Primary Culture of Porcine Retinal Pigment Epithelial Cells

Summary

Here, an easy-to-follow method to culture primary porcine retinal pigment epithelial cells in vitro is presented.

Abstract

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. Multiple functions, including phagocytosis, nutrient/metabolite transportation, vitamin A metabolism, etc., are conducted by the RPE on a daily basis. RPE cells are terminally differentiated epithelial cells with little or no regenerative capacity. Loss of RPE cells results in multiple eye diseases leading to visual impairment, such as age-related macular degeneration. Therefore, the establishment of an in vitro culture model of primary RPE cells, which more closely resembles the RPE in vivo than cell lines, is critical for the characteristic and mechanistic studies of RPE cells. Considering the fact that the source of human eyeballs is limited, we create a protocol to culture primary porcine RPE cells. By using this protocol, RPE cells can be easily dissociated from adult porcine eyeballs. Subsequently, these dissociated cells attach to culture dishes/inserts, proliferate to form a confluent monolayer, and quickly re-establish key features of epithelial tissue in vivo within 2 wks. By qRT-PCR, it is demonstrated that primary porcine RPE cells express multiple signature genes at comparable levels with native RPE tissue, while the expressions of most of these genes are lost/highly reduced in human RPE-like cells, ARPE-19. Moreover, the immunofluorescence staining shows the distribution of tight junction, tissue polarity, and cytoskeleton proteins, as well as the presence of RPE65, an isomerase critical for vitamin A metabolism, in cultured primary cells. Altogether, we have developed an easy-to-follow approach to culture primary porcine RPE cells with high purity and native RPE features, which could serve as a good model to understand RPE physiology, study cell toxicities, and facilitate drug screenings.

Introduction

The retinal pigment epithelium (RPE) is located between photoreceptors and choriocapillaris in the outer layer of the retina¹ with multiple functions, including forming the blood-retinal barrier, transporting and exchanging nutrients and retinal metabolites, recycling vitamin A to maintain a normal visual cycle, and phagocytosis and clearance of shed photoreceptor outer segments (POSs)^2,3. Since POSs require constant self-renewal to generate vision, the RPE cells need to continuously engulf detached POSs to maintain retinal homeostasis⁴. Therefore, RPE dysfunction results in many blinding eye diseases, such as age-related macular degeneration (AMD)⁴, retinitis pigmentosa (RP)⁵, Leber congenital amaurosis⁶, diabetic retinopathy⁷, etc. Till now, the exact pathogenesis of most of these diseases remains elusive. As a result, RPE cell culture is established to study RPE cell biology, pathological changes, and underlying mechanisms.

As the simplest model to study cell biology, the culture of RPE cells was started as early as the 1920s⁸. Although ARPE-19 is widely used as RPE cells, loss of pigmentation, cobblestone morphology, and, especially, the barrier functions in this cell line raise lots of concerns⁹. In comparison, the culture of primary human RPE cells offers a more realistic scenario for physiological and pathological studies⁹. However, the relatively limited availability restricts their usage and ethical issues always exist. In addition, several groups used mouse models to culture RPE cells. However, the size of the mouse eye is small, and a single culture usually requires many mice, which is not convenient⁹. Recently, scientists have developed new methods to use human embryonic stem cells or induced pluripotent stem cells to derive RPE cells. Although this technique has particular potential for the treatment of inherited RPE disorders, it is time-consuming and usually requires several months to generate mature RPE cells¹⁰. To overcome these problems, here we introduce an easy-to-follow protocol to isolate and culture high-purity RPE cells in the laboratory routinely. Under suitable culture conditions, these cells can display typical RPE functions and exhibit typical RPE morphologies. Therefore, this culture method can provide a good model to understand RPE physiology, study cytotoxicity, investigate pathological mechanisms of related ocular diseases, and conduct drug screenings.

Protocol

The use of experimental animals complied with the regulations of the Association for Research in Vision and Ophthalmology (ARVO) and was approved by the Ethics Committee of Experimental Animal Management of Xiamen University.

1. Preparation of experimental surgical devices, tissue digestion enzyme, and cell culture buffer

1. Prepare the experimental surgical devices, by cleaning and autoclaving two pairs of ophthalmic surgical scissors and forceps the day before the eyeball dissection, and afterward drying the box with surgical devices in a general protocol oven at 65 °C overnight.
2. Culture media preparation.
   1. Prepare DMEM/Basic media supplemented with 10% (v/v) fetal bovine serum, 2 mM L-glutamine, and 1% (v/v) penicillin (100 U/mL) and streptomycin (100 U/mL).
   2. Prepare DMEM/F12 media supplemented with 1% (v/v) FBS, and 1% (v/v) penicillin (100 U/mL) and streptomycin (100 U/mL).
   3. Prepare MEM-Nic¹¹, by supplementing MEM alpha with 2 mM L-glutamine, 1% FBS, 1% (v/v) penicillin (100 U/mL) and streptomycin (100 U/mL), 0.1 mM NEAA, 1% (v/v)  N1 supplement, taurine (0.25 mg/mL), hydrocortisone (20 ng/mL), triiodo-thyronin (0.013 ng/mL), and 10 mM nicotinamide.
      NOTE: To improve cell viability and proliferation, the percentage of FBS can be increased to 20% to neutralize digestion enzymes and seed the dissociated cells. For long-term cell culture, the percentage of FBS can be decreased to as low as 1%¹².
3. Thaw the tissue digestion enzyme aliquot (0.25% (w/v) Trypsin/EDTA solution supplemented with 0.91 mM EDTA, hereafter referred as Trypsin/EDTA solution) during the experiment.
   NOTE: Fresh Trypsin/EDTA solution should be used every time to obtain optimum results. Aliquot 10 mL of fresh Trypsin/EDTA solution into each 15 mL sterile centrifuge tube and freeze the aliquots at -20 °C refrigerator until use.
4. Dissection solution.
   1. Sterilize 1x Phosphate Buffered Saline (PBS) (pH 7.2) supplemented with 2% (v/v) penicillin (100 U/mL) and streptomycin (100 U/mL) by filtering the solution through a 0.22 µm syringe filter unit.
5. Coat the culture plates and transwell inserts.
   1. Wash the wells and transwell inserts with 1x PBS. Remove the PBS from wells and transwells with a pipette, and then add 1 mL of fresh 1x PBS to the lower chamber and 600 µL of 10 µg/mL of laminin solution to the upper chamber.
   2. Incubate the plates/transwell inserts in the cell culture incubator overnight at 37 °C and 5% CO₂. Remove the laminin solution from the upper chamber and wash with 1 mL of cold 1x PBS twice before seeding the cells.

2. Dissection of porcine eyeball RPE cells

1. Preparation of the instruments.
   1. Clean the laminar flow hood with 75% ethanol and UV light. Prepare three 50 mL sterile centrifuge tubes containing ~25 mL of 75% ethanol, three 50 mL sterile centrifuge tubes containing ~25 mL of 1x PBS, and three 10 cm sterile cell culture dishes containing ~15 mL of 1x PBS.
      NOTE: These settings are usually used for dissecting four porcine eyeballs. Please scale up when more eyeballs are used. To improve the cell viability, precool 1x PBS buffer on ice for at least 30 min. Both 75% ethanol and UV light are necessary to ensure that experimental instruments and cells are not contaminated. Turn on the UV lamp in advance to sterilize the entire operation table for at least 15 min.
2. Clean the porcine eyeballs.
   1. Obtain fresh eyeballs from a slaughterhouse and keep them on ice until dissection. Soak four porcine eyeballs into ~15 mL of 75% ethanol in a 10 cm Petri dish and use a pair of scissors and forceps to cut off all the residual connective tissues and muscles.
      NOTE: Remove as much tissue as possible from outside of the sclera to reduce contaminations. Do not cut the optic nerve at this time to facilitate the transfer of eyeballs during the decontamination and wash step. After this step, all the procedures should be performed in a laminar flow hood.
3. Decontaminate the porcine eyeballs by soaking and washing four porcine eyeballs in three 50 mL sterile centrifuge tubes filled with 75% ethanol in a sequential manner; each eyeball is dipped for at least 5 min in each tube. Next, wash the porcine eyeballs in three 50 mL sterile centrifuge tubes filled with 1x PBS in a sequential manner; wash each eyeball for at least 5 min in each tube (Figure 1A). Invert the tubes every minute to wash the eyeballs thoroughly.
4. Dissection of porcine eyeballs.
   1. Move the four porcine eyeballs into a 10 cm sterile cell culture dish containing 1x PBS. Trim the outer surface of each eyeball again to remove the optic nerve and small debris (Figure 1B). Use scissors to make a small cut at the intersection of the limbus and sclera, and then remove the cornea, iris, lens, vitreous body, and neural retina (for detailed structures of these tissues, please refer to Figure 1).
   2. Transfer the RPE-choroid-sclera complex into a new 10 cm cell culture dish and make four cuts to flat the eyecup in the shape of a four-leaf clover (Figure 1C).
5. Perform Trypsin/EDTA solution digestion by placing the four RPE-choroid-sclera complexes into a new 10 cm dish. Pour 20 mL of fresh Trypsin/EDTA solution to merge the RPE-choroid-sclera complexes and put the dish into the cell culture incubator at 37 °C for ~30 min.
   ​NOTE: Use fresh Trypsin/EDTA solution to dissociate the RPE cells. Carefully control the time of Trypsin/EDTA solution digestion, as a longer incubation time (more than 30 min) may increase the contamination of other types of cells.

3. Isolation and culture of porcine eyeball RPE cells

1. RPE dissociation.
   1. Take the dish out of the incubator after 30 min of incubation with Trypsin/EDTA solution and add 20 mL of prewarmed culture media (with 10% FBS) to the dish to neutralize the Trypsin/EDTA solution.
      NOTE: At this step, only DMEM/Basic media is used. When cells reach confluency, three culture medias can be used depending on the experimental conditions: DMEM/Basic media, DMEM/F12 media, and MEM-Nic media.
   2. Use a 5 mL transfer pipette to dissociate the RPE cells by gently pipetting several times. Collect cell suspensions into 15 mL centrifuge tubes. Use another 10 mL of fresh culture media (10% FBS) to wash the RPE-choroid-sclera complexes on the dish by gently pipetting to obtain as many RPE cells as possible.
      NOTE: Do not triturate the cells too vigorously. Ensure to fill and empty the pipette at a rate of about 3 mL/s. Avoid bubbling the cell suspension.
2. Collect RPE cells by centrifuging the tubes at 200 x g for 5 min at room temperature. Aspirate the supernatant using a pipette and add another 5 mL of culture media (10% FBS) to resuspend the cells and centrifuge at 200 x g for another 5 min. Decant the supernatant and resuspend the cells with 12 mL of culture media.
   NOTE: When aspirating the supernatant, leave about 1 mL of media to avoid the breaking of cell clumps.
3. Seeding RPE cells.
   1. Seed ~1-2 x 10⁵ cells/well into 12-well culture plates or transwell inserts. Usually, about 1.5 x 10⁶ RPE cells are obtained from four porcine eyeballs. Change the culture media every 2 days and reduce the serum concentration to 1% when the cells fully cover the surface of the wells/inserts.
      NOTE: Do not move the cell culture dish too frequently before cells attach to the culture dish/inserts.
4. Change the culture media every 2 days until the cells are harvested.
   ​NOTE: The concentration of FBS can be reduced after cells reach confluency, and cells can be cultured for up to several months to allow full differentiation and maturation.

4. Characterization of primary porcine RPE cells

1. Culture the cells at confluency for 1 or 2 wks, and then harvest the cells for further analysis.
2. Harvest cells for quantitative real-time PCR (qRT-PCR) analysis.
   1. Remove the culture media, wash the cells with 1 mL of cold 1x PBS, and then add 500 µL of RNA extraction solution into each well to collect cell lysate from about 1 x 10⁶ cells.
   2. Extract mRNA¹³; approximately 4 µg of RNA is extracted from each sample. Use 100 ng of mRNA to perform reverse transcription¹⁴; dilute the cDNA 40-fold for quantitative real-time PCR analysis. Primers are listed in Table 1.
3. Harvest cells for immunofluorescence staining.
   1. Remove the culture media, wash the cells with 1 mL of cold 1x PBS, and then add 500 µL of 4% (w/v) paraformaldehyde in 1x PBS to fix the cells (about 1 x 10⁶ cells/well) for 30 min at room temperature. Then, wash the cells with 1x PBS twice and perform immunofluorescence staining with primary antibodies (1:100 in 1x PBS supplemented with 1% (w/v) bovine serum albumin) at 4 °C overnight and secondary antibodies (1:200 in 1x PBS supplemented with 1% (w/v) bovine serum albumin) at room temperature for 2 h according to online protocols¹⁵.
   2. The immunofluorescence images are acquired by a confocal microscope following the online manual¹⁶.
4. Harvest cells for Western blot analysis.
   1. Remove the culture media, wash the cells with cold 1x PBS twice, and then add 80 µL of RIPA buffer (with 1x Protease inhibitor) into each well and use cell scrapers to scratch about 1 x 10⁶ cells off the dishes/inserts.
   2. Collect the cell lysate from each well/insert into a 1.5 mL microcentrifuge tube. Boil the cell lysates for 10 min and then measure protein concentrations using BCA kits; the protein concentration of each sample is about 2 mg/mL. Use 25 µg of proteins of each sample for Western blot analysis according to online protocols¹⁷.
   3. For Western blot, incubate the membranes in 5 mL of primary antibody solution (1:1,000 dilution of primary antibodies in 1x TBST solution supplemented with 5% (w/v) bovine serum albumin) at 4 °C overnight on a rotating multipurpose shaker.
   4. Then, incubate the membrane with about 15 mL of anti-rabbit or anti-mouse secondary antibody solution (1:5,000 dilution of secondary antibodies in 1x TBST solution supplemented with 5% (w/v) non-fat milk) at room temperature for 2 h on an orbital shaker, according to the source of primary antibody used.
5. Transepithelial resistance (TER) measurement.
   1. Wash the chopstick electrodes with 70% ethanol and sterile 1x PBS in a sequential way. Then place the shorter end of the electrode in the top chamber and the longer end in the bottom chamber of the transwell inserts and click the button on the epithelial voltmeter for the measurements. Record the TER measurements of each well in triplicates.

Results

The primary porcine RPE (pRPE) cells were cultured in DMEM/Basic media with 10% FBS, and cell morphology under light microscope was photographed at 2 days (Figure 2A), 6 days (Figure 2B) and 10 days (Figure 2C) after seeding. After 1 wk, a confluent monolayer of pigmented pRPE cells with cobblestone morphologies was observed.

To better characterize the primary pRPE cells, primary human RPE cells (hRPE) at...

Discussion

Here, a detailed and optimized protocol for the isolation, culture, and characterization of RPE cells from porcine eyeballs, which generates a good model for in vitro characterization of RPE cells and RPE-related disorder studies has been described. Methods for the isolation of the RPE from human, mouse, and rat eyes have been described previously^23,24,25. However, it is difficult to obtain human eyeballs in some labora...

Materials

Name	Company	Catalog Number	Comments
ARPE-19 cells	CCTCC	GDC0323
Bovine serum albumin	Yeasen	36101ES60
Confocal microscopy	Zeiss	LSM 880 with Airyscan
ChemiDoc Touch	Bio-Rad	1708370
Cell scraper	Sangon	F619301
10 cm culture dish	NEST	121621EH01
12-well culture plate	NEST	29821075P
DMEM F12 Medium	Gibco	C11330500BT
DMEM basic Medium	Gibco	C11995500BT
EVOM2	World Precision Instruments	EVOM2	For TER measurement
Fetal bovine serum	ExCell Bio	FSP500
Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488	ThermoFisher Scientific	A-11034
Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594	ThermoFisher Scientific	A-11012
Goat anti Mouse IgG (H/L):HRP	Bio-Rad	0300-0108P
Goat anti Rabbit IgG (H/L):HRP	Bio-Rad	5196-2504
hydrocortisone	MCE	HY-N0583/CS-2226
Hoechst 33342 solution (20 mM)	ThermoFisher Scientific	62249
LightCycler 96 Instrument	Roche	5815916001
Liothyronine	MCE	HY-A0070A/CS-4141
laminin	Sigma-Aldrich	L2020-1MG
MEM(1X)+GlutaMAX Medium	Gibco	10566-016
MEM NEAA(100X)	Gibco	11140-050
Millex-GP syringe filter unit	Millipore	SLGPR33RB
N1	Sigma-Aldrich	SLCF4683
NcmECL Ultra	New Cell&Molecular Biotech	P10300
Non-fat Powdered Milk	Solarbio	D8340
Nicotinamide	SparkJade	SJ-MV0061
Na+-K+ ATPase antibody	Abcam	ab76020	Recognize both human and porcine proteins
PAGE Gel Fast Preparation Kit(10%)	Epizyme	PG112
primary Human RPE cells	-	-	Generous gift from Shoubi Wang lab
Pierce BCA Protein Assay Kit	ThermoFisher Scientific	23225
Prism	GraphPad by Dotmatics	version 8.0
Protease Inhibitor Cocktails	APExBIO	K1024
PRE65 antibody	Proteintech	17939-1-AP	Recognize both human and porcine proteins
PEDF antibody	Santa Cruz Biotechnology	sc-390172	Recognize both human and porcine proteins
100 x penicillin/streptomycin	Biological Industries	03-031-1BCS
Phosphate buffered saline (PBS)	RARBIO	RA-9005
ReverTra Ace qPCR RT Master Mix	Toyobo	FSQ-201
RIPA buffer	ThermoFisher Scientific	89900
15 mL sterile centrifuge tubes	NEST	601052
50 mL sterile centrifuge tubes	NEST	602052
0.25% Trypsin-EDTA	Gibco	25200-056
Taurine	Damas-beta	107-35-7
Trizol	Thermo-Fisher	15596026	RNA extraction solution
TB Green Fast qPCR Mix	Takara	RR430A
12-well transwell inserts	Labselect	14212
VEGF antibody	Proteintech	19003-1-AP	Recognize both human and porcine proteins
VEGF ELISA kit	Novusbio	VAL106
ZO-1 antibody	ABclonal	A0659	Recognize both human and porcine proteins