Growth on the anterior surface of the intraocular lens was less prolific than on the posterior capsule. Conclusion. The protein-free model replicates many features ...
Human Lens Epithelial Cell Proliferation in a Protein-free Medium /. Michael Wormstone,* Christopher S. C. Liu *~\ Jean-Marie Rakic,X Julia M. Marcantonio* Gijs F. J. M. Vrensen,% and George Duncan*
Purpose. The ocular humors are relatively low in protein, yet cell growth in the human capsular bag still occurs after extracapsular cataract extraction (ECCE) surgery. This resilient growth gives rise to posterior capsule opacification (PCO) in a significant proportion (30%) of patients. This study compared the ability of human lens cells to proliferate in serum-supplemented and protein-free medium. Methods. Sham cataract operations were performed on human donor eyes. The capsular bag was dissected free, pinned flat on a petri dish, and incubated in Eagle's minimal essential medium (EMEM) alone or in EMEM supplemented with 10% fetal calf serum. Observations were made by phase-contrast microscopy. At the endpoint, capsules were studied by fluorescence or electron microscopy. Mitotic activity was identified using Bromo-2-deoxyuridine labeling and detection techniques. When required, an intraocular lens was implanted when surgery was performed. Results. It was found that human lens cells from a wide age spectrum of donors proliferate and migrate on the lens capsule in the absence of added protein. The rate of growth was age-dependent, such that the posterior capsule was completely confluent after 8.0 ± 0 days (n = 3) and 24.4 ± 5.3 days (n = 8) for donor lenses aged 60 years, respectively. The outgrowth of epithelial cells gave rise to capsular contraction, wrinkling, and increased light scatter. Growth on the anterior surface of the intraocular lens was less prolific than on the posterior capsule. Conclusion. The protein-free model replicates many features of clinically-observed PCO. The resilient cell growth on the natural collagen capsule explains the high prevalence of PCO, especially in younger patients, and suggests that inflammation and external growth factors are not necessary for PCO. Furthermore, the protein-free capsular bag system can be used to explore fundamental questions concerning the autocrine control of lens epithelial cell survival and growth. Invest Ophthalmol Vis Sci. 1997; 38:396-404.
.Despite improvements in surgical techniques, posterior capsule opacification (PCO) remains a major problem associated with extracapsular cataract extraction (ECCE) surgery that requires further treatment in approximately 30% to 50% of patients. PCO arises from the growth of lens epithelial cells remaining on From the *School of Biological Sciences, University of East Anglia, Norwich; f Sussex Eye Hospital, Brighton; the \Department of Ophthalmology, University of Liege, Belgium; and the ^Depart merit of Morphology, The Netherlands Ophthalmic Research Institute, Amsterdam, Netherlands. Sujtported by The Humane Research Trust and by National Eye Institute grant RO1-EY 10558. Submitted for publication August 16, 1996; revised September 24, 1996; accepted September-25, 1996. Proprietary interest category: N. Reprint requests: I. Michael Wormstone, School of Biological Sciences, University of East Anglia, Nonuich, NR4 7TJ, UK.
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the lens anterior and equatorial capsule following surgery.1'2 If it is to be overcome, it will be necessary to understand the underlying mechanisms of cell survival, proliferation, and migration within the lens capsular bag. All of the in vitro studies investigating this important problem, whether carried out on human3 or animal lens cells,4'5 have employed media supplemented with serum6'7 or defined growth factors.8 The aqueous humor in humans, however, normally contains little serum protein,9 and the use of serum-supplemented media may obscure the search into the underlying factors driving the natural growth of these cells. Ishizaki et al10 have recently shown that very young rat lens cells survive when cultured at high densities
Investigative Ophthalmology & Visual Science, February 1997, Vol. 38, No. 2 Copyright © Association for Research in Vision and Ophthalmology
Lens Cell Growth in Protein-free Medium
in protein-free media. They further reported that in lens capsule explants, epithelial cells could be seen to divide in the absence of serum or added proteins, but they did not investigate this important finding in any depth. In contrast, Reddan et al11 have reported that tissue-cultured human lens epithelial cells die in serum-free medium; it therefore seems clear that age and species differences are important. We have investigated the ability of human lens cells of a wide age range to survive and proliferate in vitro by employing a capsular bag culture system that has previously been shown to reflect many of the clinical changes seen with PCO, at least when 10% serum was used to drive growth.6 Using this model, it was possible to compare the characteristics of cell proliferation and migration within the capsular bag cultured in a protein freemedium (unsupplemented Eagle's minimal essential medium [EMEM]) with those previously obtained when the capsular bags were cultured in 10% serum. Using this system, growth could be studied on the lens capsule as well as on implanted intraocular lenses (IOL) and, hence, any influence of underlying matrix on the growth of lens cells could also be studied.
METHODS In Vitro Capsular Bag Model The capsular bag model previously described by Liu et al6 was used. After removal of corneoscleral discs for transplantation purposes, human donor eyes obtained from the East Anglian Eye Bank or the Cornea Bank Amsterdam were used to perform a sham cataract operation, including continuous curvilinear capsulorhexis and hydroexpression of the lens fiber mass, followed by irrigation and aspiration of residual lens fibers. At this stage an IOL could be implanted if required. The capsular bag was then dissected free of the zonules and secured on a sterile polymethylmethacrylate (PMMA) petri dish. Six to eight entomologic pins (Dl; Watkins and Doncaster, Kent) were inserted through the edge of the capsule to retain its circular shape. In certain cultures the anterior capsular disc, once removed, was pinned and cultured either adjacent to the capsular bag or in a separate petri dish. Capsular bags or anterior capsular discs were maintained in 1.5 ml of either of the following two media: EMEM (Sigma, Poole, Dorset) supplemented with 10% fetal calf serum (FCS); or EMEM alone. All media contained 50 mg/1 gentamicin (Sigma, Poole, Dorset). Incubation was at 35°C in a 5% CO2 atmosphere. The medium was replaced every 3 to 4 days. Ongoing observations were performed using phase-contrast microscopy. In total, 54 capsular bags were cultured in protein-free medium and 33 in 10% serum-supplemented medium. An IOL was implanted in 26 capsu-
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lar bags. The model was established and the observations replicated in two centers of study (Norwich and Amsterdam). Bromo-2-Deoxyuridine Protocol A commercial BrdU kit (Boehringer Mannheim Biochemica, Germany) was used to evaluate cell proliferation following the protocol previously described.12 In brief, after the culture period BrdU was added to the culture medium at a concentration of 10 /nmol/1. Incubation was for 2 hours followed by washing (three times) in phosphate-buffered saline (PBS). Subsequently, wholemounts were prepared on chrome aluminum-coated slides, air dried for 1 hour, treated for 30 minutes with cold ethanol (70% ethanol in glycine buffer, 5 //mol/1; pH 2.0; -20°C), and rinsed in PBS (three times). After washing, anti-BrdU was applied for 30 minutes, then the culture was washed again. Next the wholemounts were covered with alkaline phosphatase-labeled sheep anti-mouse antibody. BrdU was visualized with nitroblue tetrazolium and Xphosphate. After 10 minutes the reaction was stopped by transferring the mounts to distilled water. Counterstaining was for 1 minute with Nuclear fast red (Merck, Darmstad, Germany) and Kaiser's glycerine-gelatin was used for mounting. Scanning Electron Microscope Observations After completion of the phase-contrast observations, specimens were fixed for several days in 1.25% glutaraldehyde, 1.0% paraformaldehyde buffered with 0.08M cacodylate to pH 7.3. Specimens were dehydrated in a series of ethanols and critically point dried with CO2. The dried specimens were glued onto stubs and coated with 7 to 10 nm platinum/gold. They were inspected and photographed in a Philips SEM 505 scanning electron microscope (Philips Industries, Eindhoven, NL). Immunocytochemistry of the Capsular Bag Cytoskeletal proteins were visualized by immunocytochemistry and epifluorescence microscopy. All reagents were from Sigma (Poole, Dorset, UK) unless otherwise stated. Washes were for 3 X 15 minutes in PBS/bovine serum albumin (BSA)/Nonidet (0.02% and 0.05%, respectively). The pinned capsules were fixed for 30 minutes in 4% formaldehyde in PBS and permeabilized in PBS containing 0.5% Triton-XlOO, also for 30 minutes. Nonspecific sites were blocked with appropriate serum (1:50 in 1% BSA/PBS). Antivimentin (Clone V9) was diluted 1:100 and applied for 60 minutes at 35°C, followed by washing. Vimentin was visualized with FITC-conjugated anti-mouse serum, used at 1:64 for 60 minutes at 35°C. After extensive washing, cell nuclei were stained with DAPI at 1 mg/ml for 10 minutes at room temperature. The
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stained preparations were again washed extensively, floated onto microscope slides, and mounted in Vectashield mounting medium (Vector Laboratories, Peterborough, UK). Images were viewed with a Zeiss Standard R microscope (Zeiss, Germany) and recorded on Ektachrome 400 (Kodak UK, Hemel Hempstead, UK) with an Olympus (London, UK) OM2 camera.
RESULTS The anterior and equatorial epithelial cells that remained after the surgical manipulations not only survived without added protein, but also continued to show most of the dynamic characteristics previously observed in serum-cultured capsules.67 It was possible to study these preparations in a noninvasive manner by optical microscopy for over 100 days and to compare the characteristics of growth in the presence and absence of added proteins. Growth on the Posterior Capsule In protein-free EMEM, the first indications of activity on the posterior capsule were observed within 2 to 6 days after surgery, adjacent to the bow region. This was difficult to photograph using phase-contrast microscopy because of degradation of the image by the overlying residual fibers, capsule, and anterior epithelial cells. Unequivocal evidence of cell division and spatial distribution of mitotic activity was provided by BrdU labeling and detection of wholemount specimens.12 The highest frequency of labeled cells in early culture was always in the bow region and the same relative distribution of BrdU-labeled cells was observed in protein-free and serum-supplemented cultures. The overall incidence of positive cells, however, was consistendy higher in the presence of serum, and the difference was especially marked when comparing
the labeling of older capsules (Figs. 1A to 1C). The "wounded" area in the vicinity of the anterior rhexis, in contrast, showed no or a relatively low labeling frequency at this early stage (Fig. ID). With day-to-day observations by phase-contrast microscopy, the clearest indication of activity within the capsular bag occurred when cells emerged from beneath the anterior capsule at the rhexis region (Fig. 2a). In protein-free media, this could take more than 1 week to occur. Subsequently, growth continued across the posterior capsule until a confluent monolayer of cells was formed (Fig. 2b). In order to demonstrate more clearly the confluent nature of cells on the posterior capsule, immunocytochemical staining of vimentin filaments was performed (Fig. IE). As was the case in serum-supplemented cultures, wrinkles began to develop on the posterior capsule as cells in proteinfree medium reached confluency; these subsequently increased in number and magnitude with increasing time in culture (Fig. 2c). One significant observation made in this study was the effect of donor age on cell growth rate in proteinfree conditions (Fig. 3 and also Fig. 1). In the presence of serum, the time required for confluent coverage of the posterior capsule was only marginally longer in older capsules than in younger capsules, but in the absence of serum, growth in older capsules was greatly retarded. In fact, capsules from donors of older than 60 years took approximately three times longer to achieve confluency than did those in the younger than 40 years age group (Fig. 3). In older donors, approximately 4% of cultures failed to reach confluency in protein-free medium, whereas in all other cases confluency was always attained. Despite the slower growth rates achieved in protein-free medium, mitotic cell division was observed on the posterior capsule several days after confluency was achieved (Fig. IF). In fact, dynamic changes still occurred on the posterior capsule after confluency was reached and cells regressed
FIGURE l. BrdU labeling and detection of cells near the equatorial (Eq) region of an 83year-old donor lens cultured for three days in (A) protein-free medium and (B) Eagle's minimal essential medium supplemented with 10% fetal calf serum. (C) Increased magnification of same wholemount as (A) showing BrdU labeling of bow region cells in greater detail. (D) Absence of BrdU labeling in cells of the "wounded" rhexis area (RZ). In Figure la to Id, the cells were counterstained with nuclear fast red and the heavily stained area is of a cluster of nonmitotic cells. A neighboring area on the anterior capsule (AC) is denuded of cells; such displacement is characteristic for this type of procedure. (E) Epifluorescence micrograph of cells at the center of the posterior capsule. Vimentin was visualized with fluorescein isothiocyanate. (F) Epifluorescence micrograph of mitotic cell division on the posterior capsule of a 72-year-old donor eye visualized using DAPI. In this case confluency was reached by 19 days and this image was obtained 7 days later. The micrographs represent a fields of view of 3.2 X 2.9 mm (A,B,D), 1.3 X 1.2 mm (C), 196 X 178 nm (E), and 80 X 69 jum (F).
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from certain areas on the capsule and appeared to accumulate in others (Fig. 2c). The data shown in Figure 4 were obtained from one of the capsules (aged 78 years) that failed to reach confluency when cultured in protein-free medium. In this particular case, only 15% of the posterior capsule was covered before growth ceased. When the culture medium was supplemented with 10% FCS, immediate and rapid growth was observed across the capsule until complete cell coverage was achieved. Growth on the Intraocular Lens When a PMMAIOL was implanted, growth could also be seen on its anterior optical surface, both in the presence and absence of serum; in both cases, growth was preceded by the formation of an adherence zone6 between the rhexis edge and the IOL. In the presence of serum, there was a rapid and complete coverage of the anterior surface of the IOL, which lagged only a day or so behind the coverage of the posterior capsule (Data not shown, but see also Liu et al6). In the absence of serum, complete cell coverage of the IOL was never achieved and, in some cases, no cells were visible on the IOL, but growth still occurred on the posterior capsule. In those cases where growth on the anterior IOL did occur (approximately 80%), initial outgrowth consisted either of isolated cells or small groups at specific locations (Fig. 5a). Cells then continued to grow onto the anterior surface of the IOL for a limited period, but stopped considerably short of complete coverage (maximum ~30%) and in most cases some regression occurred (Fig. 5b). Growth From the Explanted Anterior Capsular Disc Anterior capsular discs are routinely available after cataract surgery and have been used to obtain human lens cell cultures for a variety of purposes.1314 All of the studies carried out to date have involved the use of serum or supplemented media and we have confirmed that very vigorous explant cultures can be obtained from such discs, even from older donors, in the presence of serum. What is more surprising is that relatively vigorous cultures can be obtained from discs cultured in the absence of supplements (Fig. 6). In those cultures where growth was observed on the PMMA dish (~70%), it mimicked that observed on PMMA IOLs. Adherence zones first formed between the disc edge and the dish, then cells grew out several cell diameters beyond the rhexis (Fig. 6) before regressing in prolonged culture. In some cases, although regrowth occurred on the capsular disc itself, cells failed to move onto the adjacent PMMA surface. Anterior discs were either cocultured with a capsular bag or cultured alone and in both cases the same behavior was recorded, indicating that the central epithelial
cells do not require a growth signal from bow region cells in order to proliferate. DISCUSSION Most studies concerning the growth of lens cells have not been carried out on human capsular bags, but rather on either tissue-cultured cells15 or explanted anterior epithelia1314 from a range of species. Most of these preparations fail to grow in protein-free media, although cells on the anterior capsule do survive,16 presumably because they reside on the capsule at high density.10 Interestingly, addition of fibroblast growth factor (FGF) to protein-free media bathing capsular discs removed from young rat lenses instigates both proliferation and differentiation in a dose-dependent manner.17 In tissue-cultured cells removed from the capsule, addition of growth factors to protein-free media appears to be necessary for growth.1118 The pinned capsular bag provides a stable growth substrate with the vital combination of an initial high density of cells in the anterior epithelium and the availability of free capsular matrix space onto which the cells can migrate when stimulated to grow. This study shows that, given the appropriate combination of stability, density, and space, human epithelial cells can readily proliferate and migrate in a protein-free medium and that it is the cells in the normally active equatorial region that first divide, rather than those in the "wounded" rhexis region. Ultimately, cells in this latter region do divide and migrate onto the IOL. This corresponds in time with a proliferative movement of cells off the explanted disc and onto the noncoated surface of the PMMA culture dish. Although movement onto the plastic is less vigorous than that onto the capsule, human lens cells appear to exhibit a greater migratory and proliferative capacity than very young rat lens cells, which remain strictly on the capsule, even when stimulated with FGF, unless the culture dish is precoated with laminin.16 Similarly, Ishizaki et al10 showed that explanted rat cells do not move from the disc onto uncoated plastic in protein-free media, but only migrate onto laminin-coated plastic. Several very recent studies have shown that lens cells can produce a range of growth factors19 and growth factor-associated proteins such as transferrin.20 It is likely, therefore, that such molecules are produced at a relatively high local concentration by the closely-packed cells that remain in the capsular bag after surgery, so that very active proliferation and migration can occur across the natural matrix of the capsule. The matrix can further act as a reservoir for growth factors. It has been shown, for example, that certain cells release growth factors such as FGF selectively into their extracellular matrix and not into conditioned medium.21 The importance of the matrix is
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