PDL 5% vs. co 10%, ((C), n = 3) PDL 10% vs. co 10%. Gray dots = no significant gene regulation, black dots = significant flat gene expression regulation, red dots = significant up-regulation.

4. Discussion

One of the greatest obstacles in biomedical science is the lack of translational success “from bench to bedside” [41]. A major factor in this is the choice of models that are used in biomedical research. Often, the choice is made focusing on easy reproducibility and fast significance, with less focus on biological or medical relevance. In particular, the use of immortal cell lines with their convenient handling and their fast results [42] and the use of mouse models, which do not reflect the human situation and are often chosen because of their availability [43], are part of the problem. This is even more prominent in ophthalmological research [7].

The use of porcine cell (and organ) culture is not new, and studies of porcine RPE cells are frequently published. However, there is still a lack of acceptance from the scientific community, especially considering reviewers of grant agencies and publications, but also by many researchers. The traditional use of mice is still very prominent [44], and lab material for mice (such as antibodies or primers) is highly available, while the availability of material for porcine tissue is limited. Porcine material is usually derived from animals killed for food production. These animals have a more heterogeneous background, considering both husbandry and genetic backgrounds, which will eventually lead to a higher variation of the experimental data [45]. Furthermore, cell culture techniques for porcine RPE are often traded down protocols, with various protocols in different labs that usually have not been validated for their efficiency (with some notable exceptions, e.g., [46]). In all published protocols, RPE cells of different eyes are mixed (e.g., [46,47,48,49,50]), so that cultures contain cells of different genetic origins, implying different responses in a given cell culture. Our RPE cell culture model uses a different approach. We use one eye for one culture, ensuring (1) genetic homogeneity in one culture and (2) genetic heterogeneity between cultures. This approach may lead to the need for a greater sample size to obtain significant results, but this approach ensures a higher biological relevance of the data. Indeed, it reflects the patient situation much more closely, as a patient is a genetically homogeneous entity while different patients will display different genotypes. Importantly, these cells are not passaged, avoiding the dedifferentiation that goes along with it [50,51,52]. Furthermore, we strictly evaluated the culture conditions to present a protocol based on scientific data, not a handed-down protocol. The major conditions we evaluated were PMT, coating, and serum content. The presented protocol is optimized for RPE cells grown on standard twelve-well cell culture dishes for studies not focusing on barrier functions. A protocol for RPE cells grown on transwell membranes optimized for barrier function is currently in preparation.

In our study, we could clearly show that 2 h PMT was not recommendable. Generally, a short PMT is considered beneficial for the culture, as the shorter the time, the less degeneration of the tissue has occurred; therefore, the better the quality. While this may still be true for organ cultures obtained from eyes, our results clearly show that a PMT of 2 h results in fewer successful cultures. The most likely explanation is that the RPE cells are more difficult to remove from their substrate at this early time point and that the early degenerative changes in the eye help the harvest of the cells. As the differences between 4 h and 6 h were rather small, we decided on a time window of 4 to 5 h PMT for all further experiments.

The results for the coating were rather surprising, as collagen turned out to be vastly inferior to other coatings. Collagen has been used frequently for RPE cell culture, including our own lab [53]. PDL showed to be the most appropriate coating, while no coating at all also showed very good results. PDL has been previously shown to enhance the growth of primary (bovine) RPE [54] and to enhance the attachment of (human) RPE cells compared to uncoated plates [55]. However, only a few studies concerning PDL have been published. The previous results from our group [56] indicated laminin as the most suitable coating for primary porcine RPE cells, but that study did not include PDL. As mentioned, a very interesting finding of our study is the inferiority of collagen compared to other coatings (or no coating at all). Collagen I has previously been shown to have no effect on RPE growth [54]. Another study that compared the outgrowth of human RPE did not find much difference between collagen I, collagen IV, or fibronectin, while laminin was associated with reduced growth [57]. Similarly, a recent study with primary porcine RPE did not see any effect of collagen I compared to uncoated regarding attachment, cell number, or differentiation [46]. In accordance with our findings, however, Viheriälä et al. showed that the attachment of hESC-RPE on collagen IV was poor but was enhanced if laminin was added [58].

There are several aspects that may contribute to the different suitability of the different coating, such as mechanical, the stiffness of the coating [59], or biological properties, such as their ability to facilitate adhesion of the RPE cells [58]. Substrate stiffness has been shown to influence the properties of RPE cells [60], implicated, e.g., in fibrosis formation [61] or VEGF secretion [62]. However, other studies, including our own [56,63], found little influence of stiffness variation on RPE culture. On the other hand, the ability of cells to adhere to the extracellular matrix is of pronounced importance. While PDL is so far not commonly used for RPE culture, several studies showed an enhanced adhesion of neuronal cells to PDL coatings [64,65,66]. In addition, a recent paper on iPSC-derived RPE cells described a reduced adhesion of the RPE cells on collagen compared to a better adhesion on laminin (PDL was not tested), similar to our findings [58]. A better adhesion of the RPE is, therefore, likely to strongly contribute to the better performance of PDL and laminin.

To our knowledge, none of the published studies has compared the effect of these coatings on confluency, success, and morphology as we did, so to the best of our knowledge, this study is the first to provide scientific evidence for the suitability of different collagens, fibronectin, PDL, and laminin for primary porcine RPE cells. Our data suggest the use of PDL for coating. Most importantly, the use of collagen is discouraged by the data of our study. Of note, PDL is the only coating substance that has an artificial origin and comes not from animal resources, like laminin, collagens, or fibronectin, which is also an advantage regarding animal welfare.

We also determined the effect of serum content on cell number, confluency, success, morphology, and protein expression. Generally speaking, considering cell number, confluency, and success, 1% serum was inferior to 5% or 10%. This is in accordance with previously published data. For RPE cell culture, 10% serum is not uncommon (e.g., for rat RPE [67] or for human RPE [68]) and is used as a reference concentration if serum-free options are investigated (e.g., [69]). Indeed, a recent paper suggested 10% serum as the optimal concentration for porcine RPE cell culture [46]. Even studies that switch to 1% serum in (porcine) RPE cell culture have the cells proliferate for 7 days in 10% serum [48]. In accordance with our data, a paper studying wound healing in RPE cells found no difference between 5% and 10% serum but a reduction of wound healing in less serum [70]. Of interest, however, our data show a reduction of CLDN19 expression with increasing serum content, which is in accordance with studies that highly recommend serum reduction for a good tight junction formation in the RPE [48]. On the other hand, RPE65 expression (after 14 days) is inferior in 1% serum compared to 5% or 10%. Our data indicate that the nutrient requirement of the RPE cells differ between the proliferation and maturation of the cells and is in accordance with published data on RPE cell culture, which show that RPE cells proliferate in high serum but need lower serum content in culture in order to form tight junctions [67,71]. Our data indicate that both 10% and 5% are suitable for RPE cultivation, with 5% reducing the burden (both in costs and animal suffering) of FBS use.

Finally, we presented standard values for the morphometry of primary porcine RPE cells (area, perimeter, eccentricity, form factor, radius), which, to our knowledge, has not yet been presented before, providing a reference for all users of primary porcine RPE cells. A study conducted by Bhatia and colleagues examined the morphology of human RPE cells (from human donor eyes), and compared with these data, porcine RPE cells were similar in area and eccentricity to the human RPE of the far periphery ([72]; see also [19]). It would be of high interest to conduct a morphometry analysis in the pig’s eye in situ to investigate whether the different subpopulations of RPE cells that have recently been described in the human [19,73] can also be identified in the pig.

Taken together, we provide a validated protocol (Appendix B) for the use of single-eye RPE cells, including a reference for RPE cell morphology as an offer to the scientific community for a better model to investigate RPE cells. Further research will investigate amendments to these models for specific objectives.

5. Conclusions

The aim of this study was to create basic parameters for a standardized method protocol to generate and handle porcine single-eye retinal pigment epithelium cell culture, cultivating cells from one pig eye in one well of a twelve-well plate. This guarantees genetically homogenous cells in one culture and genetically heterogenous cells between cultures, enabling a higher relevance of the experimental data. This could be used for basic research for ophthalmologic diseases, such as age-related macular degeneration. Using pig eyes, which can be obtained as waste material, this study also contributes to the 3R principle. In addition, the porcine eye is genetically and anatomically closer to the human eye than the mouse eye. Within this study, we established parameters for post-mortem time, coating, and serum quantity of the culture medium. A post-mortem time of 4 to 5 h is optimal both in outcome and practicability. Coating with Poly-d-Lysine is recommended as it contributes to a better success rate, physiological differentiation, and cell parameters. For immunofluorescence purposes, however, coating coverslips should be avoided. Also, serum can be reduced to 5% as this may benefit the confluency, protein expression, and morphology of the RPE cells with the exception of a higher interleukin 8 gene expression. Taken together, basic parameters and a detailed protocol for the preparation and cultivation of porcine single-eye retinal pigment epithelium cultures have been established.

Appendix B.1. Materials

• Freshly slaughtered pig eyes (kept on ice)
• Water bath
• Safety workbench
• Cell incubator
• Centrifuge
• 1000 µL pipettes
• Twelve-well polystyrene plate for adherent cells (e.g., Sarstedt; #83.3921)
• Optional: sterile coverslips (Th. Geyer; #CB00180RA1)
• Styrofoam box
• Crushed ice
• 500 mL sterile glass beaker
• 250 mL and 500 mL sterile plastic beakers
• Pressure seal bags (small and big)
• Sterile 15 mL and 50 mL tubes
• Underlays
• Clothes
• Sterile preparation set with

  ○

  Enucleation scissors

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  Microspring scissors

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  Long curved forceps

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  Small straight tweezers

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  Small straight tweezers with a hook

• Sterile razor blades
• Sterile medium-sized and big glass Petri dishes
• Lab bottle lids (fitting the size of one pig eye)
• 0.9% sterile NaCl (e.g., Fresenius Kabi; #04801702)
• Sterile iodine solution (e.g., Betaisodona^®; #04923204) diluted 1:3 with 0.9% NaCl
• 70% ethanol
• Poly-d-Lysine (PDL, e.g., Sigma-Aldrich; #P7886)
• Sterile trypsin solution (T, 0.25 g T/100 mL PBS, e.g., Pan-Biotech; #P10-021100)
• Sterile trypsin/EDTA solution (T/E, 0.05 g T + 0.02 g EDTA/100 mL PBS, e.g., Pan-Biotech; #P10-020100)
• Sterile Dulbecco’s phosphate-buffered saline without Ca and Mg (e.g., Pan-Biotech; #P04-53500) with 1% penicillin/streptomycin (e.g., Sigma-Aldrich; #P0781) → PBS + Pe/St
• Cell media containing

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  Dulbecco’s Modified Eagle Medium with high glucose, L-glutamine, and phenol red (DMEM, Gibco™, Thermo Fisher Scientific; #41965062)

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  1% non-essential amino acids (Pan-Biotech; #P08-32100)

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  11 mM sodium pyruvate (Pan-Biotech; #P04-43100)

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  1% penicillin/streptomycin (Sigma-Aldrich; #P0781)

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  10% or 5% fetal bovine serum (FBS, Gibco™, Thermo Fisher Scientific; #10437028)

Appendix B.2. First Things to Prepare

Working place in the lab

• Put crushed ice in the Styrofoam box and place a glass beaker filled with 250 mL of 0.9% NaCl
• Put aliquoted iodine solution on ice (50 mL tube)
• Put an underlay with an EtOH moisture cloth on it
• Have an organic waste bag ready
• Place the preparation set
• Put in a 37 °C warm water bath
• One aliquot of T (50 mL in 50 mL tube) for 12 eyes
• One aliquot of T/E (50 mL in 50 mL tube) for 12 eyes
• One aliquot of PBS + Pe/St (50 mL in 50 mL tube) for 12 eyes
• Cell media
• Bench
• EtOH moisture cloth with a sterile razor blade
• One big glass Petri dish filled with bottle lids
• One smaller glass Petri dish
• Waste bags in plastic beakers, one for organic waste and one for plastic waste
• For immune staining, add sterile coverslips into the wells of the twelve-well plate and do not coat them
• For other assays, coat the wells of the twelve-well plate with PDL as described by the manufacturer’s instructions

Appendix B.3. In the Lab

Preparation process

• Start 4 to 5 h after the first pig was slaughtered.
• Remove muscles, fat, conjunctiva, and excess connective tissue from the eyes with enucleation scissors on top of the cloth. Roughly trim the optic nerve (do not cut too close).
• Place three eyes at a time in the tube with iodine solution (on ice) for approx. 5 min.
• Transfer the eyes into cold 0.9% NaCl in a glass beaker until all eyes are fully prepared.

Appendix B.4. Under the Bench

Preparation process

5.

For the preparation process, use media with 10% FBS.

6.

Put one eye in the smaller Petri dish and cut the eye approx. 3 mm below the ciliary body with the razor blade and cut around it once with the enucleation scissors to create an eye cup.

7.

Remove the lens and the vitreous body by putting the enucleation scissors into the vitreous body, open the scissors and pull it out, so that the eye is a small open bowl.

8.

Fill the eye cup with warm PBS + Pe/St and carefully loosen the retina with the tweezers and microscissors (carefully hold the eye from the outside with the tweezer with hook and pull the retina with the other tweezer from the top to the center and cut off at the optic nerve with the microscissors). Remove the retina.

9.

Remove PBS + Pe/St and place the eye cups in the lids. Do not leave the Petri dish open for long (avoid drying out the eyes).

10.

Add approx. 3 mL T (ready aliquoted) to the eye dishes and incubate at 37 °C for 10 min in the closed Petri dish.

11.

Remove T and add approx. 3 mL T/E (ready aliquoted) to the eye cups.

12.

Incubate at 37 °C for exactly 35 min in the closed Petri dish.

13.

Dissolve the RPE cells from the choroid by pipetting up and down with the 1000 µL pipette (set to 800 µL, rinse the eye carefully exactly 30 times, dividing each time equally between the center and the edges).

14.

After all eyes have been resuspended, transfer cells into prepared 15 mL tubes (filled with 1 mL medium) and fill up to 5 mL volume with medium to neutralize the T/E.

15.

Centrifuge for 5 min at 200× g.

16.

Carefully aspirate the supernatant (keep the tube slightly tilted, the pellet is sensitive).

17.

Resuspend each pellet in 1 mL of fresh RPE medium. Fill up to a volume of 5 mL with fresh medium and centrifuge again for 5 min at 200× g.

18.

Resuspend the pellet in 1000 µL medium.

19.

Pipette the cell suspension in a circle into the respective well of the twelve-well plate. Swirl the plate once more (to ensure even distribution of the cells in the well) and place it in the incubator (humidified, 37 °C, 5% CO₂).

20.

After 3 to 4 days, change to media with 5% FBS and renew every 3 to 4 days.