Molecular Vision 2007; 13:524-533 <http://www.molvis.org/molvis/v13/a56/> Received 8 November 2006 | Accepted 2 April 2007 | Published 3 April 2007

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Optimization of culture conditions for porcine corneal endothelial cells

Stéphanie Proulx,¹ Jean-Michel Bourget,¹ Nicolas Gagnon,¹ Sophie Martel,¹ Alexandre Deschambeault,¹ Patrick Carrier,¹ Claude J. Giasson,^1,2 François A. Auger,¹ Isabelle Brunette,³ Lucie Germain¹
 
 

¹Laboratoire d'Organogénèse Experiméntale (LOEX), Hôpital du St-Sacrement du Centre Hospitalier Affilié Universitaire de Québec and Department of Oto-Rhino-Laryngology and Ophthalmology, Université Laval, Québec, Canada; ²School of Optometry, Université de Montréal, Montréal, Québec, Canada; ³Ophthalmology Research Unit, Department of Ophthalmology, Maisonneuve Rosemont Hospital, Université de Montréal, Montréal, Québec, Canada

Correspondence to: Dr. Lucie Germain, LOEX, Hôpital du St-Sacrement du Centre Hospitalier Affilié Universitaire de Québec, 1050 chemin Sainte-Foy, Québec (QC), Canada, G1S 4L8; Phone: (418) 682-7663; FAX: (418) 682-8000; email: lucie.germain@chg.ulaval.ca

Abstract

Purpose: To optimize the growth condition of porcine corneal endothelial cells (PCEC), we evaluated the effect of coculturing with a feeder layer (irradiated 3T3 fibroblasts) with the addition of various exogenous factors, such as epidermal growth factor (EGF), nerve growth factor (NGF), bovine pituitary extract (BPE), ascorbic acid, and chondroitin sulfate, on cell proliferation, size, and morphology.

Methods: PCEC cultures were seeded at an initial cell density of 400 cells/cm² in the presence or absence of 20,000 murine-irradiated 3T3 fibroblast/cm² in the classic media Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 20% fetal bovine serum (FBS). Mean cell size and bromodeoxyuridine incorporation was assessed at various passages. Growth-promoting factors were studies by seeding PCEC at 8,000 cells/cm² in DMEM with 20% FBS or Opti-MEM I supplemented with 4% FBS and one of the following additives: EGF (0.5, 5, 25 ng/ml), NGF (5, 20, 50 ng/ml), BPE (25, 50, 100, 200 μg/ml), ascorbic acid (10, 20, 40 μg/ml) and chondroitin sulfate (0.03, 0.08, 1.6%), alone or in combination. Cell number, size and morphology of PCEC were assessed on different cell populations. Each experiment was repeated at least twice in three sets. In some cases, cell cultures were maintained after confluence to observe post-confluence changes in cell morphology.

Results: Co-cultures of PCEC grown in DMEM 20% FBS with a 3T3 feeder layer improved the preservation of small polygonal cell shape. EGF, NGF, and chondroitin sulfate did not induce proliferation above basal level nor did these additives help maintain a small size. However, chondroitin sulfate did help preserve a good morphology. BPE and ascorbic acid had dose-dependent effects on proliferation. The combination of BPE, chondroitin sulfate, and ascorbic acid significantly increased cell numbers above those achieved with serum alone. No noticeable changes were observed when PCEC were cocultured with a 3T3 feeder layer in the final selected medium.

Conclusions: Improvements have been made for the culture of PCEC. The final selected medium consistently allowed the growth of a contact-inhibited cell monolayer of small, polygonal-shaped cells.

Introduction

The corneal endothelium is a single layer of flattened cells adhered to Descemet's membrane. It forms a boundary between the corneal stroma and the anterior chamber. The main role of the corneal endothelium is to maintain corneal transparency by regulating stromal hydration. Since endothelial cell density naturally decreases with age, human corneal endothelial cells (HCEC) are considered to be nonproliferative in vivo. Joyce et al. [1-3] showed that HCEC in vivo are arrested in the G₁ phase of the cell cycle, while studies by Wilson et al. [4,5] demonstrated that HCEC in vivo retain proliferative capacity.

Previous investigators have developed culture techniques and medium formulation that promote consistent culture of untransformed corneal endothelial cells from human donors [6-11] or animals [12-17]. Effects of numerous growth-promoting agents were tested using cultured HCEC. Samples et al. [18] showed the effect on cell growth of serum, epidermal growth factor (EGF), fibroblasts growth factor (FGF), nerve growth factor (NGF), bovine pituitary extract (BPE), and endothelial cell growth factor. Engelmann et al. [8] improved their previously optimized medium [7] and reduced the serum concentration down to 5% by adding ascorbic acid, insulin, selenium, transferrin, lipids, and FGF. Joyce et al. [10,11] confirmed mitotic or morphologic changes in response to serum, EGF, NGF, and BPE.

Animal models are required for preclinical studies. Because the biology of the eye of the pig is close to that of the human in many respects [19], it would be important to be able to cultivate porcine corneal endothelial cells (PCEC) in a growth medium that allows rapid proliferation while keeping a small cell size with a polygonal morphology at confluence. Several investigators used PCEC cultures for in vitro studies [20-27]. However, few have tested the stimulation of cell proliferation by growth-promoting agents. To our knowledge, only Lee et al. [14] improved the growth of PCEC cultures through their tests of different concentrations of EGF and chondroitin sulfate. Thus, the aim of this study was to further optimize the growth medium used for the culture of PCEC.

The coculture with a feeder layer may help in the initiation of cultures from a low number of cells or from cells with low proliferative ability. The first use of irradiated 3T3 as a feeder layer was reported 30 years ago, when they were shown to favor the formation of cell colonies from diluted human epidermal keratinocytes in culture [28]. This technique led to the clinical application of cultured epidermis for the treatment of burn patients [29,30]. These days, feeder layer of irradiated 3T3 cells are used to coculture many different cell types, including epithelial cells from skin [31] and cornea [32-36]. Alternatively, the growth of cells may be stimulated with additives to culture media. In this study, we evaluated the mitotic changes of PCEC in response to stimulation by growth-promoting agents, such as serum, EGF, NGF, BPE, ascorbic acid, and chondroitin sulfate.

Methods

Porcine corneal endothelial cell isolation

The use of animal cells in this study followed the guidelines set by our institution for animal cell research. Porcine eyes were obtained from a local slaughterhouse within 24 h after death. Corneas were dissected out of the globes using a curved scissors (Storz, St. Louis, MO) and washed several times in Dulbecco's Modified Eagle's Medium (DMEM; Invitrogen, Burlington, Ontario, Canada) containing 100 UI/ml penicillin (Sigma, Oakville, Ontario, Canada) and 25 μg/ml gentamicin (Schering Canada, Pointe-Claire, Québec, Canada). Corneas were incubated 30 min at 37 °C in 2.5 mg/ml dispase (Roche, Laval, Québec, Canada). The loosened Descemet's membrane was then peeled off, and endothelial cells were isolated per guidelines described in Zhu et al. [10]. Pooled cells were then centrifuged, resuspended in fresh medium, counted, and plated on dishes coated with fibronectin, collagen, and albumin (FNC; Athena Enzyme Systems, Baltimore, MD) according to the manufacturer's instructions. The FNC coating was used for primary cultures (P0) only and was not used for subsequent passaged cells. Low passaged cells were routinely cryopreserved in 90% FBS (Hyclone, Logan, UT)/10% dimethylsulfoxide (DMSO; Sigma). Unfrozen cells were allowed at least one passage before their use in experiments. After thawing, they exhibited normal cell proliferation and morphology (data not shown). For each study, a Student's t-test was performed to analyze statistical significance with p<0.001 considered to be significant.

Effect of coculture

Freshly isolated PCEC in primary culture (P0) seeded at 400 cells/cm² were cultured in DMEM 20% FBS or in the selected medium in plastic flasks with or without a feeder layer of irradiated murine 3T3 cells (20,000 3T3/cm²). Cells were trypsinized at 60-80% confluence and subcultured in the same conditions. Cell number and mean cell size was assessed using a cell counter and particle size analyzer (Beckman Coulter Z2, Mississauga, Ontario, Canada) set to evaluate mean cell size of cells counted between 7.9 and 24.1 μm. Experiments were done three times in triplicate using different pooled cells, and three different counts were done for each sample. Other cultures were maintained one week after confluence to document their morphology at post-confluence. For each condition, three other flasks were used for bromodeoxyuridine (BrdU; Sigma) analysis (Figure 1A).

Bromodeoxyuridine analysis

The 60-80% confluent cultures were exposed to medium containing 10 μM BrdU for 45 min at 37 °C. The cultures were then rinsed, trypsinized, and the cells fixed in 70% ethanol. Cells were kept at -20 °C until staining. Fixed PCEC were stained with a fluorescein isothiocyanate (FITC)-conjugated mouse anti-BrdU monoclonal antibody (clone 3D4; BD Pharmingen, Oakville, Ontario, Canada) as well as propidium iodide (Sigma) following a modified version of a method developed by Van Erp et al. [37]. Stained cells were analyzed by flow cytometry on a FACScalibur (Becton Dickinson) and analyzed using the program CellQuestPro (Becton Dickinson). For negative control, cells were stained with FITC-conjugated mouse IgG_1κ monoclonal isotype control antibody (clone MOPC-21; BD Pharmingen).

Effect of basal media

PCEC were seeded at 12,000 cells/cm² in six-well plates either in DMEM or Opti-MEM I (Invitrogen), both supplemented with 5% FBS. Triplicate wells were counted twice as previously described from day 1 to day 6 (Figure 1B). Opti-MEM I (Invitrogen) is a modification of Eagles Minimum Essential Medium, buffered with HEPES and sodium bicarbonate (2.4 g/l) and supplemented with hypoxanthine, thymidine, sodium pyruvate, L-glutamine, and trace elements. Calcium chloride (0.2 g/l) was added in the Opti-MEM I solution.

Effect of growth-promoting additives

PCEC routinely passaged at 8,000 cells/cm² were cultured in DMEM 20% FBS and switched to Opti-MEM I (Invitrogen) supplemented with 0.2 g/l CaCl₂ and 4% FBS and antibiotics overnight. PCEC were then trypsinized and plated at 8,000 cells/cm² and cultured in the presence of the following additives (used alone or in combination): EGF (0.5, 5, and 25 ng/ml; Austral Biologicals, San Ramon, CA), NGF from mouse submaxillaries (5, 20, 50 ng/ml; Biomedical Technologies, Stoughton, MA), BPE (25, 50, 100, and 200 μg/ml; Biomedical Technologies), ascorbic acid (10, 20, and 40 μg/ml), and chondroitin sulfate A sodium salt from bovine trachea (0.03, 0.08, and 1.6%; Sigma). Cell number, size and morphology were assessed on day 4. Each condition was done at least twice in triplicate using different pooled cells, and each sample was counted three times using a cell counter and particle size analyzer that was set to evaluate mean cell size of cells counted between 7.9 and 24.1 μm. Ascorbic acid and chondroitin sulfate were added fresh at every medium change. Also, the medium containing 50 μg/ml BPE, 20 μg/ml ascorbic acid, and 0.08% chondroitin sulfate was tested in the presence (4% and 8%) or absence of serum as previously described. Cells grown without serum were incubated overnight in 4% serum to allow cell adhesion (Figure 1C).

Results

Effect of coculture

We assessed the effect of coculturing PCEC with irradiated 3T3 fibroblasts on mean cell size, morphology, and proliferation using BrdU incorporation. PCEC were cultured in DMEM 20% FBS with or without a feeder layer. Endothelial cells cultured in DMEM 20% FBS with a feeder layer retained their distinctive endothelial morphology in addition to their small size longer than cultures without a feeder layer. At the low seeding density of 400 cells/cm², the cultures at passage 1 (P1) lost their uniformity in size and morphology (Figure 2A,C), whereas cells cultured with a feeder layer at the same density also at P1 retained their small size (Figure 2A) and their typical polygonal morphology (Figure 2D). BrdU analysis demonstrated that cells in both conditions incorporated BrdU at a similar percentage, indicating that the feeder layer did not affect the growth rate (Figure 2B).

Effect of basal media

We first evaluated the growth response of PCEC cultured with the classic DMEM or with the Opti-MEM I. The same cell populations and the same serum concentration used in both cases permitted a direct comparison of the number of cells after various days in culture. Opti-MEM I induced a statistically significant greater number of cells (after 2, 3, 4, 5, and 6 days) than that of the DMEM (Figure 3). Therefore, Opti-MEM I was used in other experiments for the optimization of the growth medium for PCEC.

Effect of growth-promoting additives

To study the dose-dependent effect of several growth-promoting factors, PCEC were cultured in a growth medium containing a reduced-serum concentration (Opti-MEM I 4% FBS). The effect of EGF, NGF, BPE, chondroitin sulfate, and ascorbic acid were tested on the proliferative response, morphology, and cell size of PCEC (Figure 4). Figure 4B shows that EGF at concentrations of 0.5 and 25 ng/ml and all concentrations tested for NGF had no significant stimulatory effect on the proliferation of PCEC above basal levels nor did they help maintain a small mean cell size. A concentration of 5 ng/ml had a small but statistically significant increase in cell number, however this concentration did not have a significant effect on mean cell size and morphology. Therefore, EGF and NGF were not kept for the final selected medium.

BPE shows a dose-dependent effect on PCEC growth and mean cell size as shown in Figure 4A and Figure 4B. The highest number and the smallest cells were obtained with a BPE concentration of 200 μg/ml. However, high concentrations of BPE greatly changed the morphology of PCEC. Cells at 200 μg/ml BPE had a fibroblast-like morphology (Figure 4D). In contrast, 50 μg/ml BPE preserved an adequate cell morphology. It was thus chosen as the optimal BPE concentration for PCEC cultures (Figure 4C).

Following the addition of freshly prepared solutions, ascorbic acid had a dose-dependent effect on cell growth as shown in Figure 4B. The highest concentration of ascorbic acid tested (40 μg/ml) yielded the highest cell number and the smallest cells. However, the typical endothelial cell morphology was more closely achieved with 20 μg/ml (Figure 4E).

For PCEC, 0.03 and 0.08% chondroitin sulfate reduced cell growth by, respectively 1.4 and 1.5 times, and 1.6% dramatically reduced (3.6X) the growth of PCEC compared to the control culture (Figure 4B). A concentration of 0.08% chondroitin sulfate was kept in the final selected medium because it was shown to keep cells growing in tightly packed units, thus aiding cell morphology (Figure 4F).

The medium containing the combined factors conducive to the best growth, mean cell size, and morphology was tested (Figure 5) in the presence or absence of serum. Culturing PCEC in Opti-MEM I supplemented with 0%, 4%, or 8% FBS with 50 μg/ml BPE, 20 μg/ml ascorbic acid and 0.08% chondroitin sulfate resulted in a significant increase in cell number when compared to cells grown in Opti-MEM I only (0% FBS) or with serum alone (4% or 8% FBS; Figure 5B). Adding the supplements in 0% serum resulted in a 4.1 fold increase in cell number, and the total cell number in the supplemented 0% serum medium was similar to that achieved with DMEM 20% FBS. When added in 4% and 8% serum, the supplements increased cell number 2.1 and 2.4 times, respectively. Adding the supplements also resulted in a smaller mean cell size. As shown in Figure 5C, PCEC grown in the classic DMEM 20% FBS formed a monolayer of cells harboring different shapes including some that were elongated. However, PCEC grown in our selected medium (8% serum and additives) formed a uniform monolayer of tightly packed cuboidal cells (Figure 5D), indicating that they harbor a morphology similar to the normal endothelial morphology found in vivo.

Selected medium and 3T3 coculture

We then evaluated the effect of coculturing PCEC with a feeder layer of irradiated 3T3 cells grown in this selected medium with a seeding cell density of 400 cells/cm². As shown in Figure 6A, mean cell size at P1 remained small regardless of the presence of 3T3 cells. Morphology was similar (Figure 6C, 6D), as was the percentage of BrdU incorporation (Figure 6B). Thus, no significant effect of the 3T3 feeder layer was found when PCEC were grown in our selected medium.

Discussion

Since the first cell culture of corneal endothelial cells [38], many investigators have attempted to improve growth culture conditions in order to maximally amplify cells while retaining morphology and function [6-8,10,11,17,18,39]. One way of improving culture conditions is to grow them in coculture with an irradiated feeder layer. The addition of a feeder layer of irradiated fibroblasts supports the formation of colonies from a single cutaneous epithelial cell [28,40]. This powerful method is now applied for the expansion of various cell types for in vitro purposes and clinical applications of cultured epidermis and cornea [29,30,33-36]. Since corneal endothelial cells are in close contact with the corneal stroma through Descemet's membrane, it has been speculated that keratocytes secrete growth factor or nutrients that favor the well being of corneal endothelial cells. When cells are grown in cocultures, fibroblasts are not proliferative (since irradiated) but are still able to secrete nutrients or growth factors. Their beneficial influence is well known for the culture of epithelial cells from skin [28,40] and cornea [32-36]. We have shown that coculturing PCEC with irradiated murine fibroblasts does not affect proliferation. Furthermore, a new media supplemented with serum and additives was identified which allows PCEC to retain their small cell size and morphology on plastic as well as in DMEM 20% FBS in coculture with irradiated fibroblasts.

For cells to grow in vitro, they require a complex mixture of nutrients and growth factors which contributes to an adequate physiological environment. A complete analysis demands well characterized assay systems that allow the discrimination of a single component's contribution to cell growth. In this study, we used direct cell counts to determine the relative effect of various growth-promoting agents on PCEC, as was previously done by many investigators for the optimization of corneal endothelial cell cultures [10,11].

EGF was tested because of its known positive effect on corneal wound healing in ex vivo models and in culture for human [10,11,15,41,42] and porcine [14] cells. Mixed results have been previously reported on the effect of EGF on corneal endothelial cell growth. Some studies show an increase in proliferation for bovine [12,17], rabbit [43], primates [44], and humans [18,45], while others show that EGF either has no effect [30] (human) or has an inhibitory effect [46] (rabbit) on cell growth. EGF can have a different effect, depending on the age of the donor cells. Recent studies using cultured HCEC indicated that EGF has a dose-dependent effect on proliferation in a range of 0.05 to 5 ng/ml, with a peak cell count at 5 ng/ml [10,11]. EGF moderately stimulated proliferation in cells from younger human donors, but did not consistently stimulated proliferation in cells from older donors [10,11]. Using PCEC, a previous study showed that low concentrations of EGF (10 ng/ml) had no significant stimulatory effect while high concentrations (100 ng/ml) stimulated cell growth [14]. Our results are in accordance with this study since the low concentrations of 0.5 to 25 ng/ml did not considerably increase PCEC cell numbers.

NGF is secreted in the aqueous humor [47,48]. Thus, it could influence endothelial cells since immunochemistry studies indicated that HCEC express TrkA [49], the high affinity receptor for NGF [50]. In bovine corneal endothelial cell cultures, concentrations of 1, 10, and 100 ng/ml of NGF failed to significantly stimulate DNA synthesis [17]. A study that used human cells reported that NGF (0.2-200 ng/ml) did not stimulate proliferation, but appeared to have a positive effect on cell morphology [10]. With PCEC, we did not see an effect of this growth factor on proliferation, mean cell size, or morphology.

Of the growth-promoting additives tested in this study, only BPE and ascorbic acid demonstrated a positive effect on cell growth and mean cell size. BPE is a broadly used supplement to culture a variety of epithelial and endothelial cells. It is routinely used as a mitogenic supplement in serum-free growth medium. In addition to its mitogenic activity, BPE contains a variety of growth factors and hormones with reported antioxidant activity [51]. Previous studies have shown a positive effect of BPE on cultured corneal endothelial cells from human origin [10,11]. In this study, the effective concentrations of BPE were between 25-200 μg/ml, which is consistent with a previous study done with HCEC showing that BPE induced a dose-dependent response at concentrations of 0.1 to 100 μg/ml, with a peak cell number reported at 100 μg/ml [10].

Ascorbic acid is an important water-soluble antioxidant found in numerous media. Ascorbic acid (75 μg/ml) added daily to cultured rabbit corneal endothelial cells had an inhibitory effect on cell growth [46]. However, Zhu and Joyce [10,11] routinely included ascorbic acid in their optimized growth medium for HCEC cultures. Ascorbic acid was previously found to have a dose-dependent increase in cell number on human cell cultures at concentrations of 6.25 to 25 μg/ml [8]. Further increases in ascorbic acid concentration apparently reversed this stimulation. A concentration of 20 μg/ml of ascorbic acid maximally stimulated growth [8]. Our results showed a positive effect of 10-40 μg/ml ascorbic acid on the growth of PCEC and are consistent with these previous studies.

Chondroitin sulfate is a mucopolysaccharide with an approximate molecular weight of 50,000 Da. It is found in trace amounts in the human corneal stromal layer [52]. The endothelial protective effect of chondroitin sulfate in the preservation media for cornea in eye banks has been proven by many investigators [53-58], however, reports concerning its effects on corneal endothelial cell cultures are rare. A concentration of 0.08% (0.8 mg/ml) was found to have a positive effect on proliferation and also prevented enlargement of HCEC cultures [6]. Yue et al. [39] showed that low concentrations (100 μg/ml to 1 mg/ml) of chondroitin sulfate had little effect on proliferation. However, high concentrations (13.5 and 25 mg/ml) significantly promoted HCEC growth during the 1- to 2-week incubation period. A study using PCEC demonstrated the addition of 1 mg/ml chondroitin sulfate to the growth medium had no effect on proliferation, and that higher concentrations (25 mg/ml) resulted in inhibition of proliferation [14]. In this study we showed chondroitin sulfate decreased proliferation of PCEC compared to control. However, we kept some chondroitin sulfate in the selected medium because of its beneficial aspect for overall cell morphology.

Culturing PCEC in Opti-MEM I supplemented with 8% FBS, 50 μg/ml BPE, 20 μg/ml ascorbic acid, and 0.08% chondroitin sulfate resulted in a significant increase in cell proliferation when compared to cells grown in Opti-MEM I supplemented with 8% FBS alone (Figure 2B). In confluent cultures, the shape of endothelial cells also provides important information regarding the overall health of the monolayer. Growth in our selected culture medium (Opti-MEM I supplemented with 8% FBS, 50 μg/ml BPE, 20 μg/ml ascorbic acid, and 0.08% chondroitin sulfate) consistently generated cultures of small, polygonal-shaped cells, a morphology closer to that of the endothelium monolayer in vivo. Taken together, our results present species-specific effects of various supplements. For example, 0.5 to 25 ng/ml EGF did not affect proliferation of PCEC, whereas this growth factor has a dose-dependent effect on proliferation of HCEC in a range of 0.05 to 5 ng/ml [10,11]. Such species-specific differences are frequently found in cell cultures of other cell types, such as skin epithelial cells. For instance, high concentrations of calcium inhibits proliferation of mouse epidermal cells [59], but does not affect proliferation of human epidermal cells [30].

In summary, improvements have been made for the culture of PCEC. The present study compared the effect of several growth-promoting agents on proliferation, mean cell size, and morphology of PCEC. EGF, NGF, and chondroitin sulfate did not induce proliferation above basal levels. BPE and ascorbic acid stimulated growth, the combination of which had an additive effect, significantly increasing cell numbers above that achieved with serum alone. This study showed that PCEC cultures can be initiated with a small number of viable cells, such as would be the case from a small biopsy for the culture of autologous cells. The selected medium allowed the production of a contact inhibited cell monolayer that can be grown in sufficient quantities to permit a high cell density seeding of these cells on a stroma for the reconstruction of an autologous posterior cornea in the pig model. Further studies will be required to evaluate the efficiency of the reconstructed corneas to support vision in the pig model following transplantation of these reconstructed porcine corneas before developing clinical applications.

Acknowledgements

This work was supported by the Canadian Institutes of Health Research (CIHR; L.G., I.B., F.A.A.) and the 'Réseau de Recherche en Santé de la Vision' from the 'Fonds de la Recherche en Santé du Québec' (FRSQ; L.G., C.J.G., I.B., F.A.A.). L.G. is the recipient of a Canadian Research Chair from the CIHR in Stem Cell and Tissue Engineering. N.G. held studentship from the FRSQ. S.P. held studentship from the Natural Sciences and Engineering Research Council of Canada (NSERC). The authors would like to thank Sébastien Larochelle for his technical assistance in the FACS analysis.

References

1. Joyce NC, Meklir B, Joyce SJ, Zieske JD. Cell cycle protein expression and proliferative status in human corneal cells. Invest Ophthalmol Vis Sci 1996; 37:645-55.

2. Joyce NC, Navon SE, Roy S, Zieske JD. Expression of cell cycle-associated proteins in human and rabbit corneal endothelium in situ. Invest Ophthalmol Vis Sci 1996; 37:1566-75.

3. Joyce NC. Cell cycle status in human corneal endothelium. Exp Eye Res 2005; 81:629-38.

4. Wilson SE, Lloyd SA, He YG, McCash CS. Extended life of human corneal endothelial cells transfected with the SV40 large T antigen. Invest Ophthalmol Vis Sci 1993; 34:2112-23.

5. Wilson SE, Weng J, Blair S, He YG, Lloyd S. Expression of E6/E7 or SV40 large T antigen-coding oncogenes in human corneal endothelial cells indicates regulated high-proliferative capacity. Invest Ophthalmol Vis Sci 1995; 36:32-40.

6. Engelmann K, Bohnke M, Friedl P. Isolation and long-term cultivation of human corneal endothelial cells. Invest Ophthalmol Vis Sci 1988; 29:1656-62.

7. Engelmann K, Friedl P. Optimization of culture conditions for human corneal endothelial cells. In Vitro Cell Dev Biol 1989; 25:1065-72.

8. Engelmann K, Friedl P. Growth of human corneal endothelial cells in a serum-reduced medium. Cornea 1995; 14:62-70.

9. Bednarz J, Doubilei V, Wollnik PC, Engelmann K. Effect of three different media on serum free culture of donor corneas and isolated human corneal endothelial cells. Br J Ophthalmol 2001; 85:1416-20.

10. Zhu C, Joyce NC. Proliferative response of corneal endothelial cells from young and older donors. Invest Ophthalmol Vis Sci 2004; 45:1743-51.

11. Joyce NC, Zhu CC. Human corneal endothelial cell proliferation: potential for use in regenerative medicine. Cornea 2004; 23:S8-S19.

12. Gospodarowicz D, Mescher AL, Birdwell CR. Stimulation of corneal endothelial cell proliferations in vitro by fibroblast and epidermal growth factors. Exp Eye Res 1977; 25:75-89.

13. Giguere L, Cheng J, Gospodarowicz D. Factors involved in the control of proliferation of bovine corneal endothelial cells maintained in serum-free medium. J Cell Physiol 1982; 110:72-80.

14. Lee HJ, Lin CP, Chen CW. The effects of epidermal growth factor and chondroitin sulfate on the animal corneal endothelial cell culture. Gaoxiong Yi Xue Ke Xue Za Zhi 1991; 7:614-21.

15. Schultz G, Cipolla L, Whitehouse A, Eiferman R, Woost P, Jumblatt M. Growth factors and corneal endothelial cells: III. Stimulation of adult human corneal endothelial cell mitosis in vitro by defined mitogenic agents. Cornea 1992; 11:20-7.

16. Woost PG, Jumblatt MM, Eiferman RA, Schultz GS. Growth factors and corneal endothelial cells: II. Characterization of epidermal growth factor receptor from bovine corneal endothelial cells. Cornea 1992; 11:11-9.

17. Woost PG, Jumblatt MM, Eiferman RA, Schultz GS. Growth factors and corneal endothelial cells: I. Stimulation of bovine corneal endothelial cell DNA synthesis by defined growth factors. Cornea 1992; 11:1-10.

18. Samples JR, Binder PS, Nayak SK. Propagation of human corneal endothelium in vitro effect of growth factors. Exp Eye Res 1991; 52:121-8.

19. Pond WG, Houpt KA. The Biology of the Pig. Ithaca (NY): Comstock Publishing; 1978.

20. Chang CH, Lin CP, Wang HZ. A tetrazolium-based(MTT) assay for measuring viability of cultured porcine corneal endothelium. Gaoxiong Yi Xue Ke Xue Za Zhi 1993; 9:610-5.

21. Wang HZ, Chang CH, Lin CP, Tsai MC. Using MTT viability assay to test the cytotoxicity of antibiotics and steroid to cultured porcine corneal endothelial cells. J Ocul Pharmacol Ther 1996; 12:35-43.

22. Schneider AI, Maier-Reif K, Graeve T. Constructing an in vitro cornea from cultures of the three specific corneal cell types. In Vitro Cell Dev Biol Anim 1999; 35:515-26.

23. Sobottka Ventura AC, Engelmann K, Bohnke M. Fetal calf serum protects cultured porcine corneal endothelial cells from endotoxin-mediated cell damage. Ophthalmic Res 1999; 31:416-25.

24. Wu KY, Hong SJ, Huang HT, Lin CP, Chen CW. Toxic effects of mitomycin-C on cultured corneal keratocytes and endothelial cells. J Ocul Pharmacol Ther 1999; 15:401-11.

25. Wusteman M, Wang LH. Some observations on the use of cultured corneal endothelial cells as a model for intact corneal endothelium. Cryobiology 2000; 40:376-80.

26. Wang HZ, Hong SJ, Wu KY. Change of calcium and cAMP concentration by adrenoceptor agents in cultured porcine corneal endothelial cells. J Ocul Pharmacol Ther 2000; 16:299-309.

27. Wollensak G, Sporl E, Reber F, Pillunat L, Funk R. Corneal endothelial cytotoxicity of riboflavin/UVA treatment in vitro. Ophthalmic Res 2003; 35:324-8.

28. Rheinwald JG, Green H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 1975; 6:331-43.

29. Green H. Regeneration of the skin after grafting of epidermal cultures. Lab Invest 1989; 60:583-4.

30. Germain L, Auger FA. Tissue engineered biomaterials: biological and mechanical characteristics. In: Encyclopedic Handbook of Biomaterials and Bioengineering, part B; Applications. New York, USA: Marcel Dekker, inc. Publishers; 1995. p. 699-734.

31. Michel M, L'Heureux N, Pouliot R, Xu W, Auger FA, Germain L. Characterization of a new tissue-engineered human skin equivalent with hair. In Vitro Cell Dev Biol Anim 1999; 35:318-26.

32. Lindberg K, Brown ME, Chaves HV, Kenyon KR, Rheinwald JG. In vitro propagation of human ocular surface epithelial cells for transplantation. Invest Ophthalmol Vis Sci 1993; 34:2672-9.

33. Germain L, Auger FA, Grandbois E, Guignard R, Giasson M, Boisjoly H, Guerin SL. Reconstructed human cornea produced in vitro by tissue engineering. Pathobiology 1999; 67:140-7.

34. Rama P, Bonini S, Lambiase A, Golisano O, Paterna P, De Luca M, Pellegrini G. Autologous fibrin-cultured limbal stem cells permanently restore the corneal surface of patients with total limbal stem cell deficiency. Transplantation 2001; 72:1478-85.

35. Nakamura T, Koizumi N, Tsuzuki M, Inoki K, Sano Y, Sotozono C, Kinoshita S. Successful regrafting of cultivated corneal epithelium using amniotic membrane as a carrier in severe ocular surface disease. Cornea 2003; 22:70-1.

36. Talbot M, Carrier P, Giasson CJ, Deschambeault A, Guerin SL, Auger FA, Bazin R, Germain L. Autologous transplantation of rabbit limbal epithelia cultured on fibrin gels for ocular surface reconstruction. Mol Vis 2006; 12:65-75 <http://www.molvis.org/molvis/v12/a7/>.

37. van Erp PE, Brons PP, Boezeman JB, de Jongh GJ, Bauer FW. A rapid flow cytometric method for bivariate bromodeoxyuridine/DNA analysis using simultaneous proteolytic enzyme digestion and acid denaturation. Cytometry 1988; 9:627-30.

38. Stocker FW, Eiring A, Georgiade R, Georgiade N. A tissue culture technique for growing corneal epithelial, stromal, and endothelial tissues separately. Am J Ophthalmol 1958; 46:294-8.

39. Yue BY, Sugar J, Gilboy JE, Elvart JL. Growth of human corneal endothelial cells in culture. Invest Ophthalmol Vis Sci 1989; 30:248-53.

40. Barrandon Y, Green H. Three clonal types of keratinocyte with different capacities for multiplication. Proc Natl Acad Sci U S A 1987; 84:2302-6.

41. Couch JM, Cullen P, Casey TA, Fabre JW. Mitotic activity of corneal endothelial cells in organ culture with recombinant human epidermal growth factor. Ophthalmology 1987; 94:1-6.

42. Hoppenreijs VP, Pels E, Vrensen GF, Oosting J, Treffers WF. Effects of human epidermal growth factor on endothelial wound healing of human corneas. Invest Ophthalmol Vis Sci 1992; 33:1946-57.

43. Raymond GM, Jumblatt MM, Bartels SP, Neufeld AH. Rabbit corneal endothelial cells in vitro: effects of EGF. Invest Ophthalmol Vis Sci 1986; 27:474-9.

44. Nayak SK, Samples JR, Deg JK, Binder PS. Growth characteristics of primate (baboon) corneal endothelium in vitro. Invest Ophthalmol Vis Sci 1986; 27:607-11.

45. Nayak SK, Binder PS. The growth of endothelium from human corneal rims in tissue culture. Invest Ophthalmol Vis Sci 1984; 25:1213-6.

46. Yue BY, Niedra R, Baum JL. Effects of ascorbic acid on cultured rabbit corneal endothelial cells. Invest Ophthalmol Vis Sci 1980; 19:1471-6.

47. Shelton DL, Reichardt LF. Studies on the regulation of beta-nerve growth factor gene expression in the rat iris: the level of mRNA-encoding nerve growth factor is increased in irises placed in explant cultures in vitro, but not in irises deprived of sensory or sympathetic innervation in vivo. J Cell Biol 1986; 102:1940-8.

48. Lambiase A, Bonini S, Manni L, Ghinelli E, Tirassa P, Rama P, Aloe L. Intraocular production and release of nerve growth factor after iridectomy. Invest Ophthalmol Vis Sci 2002; 43:2334-40.

49. Lambiase A, Bonini S, Micera A, Rama P, Bonini S, Aloe L. Expression of nerve growth factor receptors on the ocular surface in healthy subjects and during manifestation of inflammatory diseases. Invest Ophthalmol Vis Sci 1998; 39:1272-5.

50. Klein R, Jing SQ, Nanduri V, O'Rourke E, Barbacid M. The trk proto-oncogene encodes a receptor for nerve growth factor. Cell 1991; 65:189-97.

51. Kent KD, Bomser JA. Bovine pituitary extract provides remarkable protection against oxidative stress in human prostate epithelial cells. In Vitro Cell Dev Biol Anim 2003; 39:388-94.

52. Praus R, Brettschneider I. Glycosaminoglycans in embryonic and postnatal human cornea. Ophthalmic Res 1975; 7:542.

53. Kaufman HE, Varnell ED, Kaufman S. Chondroitin sulfate in a new cornea preservation medium. Am J Ophthalmol 1984; 98:112-4.

54. Bourne WM, Lindstrom RL, Doughman DJ. Endothelial cell survival on transplanted human corneas preserved by organ culture with 1.35% chondroitin sulfate. Am J Ophthalmol 1985; 100:789-93.

55. Stein RM, Bourne WM, Campbell RJ. Chondroitin sulfate for corneal preservation at 4 degrees C. Evaluation by electron microscopy. Arch Ophthalmol 1986; 104:1358-61.

56. Lindstrom RL, Doughman DJ, Skelnik DL, Mindrup EA. Minnesota system corneal preservation. Br J Ophthalmol 1986; 70:47-54.

57. Stein RM, Laibson PR. Comparison of chondroitin sulfate to McCarey-Kaufman medium for corneal storage. Am J Ophthalmol 1987; 104:490-3.

58. Lin CP, Bohnke M, Draeger J. Effects of fibroblast growth factor and chondroitin sulfate on predamaged corneal endothelium. An organ culture study. Ophthalmic Res 1990; 22:173-7.

59. Hennings H, Michael D, Cheng C, Steinert P, Holbrook K, Yuspa SH. Calcium regulation of growth and differentiation of mouse epidermal cells in culture. Cell 1980; 19:245-54.