The retinal pigment epithelium (RPE) is a monolayer of highly specialised pigmented cells lying between the neural retina and the Bruch′s membrane of the choroid. These cells are involved in phagocytosis of shed photoreceptor rod outer segments (ROS), metabolism of retinol, formation of blood retinal barrier, interaction with light by melanin granules, synthesis of extracellular matrix, regulation of ion and metabolite transport, etc. They are therefore critical to visual function and photoreceptor viability.1 The RPE cells are of neuroectodermal origin and lose function with time causing retinal degeneration. RPE dysfunction has also been implicated in dystrophies like retinitis pigmentosa2 and replacement of RPE in such patients has been found to rescue photoreceptors.3
Although attempts at culturing chick embryo RPE began as early as the 1920s,4 it is only in the last two decades that major breakthroughs have occurred in the understanding of the morphological and functional characteristics of these cells. The initial cultures were done using rat, bovine or porcine RPE and it was only in the early 1970s that human RPE cell cultures were taken up in earnest.5 Cells were harvested largely by enzymatic dissociation using trypsin,6 collagenase and hyaluronidase have been used occasionally, in combination or in isolation.7 Castillo et al8 used dispase on foetal RPE cells and demonstrated its superiority to trypsin in terms of preserving cell viability.9
Various substrates including plain plastic petri-plates, artificial membranes,10 cryoprecipitate membranes,11 collagen, laminin or fibrinogen coated surfaces,12 and most recently the human amniotic membrane (hAM)13 have been used to culture these cells. The anti-inflammatory, anti-apoptotic, epithelial growth promoting properties of hAM and its clinical application as a “biological dressing” make it a good substitute for a biological vehicle.14151617 With our experience in culturing limbal and conjunctival epithelium,181920 we explored the feasibility of culturing human RPE cells on hAM. We used both the enzymatic and mechanical techniques of RPE cell isolation from adult cadaver eyeballs and compared the outcomes- an area hitherto unexplored.
Materials and Methods
Adult cadaver eyeballs (screened serologically for viral infections) were obtained from the Ramayamma International Eye Bank after corneal trephination other reagents/instruments included DMEM and Hams F-12 in the ratio of 1:1 supplemented with NaHCO3, insulin, glutamine and antibiotics (penicillin 50 U/ml, streptomycin 0.1mg/ml and amphotericin B 0.0025 mg/ml), 10% foetal calf serum and epithelial growth factor (0.1ul/ml); Calcium Magnesium free Hanks Balanced Salt Solution (CMF HBSS) with added antibiotics (as in media); 0.25% Trypsin /0.2% EDTA for preparation of amniotic membranes and 0.25% Trypsin/0.02% EDTA for dissociation of RPE cells; Ficol-Hypaque 40% with 0.01M Na2PO4 and 0.15 M NaCl; Trypan blue; 65 mm plastic petri plates (Cellstar); sterile blunt and fine forceps; sterile fine scissors; amniotic membranes 2.5-5 cm x 5 cm in size; glass slides cut to size; cell scrapers or glass slides. All media and reagents were obtained from Sigma-Aldrich (85 gms - Aldrich Chemicals Pvt Ltd, USA).
RPE cell harvesting
Sixteen cadaver eyeballs were obtained from the eye bank and processed immediately. Ten eyeballs were processed mechanically and 6 were processed enzymatically.
Mechanical isolation (group 1)
The iris and lens were removed. The retina along with the vitreous was separated from the underlying RPE. The choroid was held at its edge with the blunt forceps and the retina grasped with the fine forceps and gently peeled off. The retina came out en bloc except for a small defect at the site of attachment to the optic nerve. The eyecup was washed once with CMF HBSS to remove any residual retinal or vitreous tissue. Following this the entire eyecup was opened out with four radial incisions to expose the inner surface of the choroid and immersed in antibiotic-rich CMF HBSS for at least 10 minutes to effectively decontaminate the tissue. The RPE cells, which appear as a layer of brown over a white choroid, were gently scraped off with a 22-G needle and the cell material thus collected was explanted onto the amniotic membrane after a Trypan blue viability test.
Enzymatic isolation (group 2)
After removal of its contents the eyecup was incubated with 1-2 ml of 0.25% Trypsin/0.02% EDTA for 10 minutes. A drop of FCS was used to inactivate the trypsin at the end of incubation. The solution collected subsequently from the eyecup was centrifuged at 500 rpm for 30 minutes over a cushion of Ficol-Hypaque (density gradient centrifugation) to pellet the pigmented cells.20 The cell pellet was resuspended in media and assessed for viability using Trypan blue in a dilution of 1:1 after which it was plated onto the amniotic membrane.
Preparation of amniotic membranes
Amniotic membranes were obtained during Caesarean sections (donors screened serologically for TORCH, HIV and Hepatitis B) and cut to size, layered onto nitrocellulose paper and stored in the eye bank in DMEM at -800C. Prior to use for culture they were thawed; separated from the nitrocellulose paper, treated with a few drops of 0.25% Trypsin/0.2% EDTA at 370C for 30 minutes and scraped with a glass slide to remove their native epithelium. The denuded membrane was cleared with phosphate buffer it and tucked securely around a glass slide cut to size. This surface was then used to culture the cells.
The needle-picked RPE cell patches or the cell suspensions were placed onto the amniotic membrane. Two drops of media were added and the plates incubated after assessing yield and appearance at explantation under the phase contrast microscope. The RPE patches appear as a collection of 30-50 pigmented polygonal cells arranged in a honeycomb pattern [Figure 1]. The cells showed varying degree of cytoplasmic pigmentation, obscuring the nuclear details in some cells. Special attention was given to the edge of the explant, which showed the cell growth initiation in explant culture. The single cell suspensions showed similar morphology but without any definite pattern; the cells were scattered over the surface either uniformly or in aggregates.
To allow cell adhesion the plates were allowed to stay undisturbed in the incubator for 48 hours. Media was then added gradually and made up to 4 ml. Thereafter growth was assessed every alternate day under the phase contrast microscope and half the media changed every third day. The cells were followed in culture for 10 weeks.
Whole mount preparation and staining
Growth was terminated and the membranes along with the cells were fixed using 10% formalin for 4 hours. They were air-dried and then rehydrated before staining with haematoxylin and eosin (H/E). The total area of the monolayer was measured on the whole mount taking the maximum diameter in two directions. Paraffin sections of the monolayer on the membrane were stained with H/E and per-iodic acid Schiffs (PAS) stains.
A students′ ′t′ test was used to compare the time taken for onset of growth in the 2 groups, other parameters affecting growth, like donor age; time interval from death to processing and viability at the time of explantation, being matched.
The mean donor age was 57.3 years. The eyeballs were processed at an average of 50 hours from the time of death. Both groups showed similar viability in the range of 60-78% at the time of explantation. Successful pigmented cell cultures were established in 11 (68.75%) of 16 samples, of which 7 were isolated by mechanical dissociation (group 1) and 4 were isolated by the enzymatic method (group 2). Monolayers of small pigmented hexagonal cells were seen arising from the edge of the explants and spreading away from them [Figures 2 and 3]. The cells became progressively pigmented and formed a confluent monolayer of epithelial cells [Figure 4] arranged compactly like cobblestones on a pavement [Figure 5]. Whole mount preparations showed growth of 100-120 mm2 over the membranes. The individual cells maintained their epithelial morphology and showed densely pigmented cytoplasm, obscuring the nuclear details [Figure 6]. Paraffin sections showed cuboidal cells with dispersed intracellular and extracellular pigment granules [Figure 7].
The morphological characteristics of the cultures in the two groups were similar but the time frame in which these were established differed. Mechanically isolated cells showed onset of growth at an average of 11.5 days (6-23 days), forming confluent monolayers by 22-34 days. Trypsinised samples showed growth by day 31.6 on an average (29-35 days) and formed confluent monolayers by 41-57 days. The difference was found to be significant (P < 0.025).
We evaluated the use of hAM with the purpose of standardising the culture technique and obtaining RPE cells that preserve epithelial cell morphology in vitro. The other surfaces that are likely to prove equally effective would include laminin, fibrinogen or collagen coated petri-plates.12 These would also permit subculture and passaging of cells, which can be cumbersome when working with amniotic membranes.
We also used both mechanical dissection and enzymatic digestion to harvest the cells. Mechanical separation of the cells from their basement membrane resulted in good cell yield with rapid onset of growth. Attempts at using trypsin to harvest the cells also resulted in successful cultures though the time required for the onset of growth was significantly longer (P < 0.025) than that required by the mechanically isolated cells. Though the numbers are small, these findings suggest that mechanical harvesting retains the proliferative potential of adult RPE cells better than enzymatic dissociation.
Previously described dissection techniques required the use of fine jewel-tipped forceps, to peel the RPE from the choroid under dissection microscope or an operating stereo microscope.22 Our technique obviates the need for this equipment. The problem of other cellular contaminants is also eliminated since only the RPE patches are needle-picked and placed on the amniotic membrane.
Human RPE cultures have proved invaluable in understanding the structure and function of these cells. Eventually it should be possible to culture these cells for clinical application, using the most compatible substrate. The RPE layer plays a crucial role in the development of neuroretina from the neuroectoderm during embryonic stages through various factors like fibroblast growth factor (FGF), microphthalmia transcription factor (MITF) etc.23 In future, these cells or the factors derived from these cells may also help induce photoreceptor differentiation in neural cells or other primitive undifferentiated cells in vitro. We believe that new techniques of culturing RPE cells will improve our understanding of their pathophysiology and may serve as a good research tool.
In conclusion primary adult human RPE cell cultures, which retain their epithelial morphology in vitro, may be established from cadaver eyeballs using human amniotic membranes as substrate. This study also proves that the cells isolated by mechanical dissociation yield significantly earlier growth in cultures compared to the isolation by enzymatic digestion.
We thank Dr. Bienvenido V. Castillo for his invaluable advice and guidance. We are also grateful to the Ramayamma Eye Bank for providing us the preserved human amniotic membranes
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Financial Support: This work was funded by the Hyderabad Eye Research Foundation
Proprietary Interest: None