Cultivation of Rabbit Corneal Epithelial Cells in Serum-Free Medium

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1 Cultivation of Rabbit Corneal Epithelial Cells in Serum-Free Medium Federico Castro-Munozledo, Conception Valencia-Garcia, and Walid Kuri-Harcuch Purpose. To establish conditions for cultivation, serial growth, and normal differentiation of corneal epithelial cells in serum-free medium (SFM). Methods. Rabbit corneal epithelial cells were co-cultured with lethally treated 3T3-cell feeder layers. Instead of serum, medium was supplemented with serum albumin, hormones, and other additives. Cell growth was quantitated spectrophotometrically with a new rhodamine-b staining protocol with a sensitivity range of 5 X 10 3 to 1 X 10 5 cells/cm 2. Keratin expression was analyzed by immunostaining or sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell extracts. Results. In SFM, without growth factors, cells grew no more than six to eight doublings, but when 10 ng/ml epidermal growth factor were added, serial transfer was possible, and epithelial cells grew to up to 18 to 20 doublings (three cell passages). Two cell colony types were seen: One type was composed of nonstratified proliferating cells, and the other of stratified cells expressing high levels of the differentiation-linked keratins K3 and K12. Confluent cultures formed a four- tofive-layerstratified epithelium whose suprabasal cells were stained with anti- K12 antiserum. Acidic and basicfibroblastgrowth factors and epidermal growth factor reduced the expression of keratins K3 and K12. Transforming growth factor-a and epidermal growth factor led to the highest stimulation of cell proliferation. Limbal, peripheral, and central corneal epithelial cells showed similar clonal growth abilities, but colony size was larger for cells derived from limbal epithelium. Conclusions These SFM conditions support the serial transfer, normal differentiation, and formation of typical corneal epithelium by cultured corneal epithelial cells and are useful in studying and assaying a variety of cytokines and compounds that modulate corneal epithelial cell proliferation and differentiation. Invest Ophthalmol Vis Sci. 1997;38: he effects of growth factors on corneal regeneration and epithelial cell proliferation have been studied in assays in vivo, 1 " 3 in organ, 1 and in cell cultures. 4-6 It has been suggested that corneal epithelial cell proliferation is regulated by such growth factors as epidermal growth factor (EGF) 1478 and fibroblast growth factors (FGF). 2 ' 4 ' 6 However, most of these results come from studies in vivo, requiring high protein concentrations to obtain significant stimulation, and from clini- From the Department of Cell Biology, Center of Research and Advanced Studies of the National Polytechnical Institute, Mexico City. Supported in part by grants from National Council of Science and Technology (U65-N9202 and 30I0M) and from RkardoJ. Zevada Foundation (15/93 and 13/94). Submitted for publication September 5, 1996; revised May 8, 1997; accepted June 2, Proprietary interest categoiy: N. Rejmnt requests: Federico Castro Mufwzledo, Department Cell Biology, Centro de Investigacidn y de lisludios Avanzados del I.P.N., Apdo. Postal , Mexico, D.F , Mexico. cal studies, which have shown variable results. 9 " 1 ' 1 Because of variability of results, growth factor application protocols, and the high doses required, the role of cytokines as modulators of corneal epithelial differentiation and proliferation has not yet been understood. A possible approach to studying corneal epithelial cell growth regulation by cytokines and growth factors could involve cell culture, immunochemical analysis, and recombinant DNA techniques. Previous work has led to the establishment of conditions for serial culture of corneal epithelial cells in media containing serum, but cultivation in serum-free media (SFM) should bring a better understanding of growdi control in such cells. During the last few years, human epidermal keratinocytes were cultured in medium whose serum supplement and feeder-layer dependence were eliminated by addition of crude bovine hypothalamic extract (BPE), trace elements, and low Ca con Investigative Ophthalmology & Visual Science, October 1997, Vol. 38, No. 11 Copyright Association for Research in Vision and Ophthalmology

2 Serum-Free Medium for Corneal Epithelial Cells 2235 centrations. These conditions do not support keratinocyte differentiation and stratification or the formation of typical epithelial layers. Later, media based on MCDB-153 formulation and low Ca 2+ concentrations were also developed, 19 " 21 but they had the same limitations as the SFMjust described. In all cases BPE, which is a highly complex mixture of proteins and growth factors, similar to animal serum, was used as a supplement. Recently, other investigators have cultured corneal epithelial cells in a modified MCDB-151 SFM, which does not require the addition of BPE and does not depend on a 3T3-cell feeder layer 722 ' 23 but also has low Ca 2+ concentrations. 22 However, a stratified epithelial organization could not be obtained, and serial transfer of the cultured cells was not attempted. 7 ' 22 ' 23 In contrast, when corneal epithelial cells were grown under such conditions, basal cells stained positive for such terminal differentiation markers as K3 keratin, 7 ' 22 ' 23 which should be absent from the basal layers. Also, the formation of cornified envelopes was seen when normal Ca 2+ concentrations were provided to the cultured cells. 22 ' 23 These results suggest that cells grown with the modified MCDB-151 SFM express an abnormal differentiation and do not form typical epithelial layers. Therefore, we carried out experiments for cultivation and serial transfer of rabbit corneal epithelial cells under serum-free culture conditions with normal (1.2 mm) Ca 2+ concentrations, and in the presence of lethally treated 3T3-cell feeder layers to obtain a normally differentiated and well-organized epithelium. The 3T3 feeders are not only necessary to support keratinocyte growth but also to allow the formation of well-defined keratinocyte colonies, 24 " 26 and organization of stratified epithelia. Evaluation of cell culture conditions was made through plating cells at high densities, plating cells at low densities similar to those used for colony-forming ability measurements, and keratin expression. These parameters should give important and complementary information on the states of differentiation and proliferation abilities of cell populations. Under these conditions, we have serially grown corneal epithelial cells in up to three subcultivations (18 to 20 generations). The response of cells to growth factors was also easily assayed, because growth was dependent on addition of the growth factor tested. Moreover, a well-organized four- to fivelayer epithelium was obtained, and the terminal differentiation marker K3-K12 keratin pair was only expressed at suprabasal layers in the stratified colonies. MATERIALS AND METHODS Materials Hepes, bovine serum albumin (BSA), insulin, hydrocortisone, human transferrin, cholera toxin, L-triiodothyronine (L-T 3 ), and mitomycin C were obtained from Sigma (St. Louis, MO); fetal bovine serum (FBS) from HyClone Laboratories (Logan, UT); biotinylated horse antimouse immunoglobulin (Ig) G, goat antirabbit IgG and fluorescein isothiocyanate (FITC)- streptavidin from Vector (Burlingame, CA); Gelvatol from Monsanto (St. Louis, MO); recombinant human acidic and basic fibroblast growth factors (afgf and bfgf) from Upstate Biotechnology (Lake Placid, NY); recombinant human transforming growth factor-a (TGF-a) from R & D Systems (Minneapolis, MN); and recombinant human epidermal growth factor (EGF) from Mallinckrodt (Chesterfield, MO). The R167 antiserum, which is specific for K12 keratin, and the monoclonal antibodies AE5, AE1, and AE3 were the generous gift of Dr. Tung-Tien Sun, New York University School of Medicine, New York, NY. All other reagents used were of analytic grade. Cell Culture Male albino rabbits, weighing 2 to 2.5 kg each, were handled according to the NIH Guide for the Care and Use of Laboratory Animals, NIH Publication No (revised 1985), and in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Rabbits were killed by intravenous injection of sodium pentobarbital solution (100 mg/ kg). Corneas were excised, and corneal epithelial cell suspensions were obtained as previously described. 17 Disaggregated epithelial cells were plated at 2.7 X 10 3 cells/cm 2 together with mitomycin C-treated 3T3 cells at 2.2 X 10 4 cells/cm 2, using a (3:1) Dulbecco's modified Eagle's medium (DMEM)-F12-Ham nutrient mixture, supplemented with 10% (vol/vol) FBS, plus 5 //g/ml insulin, 5 /i,g/ml transferrin, 0.4 //g/ml hydrocortisone, 2 X 10" 9 M L-T 3, 1 X 10" 10 M cholera toxin, and 24.3 mg/1 adenine. One day after plating, cultures were rinsed three times with DMEM, without supplements, and changed to SFM. Serum-free medium consisted of a (3:1) DMEM-F12-Ham mixture, containing the same supplements as the solution without serum but with 10 mg/ml BSA. For subculturing, 6 days after plating, cultures were incubated with a (1:1) mixture of 0.15% trypsin and 0.02% EDTA for 20 minutes at 37 C. 17 Cells were always plated with 3T3-cell feeder layers, as described, and changed to SFM 1 day after plating. For colony-forming efficiency, cells were plated at 1000 cells/60-mm culture dish, together with mitomycin C-treated 3T3 cells in serumsupplemented medium and were changed to SFM as described. Cells were fed again every 3 days. For experiments with the RCE1 cell line, which undergoes corneal epithelial differentiation, 17 cells were grown under SFM conditions, as described.

3 2236 Investigative Ophthalmology & Visual Science, October 1997, Vol. 38, No. 11 Assay of Growth Factors To assay for growth factor activity, primary corneal epithelial cell cultures or RCE1 corneal epithelial cells, 17 were plated at 6.75 X 10 2 cells/cm 2, together with mitomycin C-treated 3T3 cells at 2.2 X 10 4 cells/ cm 2, in 24-well culture plates (Linbro, Horsham, PA). One day after plating, wells were rinsed three times with DMEM, without additive, and cells were changed to SFM without 1 X 10" l0 M cholera toxin, containing increasing concentrations of the tested cytokine as indicated. Medium was changed every other day. Ten days later, cultures were fixed and cell growth was quantitated by two stainings. Epithelial Cell Quantitation by Staining of Cell Cultures With Rhodamine B It is widely known that rhodamine B specifically stains keratinocytes but does not stain fibroblasts. 25 ' 26 We took advantage of this property to quantitate the number of epithelial cells in culture. The 24-well tissue culture plates were fixed with 10% (vol/vol) formalin in phosphate-buffered saline. After 2 hours, cultures were rinsed with water and stained with a 2% (wt/vol) rhodamine-b aqueous solution for 30 minutes. After staining, cultures were exhaustively washed with 0.2 N HC1 to eliminate excess rhodamine B. The rinsing solution was removed, and residual excess was evaporated by placing the stained cultures at ~32 C. To determine the number of epithelial cells, 1 ml 0.2 N NaOH was added to the stained culture wells and incubated for 10 minutes at room temperature. The extracted dye was gently removed by pipette and its absorbance determined at 550 nm. Standard and cell proliferation curves were prepared after plating increasing concentrations of epithelial cells in 24-well culture plates. Nonspecific staining of culture wells and 3T3 fibroblasts was subtracted from absorbance values of the extracted dye and was never higher than 0.2% of the values obtained from epithelial cell staining. This method showed a linear correlation between absorbance and cell number per well when epithelial cells were at densities in the range of 5 X 10 3 to 1 X 10 5 cells/cm 2 (Fig. 1). Higher cell densities, similar to those found in subconfluent and confluent cultures, were not quantifiable. Growth factor assays were carried out as described, and absorbance was determined when cell numbers per well were at the indicated range of cell densities. Keratin Extraction After 3T3 cells were removed with EDTA, epithelial cells were extracted with 25 mm Tris-HCl (ph 7.5) containing 1% Triton X-100, 1 mm EDTA, 1 mm EGTA, 1 mm phenylmethylsulfonyl fluoride, 87 mg/ ml aprotinin, and 0.5 mg/ml leupeptin, to remove E s CO Cell number/well (x 10" 4 ) FIGURE l. Cell number quantitation by rhodamine-b staining. Corneal epithelial cells were seeded at the indicated cell densities in 24-well tissue culture plates. One day later, cultures were fixed and stained with 2% (wt/vol) rhodamine B in aqueous solution. Absorbance of the extracted dye was determined at 550 nm and plotted versus the number of epithelial cells per well. The absorbance by 3T3 feeder layers was used as a blank, and these values were subtracted from absorbance values of the extracted dye. The absorbance background from 3T3 cells was never higher than 0.2% of the values obtained from epithelial cell staining. Arrows (1) show the linear range of the standard curve. the water-soluble proteins. The remaining cytoskeletal elements were solubilized by heating at 95 C for 5 minutes in 1% (wt/vol) sodium dodecyl sulfate and 25 mm Tris-HCl (ph 7.4) P Immunofluorescence Staining Epithelial cells were grown on 18 X 18 mm glass coverslips. After fixation and permeabilization, they were stained with R167 antiserum (which is specific for K12 keratin) or with monoclonal antibodies AE1 and AE3 (which are specific for acidic and basic keratins, respectively) or AE5 (which is specific for K3 keratin), as described. 28 Primary antibodies were detected with FITC-streptavidin and biotinylated horse antimouse IgG or goat antirabbit IgG as secondary antibodies. RESULTS Growth of Corneal Epithelial Cells Under Low or Nonserum Conditions It has been reported that corneal epithelial cells could be cultured under serum-free conditions that do not require addition of BPE and do not depend on a 3T3-

4 Serum-Free Medium for Corneal Epithelial Cells 2237 \ FIGURE 2. Growth of corneal epithelial cells supplemented with different serum concentrations. The epithelial cells were seeded at 2.7 X 10 3 cells/cm 2, together with mitomycin C-treated 3T3 cells (see Materials and Methods). One day after plating, cultures were rinsed and changed to a 3:1 Dulbecco's modified Eagle's medium-f12-ham nutrient mixture, containing the supplements described in Materials and Methods and the indicated varying concentrations of fetal bovine serum. Eight days later, when cells grown in the presence of 10% fetal bovine serum reached confluence, cultures were fixed and stained with rhodamine B. cell feeder layer, but require low Ca 2+ concentrations. 722 ' 23 However, cultured cells expressed abnormal differentiation, and organization of stratified epithelia and serial transfer were not demonstrated. 22 ' 23 Hence, we carried out experiments to establish serumfree conditions for cultivation, serial transfer and normal differentiation, and stratification of rabbit corneal epithelial cells in the presence of lethally treated 3T3- cell feeder layers and normal Ca 2+ concentrations. First, we tested whether rabbit corneal epithelial cells were able to grow when serum concentration in culture medium was significantly reduced. Rabbit corneal epithelial cells were seeded in 24-well tissue culture plates at 2.7 X 10 3 cells/cm /2, as described in Methods. One day after plating, cells were rinsed and changed to a (3:1) DMEM-F12-Ham nutrient mixture, containing 5 //g/ml insulin, 5 A*g/ml tranferrin, 0.4 jug/ml hydrocortisone, 2 X 10~ 9 M L-T 3 ; 1 X 10" 10 M cholera toxin, 24.3 mg/1 adenine, and the indicated varying concentrations of FBS. Eight days later, when cells grown in the presence of 10% FBS reached confluence, cultures were fixed and stained with rhodamine B. Results showed that rabbit corneal epithelial cells reached similar cell densities when supplemented with serum concentrations at the range of 2% to 10% (vol/vol; Fig. 2). When cells were fed with medium containing 1% (vol/vol) FBS, growth rate was reduced, and confluence was reached approximately 1 day after confluence was attained in those grown with higher serum supplements (Fig. 2). Because the results show that corneal epithelial cell growth is supported with medium supplemented with low serum concentrations, we tested whether corneal epithelial cells or RCE1 cells 17 could grow with medium whose serum supplement was replaced with serum albumin to a final concentration of 1% (wt/ vol). The addition of serum albumin is well known for serum-free cultivation of various cell types, in that it facilitates the adsorption and transport of fatty acids. Corneal epithelial cells and RCE1 cells showed the same growth when supplemented with medium containing 1% (wt/vol) serum albumin, compared with cells supplemented with 1% (vol/vol) FBS. Cells remained healthy for at least 2 weeks, forming small, highly differentiated colonies. These results were obtained only in the presence of a 3T3 feeder layer; if 3T3 cells were absent, epithelial cells were unable to grow, and cell population was lost after a few days in culture (data not shown). Thus, minimum requirements for survival and limited growth of corneal epithelial cells in a serum-free environment consists of a. (3:1) DMEM-F12-Ham nutrient mixture containing 5 Mg/ml insulin, 5 /Ltg/ml transferrin, 0.4 //g/ml hydrocortisone, 24.3 mg/1 adenine, 2 X 10" 9 M L-T H ; 1 X 10" 10 M cholera toxin, plus 1% (wt/vol) serum albumin and a 3T3-cell feeder layer. Serial Transfer of Corneal Epithelial Cells in Serum-Free Medium Having observed in other serum-free media formulations that serial transfer of corneal epithelial cells was not demonstrated, we also made experiments to determine whether corneal epithelial cells could be serially transferred under the serum-free conditions described. Primary cultures of rabbit corneal epithelial cells were plated and changed to SFM, with or without 10 ng/ml EGF (see Materials and Methods). By the sixth day, cultures were subconfluent and were trypsinized for subcultivation. Serial transfer of epithelial cells was carried out as many times as possible, and the number of population doublings was calculated after each transfer. Primary cultures kept in SFM without EGF did not grow to more than 6 to 8 doublings, and subcultured cells did not grow further; but when corneal epithelial cells were supplemented with SFM containing EGF, they were serially transferred, and by the third subcultivation, the growth rate was significantly decreased (Figs. 3A, 3B). In contrast, cultures maintained with medium containing high serum concentrations were subcultured up to 14 times, also

5 2238 Investigative Ophthalmology & Visual Science, October 1997, Vol. 38, No. 11 Passage number FIGURE 3. Serial transfer of corneal epithelial cells grown under serum-free medium conditions. (A) Epithelial cells were fed with serum-free medium, containing 1 X 10" 10 M cholera toxin and 10 ng/ml epidermal growth factor ( ) or serum-supplemented medium (O) and were subcultured every 6 days until cell population senescence. After each transfer, cell generations were calculated. The average from three experiments for each culture condition is shown. (B) Change in growth rates during culture in serum-free medium for three independent experiments (, A, ) or senim-supplemented medium (O). showing a continuous decrease in growth rate (Fig. 3B). 17 These results demonstrate that serial transfer of corneal epithelial cells in the SFM we describe was possible only when EGF was present, suggesting that these SFM conditions should be adequate to assay for growth factor-cytokine activities on corneal epithelial cell proliferation and differentiation. Corneal Epithelial Cells Cultured With Serum- Free Medium Show Normal Differentiation and Form Stratified Epithelia The results demonstrated the cultivation and serial transfer of corneal epithelial cells in SFM. Thus, we studied whether our SFM supported the normal differentiation and organization of stratified epithelia, in contrast to the serum-free conditions described by others. In our earlier experiments, results suggested that cells grown under serum-free culture conditions displayed various morphologies of cell colonies. We made a closer examination of cultures maintained with SFM and compared them with cells supplemented with FBS. Cultures were plated at 1 X 10 3 cells/60-mm dish; and after 10 days, all cell cultures were fixed, and colonies were photographed. Figure 4 shows that cultivation of epithelial cells with SFM led to formation of two cell colony types: first, looseappearing colonies containing mitotically active round cells and cells with typical epithelial morphology and evident intercellular spaces (Figs. 4A, 4B) and second, tight, highly stratified colonies with intercellular spaces not easily distinguishable (Figs. 4C, 4D). The first type of colony was similar to those found in control cultures supplemented with medium containing serum. When colonies were analyzed for expression of differentiation-linked keratins, we found that the first type of colony was immunostained with monoclonal antibodies against acidic or basic keratins (Fig. 5H) but not with antiserum against the terminal differentiation-associated keratin K12 (Fig. 5B). The second type of colony was positively stained with antibodies raised against K3 or K12 keratins found only in suprabasal cells (Fig. 5D); therefore, this colony type seemed to be derived from cells near terminal differentiation. This conclusion was supported by cultivation of cells for long culture periods, in that all colonies with loose-appearing morphology slowly converted into the tight and stratified colonies that were immunostained with anti-k3 or anti-k12 antibodies. When cells were plated at 2.7 X 10 3 cells/cm 2 and further maintained with SFM plus 10 ng/ml EGF, they reached confluence and organized into a 4- to 5-layer epithelium (Fig. 5E). This epithelium was positively immunostained with anti-k3 or anti-k12 antibodies at the suprabasal layers (Fig. 5F). Together, these results show that corneal epithelial cells cultured in SFM, in the presence of 3T3-cell feeder layers, were able to grow and to form a stratified epithelium with normal differentiation characteristics. Limbal, Peripheral, and Central Corneal Epithelial Cells Show Similar Clonal Growth When Cultured With Serum-Free Medium It was previously shown that limbal corneal epithelial cells cultured with modified MCDB-151 serum-free medium have lower clonal growth capacities than do peripheral and central corneal cell subpopulations. 22 This seems surprising, because the highest growth capacity should be found in the limbal cells, where the

6 Serum-Free Medium for Corneal Epithelial Cells 2239 FIGURE 4. Keratinocyte colony types found under serum-free medium cultivation. (A, B) Loose colony containing proliferating cells. Note the intercellular spaces that confer the loose appearance to the colony. (C, D) Differentiated colony, with cells tightly organized and stratified. Photographs show 10-day-old colonies. Bar =100 fj.m. corneal epithelial stem cells should be located. 29 Therefore, the proliferative capacity of corneal epithelial cell subpopulations grown in our SFM conditions was assayed by colony-forming efficiency (CFE), a parameter that reflects proliferative potential of single cultured cells. After isolation of limbal, peripheral, and central corneal epithelium, epithelial cells were plated at 1 X 10 3 cells/60-mm dish. One day after seeding, cultures were changed to SFM. Ten days later, cultures were fixed and stained with rhodamine B, and CFE was quantitated. As demonstrated in Table 1, all epithelial cell subpopulations showed CFE values of approximately 22% to 25%, in spite of their origin and different proliferative potential. However, although no significant differences were found between CFEs of cell populations derived from the corneal surface regions, as expected, we observed a higher colony size for those cultures obtained from limbal epithelium. Response of Corneal Epithelial Cells to Growth Factors in Serum-Free Medium We made experiments to develop an assay for growth factor-cytokine effects on corneal epithelial cell proliferation and differentiation. Because our preliminary results showed that cholera toxin added to the serum-free medium made growth factor effects imperceptible in cultured corneal epithelial cells, we assayed growth factor effects in SFM without cholera toxin. The rabbit corneal epithelial cells or corneal RCEl cells, a cell line that undergoes corneal epithelial cell differentiation," were plated at 6.75 X 10 2 cells/cm 2 and maintained with SFM without cholera toxin, as described in Materials and Methods. Various concentrations of growth factors EGF, TGF-«, and basic FGF, were added to the medium. Ten days later, cultures were fixed and stained with rhodamine B, and cell growth was quantitated spectrophotometrically (see Materials and Methods). Figure 6 shows that in both cell types, growth factors significantly stimulated corneal cell proliferation. Epidermal growth factor and TGF-a exerted the largest effect, with approximately a 10- to 15-fold increase in RCEl cell number per culture, compared with a two- to threefold increase with basic FGF (Fig. 6A). Similar results were observed for primary corneal epithelial cells (Fig. 6B), but their total growth capacity was lower than that of RCEl cells. These results show that the SFM conditions we have described might be useful to study growth factor effects in corneal epithelium not only with the RCEl cell line, which seems to be an adequate model to understand regulation of corneal cell differentiation by cytokines and other agents, but also with primary or early culture passages of corneal epithelial cells. In other experiments, we tested whether growth factor modulated clonal growth capacity and keratin expression of corneal epithelial cells. The RCEl cells or corneal epithelial cells were seeded at 1.1 X 10 2 cells/cm 2 and were changed to SFM, without cholera

7 Investigative Ophthalmology & Visual Science, October 1997, Vol. 38, No. 11 fed with medium containing EGF or basic FGF and was the lowest with acidic FGF. In acidic-fgf-treated cultures, die differentiated colonies were more prevalent. In control cultures, which were not stimulated with growth factors, most colonies were highly differentiated. These results also showed a high correlation with keratin expression. The keratin pair K3-K12, which is characteristic of suprabasal differentiated cells, was expressed at low levels in those cultures grown in the presence of EGF or basic FGF (Fig. 7), but it was expressed at high levels in those cultures treated with acidic FGF. Even a higher level of expression was observed in cultures maintained with SFM without growth factors. Our results suggest that EGF and basic FGF reduce the expression of differentiation-linked keratins and promote a proliferating phenotype when assayed under the SFM conditions we describe. FIGURE 5. Immunostaining for K12 keratin (B, D, F) and the corresponding phase-contrast fields (A, C, E). Loose (A, B) and differentiated colonies (C, D) from 10-day-old cell cultures maintained in serum-free medium. Bar = 30 //.m. Frozen sections (E, F) from a cultured epithelium obtained in serum-free medium. Note the suprabasal (not basal) expression of K12 (F). Dashed line at F corresponds to the basal side of the epithelium. Bar = 15 yxn. A loose colony stained with a (1:1) AE1-AE3 mixture for acidic and basic keratins (G, H). Bar = 30 ^ra. toxin, that contained varying concentrations of EGF, basic FGF or acidic FGF. Some control cultures were maintained with medium containing 10% (vol/vol) FBS. After 10 days, cultures were fixed to quantitate CFE and to examine the structure of colonies; or were extracted to analyze keratin expression. In the presence of EGF, acidic FGF, or basic FGF, the CFE of RCE1 cells was increased (Fig. 6C). The largest stimulation was observed in cultures fed with serum-free medium containing EGF. This growth factor increased CFE values 2 to 3 times when added at a final concentration of 10 ng/ml (Fig. 6C); those cell cultures stimulated with basic or acidic FGFs showed only a 1.4- to 1.5-fold increase in CFE values (Fig. 6C). When we studied colony morphology in the presence of EGF, acidic FGFj or basic FGF, most rabbit corneal epithelial cell colonies were nondifferentiated (discussed earlier; Fig. 4A, 4B); die largest frequency of nondifferentiated colonies was attained when cultures were DISCUSSION To allow a simpler design and interpretation of experiments, and to reduce variability of results in cell culture, different approaches have been taken to eliminate the requirement for serum supplement. Some investigators have attempted to adapt cells to media without serum, or to purify those factors responsible for cell growth and differentiation. Others have searched for nutrient combinations that would support cell growth and differentiation, 30 or alternatively, have tried media supplementation with hormones or other macromolecular fractions. 31 From results reported in these studies, it can be concluded that each cell type shows unique requirements. Several investigators have attempted different serumfree formulations for cultivation of epithelial cells. For example, transformed cells, including human colon carcinoma-derived cells, 32 ' 33 A431 cells, 34 bronchogenic epidermoid carcinoma cells, 35 LLC-PK], and MDCK cells 36 have TABLE i. Colony Forming Efficiency of Corneal Epithelial Cell Subpopulations Supplemented With Serum-Free Medium Corneal Epithelial Cell Subpopulation Limbal Peripheral Central Colony Forming Efficiency (±SD) 24.6 ± ± ± 1.24 After isolation of corneal tissue including conjunctival epithelium, corneal subpopulations were dissected under a SMZ- 1B Nikon stereomicroscope. Then epithelial cells were isolated and plated in serum-supplemented medium. One day after plating, cultures were rinsed and changed to serum-free medium. Ten days after, cultures were fixed and stained with rhodamine B for colony quantitation.

8 Serum-Free Medium for Corneal Epithelial Cells r Concentration (ng/ml) Recently, the use of serum-free formulations based in MCDB or MCDB nutrient mixtures, both without BPE addition, were also reported for corneal epithelial cell cultivation. Using the MCDB-151 nutrient mixture, Kruse and Tseng have obtained the better results, showing clonal growth of corneal epithelial cells plated at low densities, and assaying growth factors and calcium effects on corneal epithelial cell growth. 7 * 2223 However, serial transfer and organization of stratified epithelia were not demonstrated, and even cells localized at the basal compartment were stained positive for such terminal differentiationlinked keratins as the K3 keratin. 7 ' 22 ' 23 Moreover, when cell cultures were maintained at normal Ca 2+ concentrations, cornified envelopes were observed. 21 ' This seems to be part of an abnormal differentiation pattern, in that corneal epithelial cells do not form such structures. 23 These results suggest that corneal epithelial cells cultured in serum-free media express a lim Concentration (ng/ml) FIGURE 6. Response of (A) RCE1 cells and (B) primary cornea! epithelial cells to growth factors. Cells were plated at 6.75 X 10 a cells/cm 2 and maintained with serum-free medium, containing epidermal growth factor (O), transforming growth factor ( ), or basic fibroblast growth factor ( ). After 10 days, cultures were fixed and stained with rhodamine B, and cell growth was quantitated by extraction of the dye. (C) Colony-forming efficiency of 10-day-old RCE1 cell cultures, supplemented with serum-free medium containing epidermal growth factor (O), acidic fibroblast growth factor (A), or basic fibroblast growth factor ( ) : a r a been grown with a DMEM-F12-based mixture, with several supplements. Human epidermal keratinocytes, however, have been grown under low-serum 3337 ' 38 or serum-free culture conditions with low Ca 2+ concentrations. 18 ' 39 For human epidermal keratinocytes, Medium 199 was supplemented with several additives, 18 and MCDB-151 formulation was modified to obtain the MCDB-152 medium. 39 Conversely, cultivation of keratinocytes without a feeder layer and low Ca 2+ concentrations seemed possible by addition to MCDB-153 medium of trace elements, ethanolamine, phosphoethanolamine, triiodothyronine, hydrocortisone, EGF, and, most importantly, BPE. 18 " 21 ' 40 ' 41 Currently, the MCDB- 153 formulation is one of the most commonly used media to grow keratinocytes in a serum-free environment; 19 " 21 ' however, the BPE used as a supplement is a highly complex mixture of proteins and growth factors and raises similar objections to those raised by the use of animal serum. In view of these results, several laboratories have attempted to describe serum-free culture conditions for corneal epithelial cell growth and differentiation. FIGURE 7. Keratin expression during stimulation of cultures with epidermal growth factor, acidic fibroblast growth factor and basic fibroblast growth factor in serum-free medium. Cells were cultured in serum-free medium (lane 2, control), with 10 ng/ml epidermal growth factor (lane 3), 10 ng/ml acidic fibroblast growth factor (lane 4), or 10 ng/ml basic fibroblast growth factor (lane 5). Other cultures were maintained widi medium containing 10% fetal bovine serum (lane 6, standard conditions). Arrows show the keratin pairs K3-K12 and K5-K14 and keratin K19. Molecular weight markers (lane 1): bovine serum albumin (66 kda), ovalbumin (45 kda), glyceraldehyde-3-phosphate dehydroge-, nase (36 kda), carbonic anhydrase (29 kda), trypsinogen (24 kda), trypsin inhibitor (20.1 kda), and a-lactalbumin (14.2 kda).

9 2242 Investigative Ophthalmology 8c Visual Science, October 1997, Vol. 38, No. 11 ited proliferative potential and abnormal differentiation. Therefore, we cultured the corneal epithelial cells under SFM conditions, with lethally treated 3T3 cells as feeder layers. Lethally treated fibroblasts were not only necessary to support epithelial cell growth as described earlier, 25 ' 26 but also allowed the formation of well-defined epithelial cell colonies and as we expected, the organization of a four- to five-layer stratified epithelia (see Fig. 5E), and the normal expression of the terminal differentiation-linked K3-K12 keratin pair (Figs. 5D, 5F). Moreover, under these SFM conditions, we did not observe microscopically the formation of cornified envelopes. The presence of 3T3-cell feeder layers, also allowed plating of the corneal epithelial cells at low cell densities. For instance, we plated and grew one sixth of the number of cells used by Wilson et al 42 to assay growth factors; and to analyze clonal growth, corneal cells were plated at 18 to 36 cells/cm 2, in a method similar to that of Kruse and Tseng. 7 ' 22 ' 23 Furthermore, in contrast with results previously reported, we show that corneal epithelial cells were susceptible to serial transferal with SFM containing the 3T3 cells and 10 ng/ml EGF. Previously, serial transfer was only demonstrated for epidermal keratinocytes and other epithelial cells when calcium concentration in culture medium was reduced at the 0.06-mM: range, 18 " 21 ' 23 ' 40 or when plating was done at very high cell densities. Ours the first work in which the cultivation of corneal keratinocytes under serum-free conditions has been reported at calcium concentrations of ~ 1.2 mm in which growth, organization of a four- to five-layer epithelium, long-term serial transfer, and expression of differentiation markers in suprabasal cells (not in basal cells), were obtained. The culture conditions described in this work, allowed us to assay for various growth factor activities EGF, TGFa, basic FGF, and acidic FGF to stimulate cell proliferation. The results show that SFM is useful for studying growth factor effects in cell culture, in that the growth factors tested significantly increased cell proliferation of the corneal epithelial cells. Acidic FGF, basic FGF, or EGF induced a colony structure that could be related with a migratory phenotype, as previously suggested, 8 and a reduction in the expression of the differentiation-linked keratin pair K3-K12. Similar to results obtained by Kruse and Tseng, 22 we also show that EGF induced a higher effect than acidic FGF and basic FGF. We must remark that, under the SFM conditions we describe, response to growth factors was several times higher than those results described by Kruse and Tseng 7 and by Wilson et al. 42 Also, we show that the RCE1 cell line 17 cultured with the serum-free conditions described here, should be valuable in evaluating growth factor activity on corneal epithelial cell populations. We were also able to grow corneal epithelial subpopulations. Kruse and Tseng 22 showed that corneal epithelial cells have different clonal growth capacities in primary cultures, with CFE values of 4% for limbal and 12% for peripheral and central corneal epithelial cells. This seemed surprising, because it is expected that limbal cells should have the largest growth potential when compared with that of the other corneal epithelial cells, because the stem cells of the cornea are located in the limbus. 29 ' 43 In our case, limbal, peripheral, or central corneal epithelial cells showed similar clonal growth abilities; but as expected, limbal cells gave rise to larger colonies. In view of this, we think that our culture conditions are less restrictive for the expression of growth potential, when compared with those used by Kruse and Tseng 7 ' 22 ' 23 ; therefore, they support a higher CFE. In comparison with results in Kruse and Tseng, 2223 we obtained approximately four to five times higher CFE values for limbal cells and approximately two times higher for the other two corneal epithelial cell populations. These differences clearly show that our serum-free media conditions support better growth of the corneal cells. It can be expected that during serial transfer, the clonal growth of these three types of cells may differ, with the limbal cells showing the largest growth capacity. Our results demonstrate that the serum-free conditions described here would be useful in assaying a variety of cytokines, growth factors, hormones, and compounds that modulate proliferation and differentiation of epithelial cells isolated from different corneal subregions. Because the proliferative potential of isolated single cells is easily assayed in the presence of feeder layers, 44 these serum-free culture conditions will help to characterize stem cells from epithelial tissues and their response to growth factors, cytokines, and hormones. Key Words cell culture, corneal epithelium, growth factors, keratins, serum-free medium References 1. Savage CR, Cohen S. Proliferation of corneal epithelium induced by epidermal growth factor. Exp Eye Res. 1973;15: Thompson P, Desbordes JM, Giraud J, et al. 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