Experimental Eye Research 122 (2014) 132e140
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Effect of amniotic fluid on the in vitro culture of human corneal endothelial cells Sepehr Feizi a, *, Zahra-Soheila Soheili b, Abouzar Bagheri a, Sahar Balagholi a, Azam Mohammadian b, Mozhgan Rezaei-Kanavi a, Hamid Ahmadieh a, Shahram Samiei c, Kambiz Negahban d a
Ophthalmic Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran National Institute of Genetic Engineering and Biotechnology, Tehran, Iran Iranian Blood Transfusion Organization Research Center, Tehran, Iran d Department of Ophthalmology, Boston University School of Medicine, Boston, USA b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 September 2013 Accepted in revised form 1 April 2014 Available online 12 April 2014
The present study was designed to evaluate the effects of human amniotic fluid (HAF) on the growth of human corneal endothelial cells (HCECs) and to establish an in vitro method for expanding HCECs. HCECs were cultured in DMEM-F12 supplemented with 20% fetal bovine serum (FBS). Confluent monolayer cultures were trypsinized and passaged using either FBS- or HAF-containing media. Cell proliferation and cell death ELISA assays were performed to determine the effect of HAF on cell growth and viability. The identity of the cells cultured in 20% HAF was determined using immunocytochemistry (ICC) and realtime reverse transcription polymerase chain reaction (RT-PCR) techniques to evaluate the expression of factors that are characteristic of HCECs, including Ki-67, Vimentin, Naþ/Kþ-ATPase and ZO-1. HCEC primary cultures were successfully established using 20% HAF-containing medium, and these cultures demonstrated rapid cell proliferation according to the cell proliferation and death ELISA assay results. The ICC and real time RT-PCR results indicated that there was a higher expression of Naþ/Kþ-ATPase and ZO-1 in the 20% HAF cell cultures compared with the control (20% FBS) (P < 0.05). The 20% HAFcontaining medium exhibited a greater stimulatory effect on HCEC growth and could represent a potential enriched supplement for HCEC regeneration studies. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: human corneal endothelial cells human amniotic fluid in vitro culture
1. Introduction Human corneal endothelial cells (HCECs) help to maintain corneal clarity by serving the following two functions: a pump function that drives water from the corneal stroma to the aqueous humor and a barrier function that involves focal tight junctions. Therefore, these cells regulate the corneal stromal hydration state, which is critical for corneal transparency (Dikstein and Maurice, 1972). The density of HCECs decreases with age and in various diseases, such as bullous keratopathy and Fuchs’ endothelial dystrophy. This cell decrease is predominantly compensated by cell migration and enlargement, rather than by cell division, to replace dead cells (Joyce, 2004).
* Corresponding author. Ophthalmic Research Center, Department of Ophthalmology, Labbafinejad Medical Center, Boostan 9 St., Pasdaran Ave., Tehran 16666, Iran. Tel.: þ98 21 2258 4733/2259 1616; fax: þ98 21 2256 2138. E-mail address: [email protected] (S. Feizi). http://dx.doi.org/10.1016/j.exer.2014.04.002 0014-4835/Ó 2014 Elsevier Ltd. All rights reserved.
Corneal transplantations for endothelial dysfunction have evolved over the last decade from using penetrating keratoplasty to posterior lamellar keratoplasty techniques (Patel, 2007). The recently introduced posterior lamellar keratoplasty techniques, such as Descemet stripping endothelial keratoplasty (DSEK), are an effective therapeutic approach for these conditions. These techniques, however, are dependent on the availability of a good quality donor corneal tissue for transplantation, which can be scarce in some regions of the world because of a lack of donors. Tissue-engineered corneal endothelium is a novel approach that addresses this issue (Joyce and Zhu, 2004; Lai et al., 2006). HCECs do not proliferate in vivo because the cell cycle is arrested in the G1 phase (Chen et al., 2003; Kim et al., 2001; Paull and Whikehart, 2005). HCECs, however, retain the ability to proliferate in vitro under the proper culture conditions that include growth factors and eliminate suppressing factors from the culture medium (Ishino et al., 2004; Li et al., 2007; Zhu and Joyce, 2004; Engelmann and Friedl, 1989; Nakahara et al., 2013).
S. Feizi et al. / Experimental Eye Research 122 (2014) 132e140
Previously, several techniques and culture media have been developed and optimized using numerous agents and growth factors, including epidermal growth factor (EGF), fibroblast growth factor (FGF), nerve growth factor (NGF), and bovine pituitary extract (BPE) (Ishino et al., 2004; Li et al., 2007; Zhu and Joyce, 2004; Engelmann and Friedl, 1989; Joyce, 2004). Human amniotic fluid (HAF) is enriched with a variety of growth factors and nutrients, and different reports have shown that it is necessary for embryonic cell proliferation, differentiation and dedifferentiation (Hirai et al., 2002). In addition, HAF has also been found to be an effective stimulator of retinal progenitor cell development from the retinal pigment epithelium (Ghaderi et al., 2011; Sanie-Jahromi et al., 2012). The purpose of this study was to evaluate the effect of adding HAF to a culture system for the isolation and cultivation of HCECs. 2. Materials and methods 2.1. Tissue preparation Ninety-six human corneas, of which 95.8% were cultured successfully, were obtained from 75 donors between the ages of 5 and 51 years old (mean age, 35 6 years). All of the donor human corneas were obtained from the Central Eye Bank of Iran, preserved at 4 C (Optisol-GS preservative; Chiron Vision, Irvine, CA, USA) and used within 1 week of collection. All of the corneas had an endothelial cell count >2500 cells/mm2 but were deemed unsuitable for clinical use. The study was approved by the ethics committee of the Ophthalmic Research Center. The corneas were transferred to a plastic dish with the epithelium facing down. Then, under an operating microscope and sterile conditions, a 27-gauge needle attached to a 5-cc syringe (bevel facing up) was inserted into the stroma beneath Descemet’s membrane (DM) and up to the center of the cornea. Balanced salt solution was gently injected to detach DM and the endothelial cells from the stroma to Schwalbe’s line at 360 when possible. The samples were transferred to the laboratory in phosphate buffered saline (PBS) solution. 2.2. Human amniotic fluid preparation Ten ml HAF samples were collected from pregnant women who underwent amniocentesis to assess for genetic deficiencies during the first trimester of gestation. The amniotic fluid cells were collected for karyotype analysis. The supernatants from the samples showing no evidence of chromosomal abnormalities were pooled and used in our experimental procedures. The procedure used to collect these samples was approved by the ethics committees of the National Institute of Genetic Engineering and Biotechnology (NIGEB) and the Ophthalmic Research Center of Iran. All of the donors signed the informed consent document after the investigators explained the purpose of the study. The HAF samples were centrifuged at 300 g for 5 min at 4 C, and the resulting supernatants were then sterilized using a 0.2 mm membrane filter (Orange Scientific, Belgium) and stored at 70 C until further analysis. 2.3. Ex vivo cell culture The separated DM and endothelial cells were incubated in 3.5 mg/mL collagenase A (Roche, USA) for 50 min and then cultured in DMEM:F12 at a 1:1 (v/v) ratio (Sigma, Germany) and supplemented with 20% fetal bovine serum (FBS), 120 mg/ml penicillin (Sigma, Germany), and 220 mg/ml streptomycin (Sigma, Germany) on 6-well plates coated with 20 mg/ml of type IV collagen (Sigma,
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USA). All cultures were carried out in an incubator at 37 C with a humidified atmosphere of 5% CO2. The culture medium was first changed with DMEM:F12 1:1 supplemented with three different concentrations (10%, 20% and 30%) of HAF on the seventh day and subsequently replaced every 6 days. To potentially preserve any secreted growth factors, the culture medium was only partially replaced. Upon reaching 80% confluency, the cells were subcultured by treating with trypsin/EDTA. The trypsinized cells were gently centrifuged (5 min at 300 g), the supernatant was discarded and the residual precipitates were re-suspended in complete medium supplemented with 10%, 20% or 30% HAF and seeded on new plates. Seeding density was approximately 1 104 cells/cm2. Control samples were exposed to the same procedure and incubation conditions, however, the culture medium was supplemented with 20% FBS instead of HAF. 2.4. Purification of endothelial cells The presence of spindle shaped cells in the primary culture was indicative of stromal fibroblast contamination, and the colony of endothelial cells was transferred to a new 6-well plate using a cell scraper. This procedure was repeated as necessary until no fibroblasts were observed in any of the culture dishes. 2.5. Cell identification The characteristics of the cultured HCECs were determined and verified based on the morphology and the expression of molecular markers. In terms of morphology, the appearance of hexagons or “cobblestone”, which is characteristic of HCECs, was used to differentiate these cells from human corneal stromal fibroblasts, which have an elongated and spindle-shaped appearance (phase-contrast microscope, Olympus IX71). Cell morphology was quantitatively evaluated using morphometric analyses to compare cell size, coefficient of variance, as well as cell circularity between the different groups. For this purpose, morphometric data of the area and perimeter of randomly selected cells from phase contrast images of each culture medium (when cells became confluent) was manually outlined by pointto-point tracing of the cell borders using ImageJ software (Teo et al., 2012; Peh et al., 2013). When cultures reached a sufficient confluency, the cells were trypsinized and cultured in a 24-well plate. The expression level of the molecular markers was detected using immunocytochemistry (ICC). Furthermore, the RNA was extracted and reverse transcribed using a cDNA synthesis kit and subjected to amplification by realtime reverse transcription polymerase chain reaction (RT-PCR). Detailed procedures for the ICC and real time RT-PCR were provided by the product manufacturers. 2.6. Cell proliferation and death ELISA assays HCECs were plated on 96-well plates at 5000 cells/well in 200 ml complete medium. Next, the medium was replaced with 100 ml of fresh serum-free medium plus the indicated concentration of HAF. To determine whether the HAF altered cell proliferation, bromodeoxyuridine (BrdU) was added after 6 days of treatment, and the proliferation assay was performed according to the manufacturer’s instructions (Cell Proliferation assay kit; Roche, USA). The cytotoxicity was evaluated after 6 days of treatment using the Cell Death assay kit (including positive control; DNA-histone complex) according to the manufacturer’s instructions (Roche, USA).
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2.7. Immunocytochemistry (ICC)
3. Results
Cultured cells from passage 3 in 24-well plates were first fixed by incubating in 10 C methanol for 10 min. The cells were permeablized using Triton X-100 (0.25%) and then blocked in 1% bovine serum albumin (BSA) in PBS for 1 h at room temperature. Specific primary rabbit anti-human polyclonal antibodies were used to detect the Ki-67 (Rabbit anti-Ki67 H-300, Santa Cruz, USA, sc-15402, 60 min, 1:400), Vimentin (Rabbit anti-Vimentin H-84, Santa Cruz, USA, sc-5565, 60 min, 1:800), Naþ/Kþ-ATPase (Rabbit anti-Naþ/Kþ-ATPase H-300, Santa Cruz, USA, sc-28800, 90 min, 1:500) and ZO-1 (Rabbit anti-ZO-1 H-300, Santa Cruz, USA, sc10804, 90 min, 1:400) protein levels. Secondary fluorescein isothiocyanate (FITC) conjugated antibody (Goat anti-rabbit IgG-FITC, Santa Cruz, USA, sc-2012) against the primary antibodies were diluted (1:200) and used to detect the immunoreactivity of the cultures to the primary antibodies. All of the antibodies were diluted in PBS. After the final wash, the slides were incubated in mounting medium containing 4,6-diamidino-2-phenyindole dihydrochloride (DAPI) (1.5 mg/mL, Santa Cruz, USA, sc-24941) for 10 min to counterstain the nuclear DNA. The slides were examined using a fluorescence microscope (Olympus IX71) equipped with a 460 nm filter for the DAPI dye and a 520 nm filter for the FITC conjugated antibodies. To determine the specificity of our antibodies, we used a negative control for each marker that underwent the same procedure and used all of the same materials except for the primary antibody.
3.1. Endothelial cell morphology and purification Compared with the cells grown in 10% HAF, 30% HAF and 20% FBS, the cells grown in 20% HAF demonstrated a more uniform distinctive corneal endothelial morphology, which was very similar to the hexagonal mosaic appearance of corneal endothelial cells in vivo, produced a higher density in culture and reached complete confluency earlier (Fig. 1). Morphologic assessment demonstrated that HCECs cultured in 20% HAF were distinctly the most compact (2599.91 528.54 mm2), most homogeneous (coefficient of variation: 0.20) and hexagonal in shape as suggested by their cellular circularity index of 0.88 0.04 (Table 1). As shown in Fig. 1E, the 20% HAF-supplemented medium yielded a cell density that was 7-, 3-, and 1.5-times greater than the cell density achieved using 20% FBS, 10% HAF, and 30% HAF, respectively (P < 0.05 for all comparisons). Although the cells cultured in the 20% HAF-treated medium retained the morphology of endothelial cells, some vesicles began to grow and the HCECs became wider and lost their hexagonal shape after passage 4 and died after several weeks (Fig. 2A). In the beginning, the rate of fibroblast fusiform cell contamination in the cultures was 5%. Using a cell scraper to isolate the HCECs from the stromal fibroblasts (mainly performed at the first passage), no spindle shaped cells were observed after two passages (Fig. 2B). 3.2. Cell proliferation and death ELISA assays
2.8. Real-time reverse transcription polymerase chain reaction The total RNA was extracted from passage 3 cultured cells using QIAzolLysis Reagent (Qiagen, Germany). The concentration and purity of the isolated RNA were determined using a NanoDrop, and the integrity of the RNA was verified using agarose gel electrophoresis and ethidium bromide staining. Reverse transcription was performed using oligodT primers and the SuperScript reverse transcriptase kit (Invitrogen, USA). Real time RT-PCR was then performed using the QuantiFast SYBR Green PCR Kit (QIAGEN, Germany, 204054). To perform PCR, specific primers for Ki-67 (Qiagen, Germany, QT00014203), Vimentin (Qiagen, Germany, QT00095795), Naþ/Kþ-ATPase (Qiagen, Germany, QT00059808), ZO-1 (Qiagen, Germany, QT00077308) and GAPDH (QIAGEN, Germany, QT01192646), as a housekeeping gene control, were used. The PCR parameters were an initial denaturation (one cycle at 95 C for 10 min); denaturation and annealing/amplification at 95 C for 10 s and 60 C for 30 s, respectively, for 40 cycles; and a melting curve, 72 C, with the temperature gradually increasing (0.5 C) to 95 C.
2.9. Statistical analysis Real time RT-PCR, ICC and cell counts were performed in triplicate and ELISA tests were performed in duplicate. The morphometric parameters and cell density resulting from specific culture media as well as cell proliferation and death (determined by ELISA assay) were quantitatively compared between the control and experimental conditions (various concentrations of HAF) using a one-way ANOVA test. Differences in the expression levels of the different markers (determined by ICC and RT-PCR) were quantitatively evaluated between the control and the 20% HAF-treated cells using Student’s t-test (SPSS 16.0; IBM Corp., USA). A P < 0.05 was considered statistically significant.
The ELISA test results showed that the concentrations of HAF and FBS used to supplement the media had no cytotoxic effects on the HCECs compared with the positive control sample included with the kit (DNA-histone complex) (Fig. 3A). The HAF at all of the concentrations tested increased the cell proliferation rate (1.5 to two fold) compared with the control cultures grown in FBScontaining medium (P < 0.05). The greatest effect was observed in the cells cultured in 20% HAF-containing medium compared with the 20% FBS-, 10% HAF-, and 30% HAF-containing media (P < 0.05 for all comparisons) (Fig. 3B). 3.3. Demonstrative markers for characteristics specific to corneal endothelial cells 3.3.1. Immunocytochemistry The ICC results revealed that there were Vimentin, Ki-67, Naþ/ Kþ-ATPase and ZO-1 positive cells in the cultures (Fig. 4). The expression of the Naþ/Kþ-ATPase and ZO-1 proteins confirmed the identity of the isolated cells as corneal endothelial cells. For the HCECs cultured in 20% HAF, more than 98% of the cells expressed Vimentin and more than 95% of the HCECs expressed ZO-1. Furthermore, Naþ/Kþ-ATPase expression was detected in nearly 100% of the HCECs. Taken together, these results indicate that the HCECs retained the original characteristics of corneal endothelial cells. Additionally, more than 70% of the HCECs expressed the nuclear Ki-67 cell proliferation index, which indicates that the isolated cultivated cells also retained their proliferative ability in vitro. The negative controls (ICC test without primary antibody) did not exhibit any relative FITC signal. Compared with the control, the HCECs cultured in 20% HAFcontaining medium demonstrated a 3.5-fold, fivefold and threefold increase in the number of Naþ/Kþ-ATPase, Vimentin and ZO-1 positive cells, respectively (P < 0.05 for all comparisons) (Fig. 5). The number of cells positive for Ki-67, however, did not differ
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Fig. 1. Human corneal endothelial cell (HCEC) cultures in control serum (20% fetal bovine serum; A), 10% human amniotic fluid (HAF; B), and 10% HAF (C). The HCECs grown in 20% HAF (D) demonstrate a more uniform distinctive corneal endothelial morphology and produce a higher density, (phase-contrast microscope, Olympus IX71, 200). (E) HCEC numbers after 4 weeks of treatment with 10%, 20%, 30% HAF and 20% FBS as a control. The 20% HAF-supplemented medium yielded a cell density that was 7-, 3-, and 1.5-times greater than the cell density achieved using 20% FBS, 10% HAF, and 30% HAF, respectively (*P < 0.05 for all comparisons). Table 1 Cell size, coefficient of variation and cellular circularity of corneal endothelial cells cultured in different media. Culture medium Cell size (mm2) 20% FBS 10% HAF 20% HAF 30% HAF P value
6484.70 4025.22 2599.91 3372.88 0.05). Compared with the control, the number of cells positive for Naþ/K þ -ATPase (B), Vimentin (C) and ZO-1 (D) was significantly higher in the 20% HAF-treated HCECs (3.5-fold, fivefold and threefold increase, respectively, *P < 0.05 for all comparisons).
for the ex vivo expansion of RPE cells and the trans-differentiation of these cells into differentiated neurons (retinal ganglion cells and rod photoreceptors) (Ghaderi et al., 2011; Sanie-Jahromi et al., 2012). Currently, the collective profile of AF proteins, the precise interactions between these proteins and their potential beneficial effects remain to be determined. Furthermore, potential variability can exist between samples obtained from different donors and at different gestational ages (Michaels et al., 2007). More than 98% of HAF is composed of water. Electrolytes, proteins, peptides, carbohydrates, lipids, hormones and various growth factors, including epidermal growth factor (EGF), fibroblast growth factor (FGF), nerve growth factor (NGF), hepatocyte growth factor (HGF), transforming growth factor alpha (TGF-a), transforming growth factor beta (TGF-b1), insulin-like growth factor-I (IGF-I), insulinlike growth factor-II (IGF-II), erythropoietin (EPO), granulocyte colony stimulating factor (G-CSF), and macrophage colony stimulating factor (M-CSF), make up the remaining portion (Hirai et al., 2002; Colombo et al., 1993; Marx et al., 1999; Kimble et al., 1999). Cho et al. (2007) identified the 15 most abundant proteins in HAF at the gestational ages between 16 and 18 weeks, which include albumin, fibronectin, serotransferrin, complement C3, and ceruloplasmin. Complement C3 is a factor in HAF that has been found to be responsible for the regeneration of damaged tissue (Kimura et al., 2003; Reca et al., 2003). Plasminogen is a factor that is also involved in cell proliferation and wound healing. Ceruloplasmin, a1 microglobulin, serotransferrin, apolipoprotein A and
albumin are other HAF proteins that are essential for cell homeostasis and transport. The hyaluronic acid in HAF fosters an extracellular environment that is permissive for cell motility and proliferation (Longaker et al., 1989; Siebert et al., 1990). These factors may work in a synergistic manner and may play a more important role in tissue development when acting in combination (Bry and Hallman, 1992; Wilkinson et al., 1994). The results of this study suggest that HAF is a valuable compound because it contains all of these aforementioned factors, and therefore, it should be considered a potential substantial supplemental medium. Based on the morphometric analysis, HCECs demonstrated more organized alignment and higher density in the 20% HAF culture media. After passage 4, however, the HCECs cultured in 20% HAF demonstrated some vesicles and became wider suggestive of senescence. A higher concentration of HAF (30%) repressed further cell proliferation. It is possible that the proliferative effects of the growth factors are overshadowed by the inhibitory factors at this higher concentration. For example, TGF-b, an ingredient in HAF, has been found to suppress S-phase entry in cultured corneal endothelial cells (Chen et al., 1999). To prevent stromal fibroblast contamination, we attempted to strip only the endothelial cells along with DM. Some posterior stroma (between 6 and 20 microns), however, inevitably remain attached to the underlying DM, no matter how precisely DM is removed (Jafarinasab et al., 2010). Therefore, the explant in vitro will normally give rise to a mixed cell population that contains stromal fibroblasts and endothelial cells. Because stromal
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Fig. 6. Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) analysis of the Ki-67, Naþ/K þ -ATPase, Vimentin and ZO-1 gene expression in cultured cells treated with 20% human amniotic fluid (HAF). The mRNA levels were normalized to GAPDH and are presented as a percentage of the control value. Compared with the control, the HAF treatment increased the Naþ/K þ -ATPase and ZO-1 gene expression by 400-fold and 600-fold, respectively (*P < 0.05). There appeared to be no significant difference in the expression levels of Ki-67 and Vimentin between the HAF-treated cells and the control (P > 0.05).
fibroblasts have a faster growth rate, they can outnumber the endothelial cells after 10 days of culture (Hyldahl, 1984). To eliminate the contaminating fibroblasts, a cell scraper was used on the primary culture as soon as a few fibroblasts were visible to transfer only the endothelium to a new culture vessel. The results of the present study demonstrate that the method of DM removal from the donor corneas and the use of a cell scraper to remove the fibroblasts at a very early passage of cell culture is an effective method for eliminating irrelevant cellular contamination. Some investigators have used a selective medium, containing D-valine instead of L-valine, in which fibroblastic cells are unable to grow (Engelmann et al., 1988) to eliminate unrelated cellular contamination. Alternatively, contaminating stromal fibroblasts can effectively be depleted from HCEC cultures through a negative celleselection strategy by magnetic affinity cell separation using antifibroblast magnetic microbeads. Using this technique, Peh et al. (2012) reported a separation efficacy of 96.88%. Additionally, a positive selection strategy recruiting two monoclonal antibodies (anti-GPC4 and anti-CD200) has been adopted to isolate HCECs from a mixed population of HCECs and stromal fibroblasts by fluorescence-activated cell sorting (Cheong et al., 2013). In conclusion, to the best of our knowledge, this study is the first to report that 20% HAF significantly promotes the proliferation of and maintains the morphology of HCECs. The data presented in this study suggest that HAF possesses an invaluable stimulatory effect on HCEC growth and should be considered as a potential novel enriched supplement that can be used in serum-free media for the in vitro propagation of these cells.
Financial propriety There is no financial or propriety interest in any of the materials used in this manuscript.
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factor in fetal serum that stimulates deposition of hyaluronic acid. J. Pediatr. Surg. 24, 789e792. Marx, C.E., Vance, B.J., Jarskog, L.F., Chescheir, N.C., Gilmore, G.H., 1999. Nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3 levels in human amniotic fluid. Am. J. Obstet. Gynecol. 181, 1225e1230. Michaels, J.E., Dasari, S., Pereira, L., Reddy, A.P., Lapidus, J.A., Lu, X., Jacob, T., Thomas, A., Rodland, M., Roberts Jr., C.T., Gravett, M.G., Nagalla, S.R., 2007. Comprehensive proteomic analysis of the human amniotic fluid proteome: gestational age-dependent changes. J. Proteome Res. 6, 1277e1285. Nakahara, M., Okumura, N., Kay, E.P., Hagiya, M., Imagawa, K., Hosoda, Y., Kinoshita, S., Koizumi, N., 2013. Corneal endothelial expansion promoted by human bone marrow mesenchymal stem cell-derived conditioned medium. PLoS One 8, e69009. Newsome, D.A., Gross, J., Hassell, J.R., 1982. Human corneal stroma contains three distinct collagens. Investig. Ophthalmol. Vis. Sci. 22, 376e381. Park, S.J., Yoon, W.G., Song, J.S., Jung, H.S., Kim, C.J., Oh, S.Y., Yoon, B.H., Jung, G., Kim, H.J., Nirasawa, T., 2006. Proteome analysis of human amnion and amniotic fluid by two-dimensional electrophoresis and matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry. Proteomics 6, 349e363. Patel, S.V., 2007. Keratoplasty for endothelial dysfunction. Ophthalmology 114, 627e628. Paull, A.C., Whikehart, D.R., 2005. Expression of the p53 family of proteins in central and peripheral human corneal endothelial cells. Mol. Vis. 11, 328e334. Peh, G.S., Lee, M.X., Wu, F.Y., Toh, K.P., Balehosur, D., Mehta, J.S., 2012. Optimization of human corneal endothelial cells for culture: the removal of corneal stromal fibroblast contamination using magnetic cell separation. Int. J. Biomater. 2012, 601302. Peh, G.S., Toh, K.P., Ang, H.P., Seah, X.Y., George, B.L., Mehta, J.S., 2013. Optimization of human corneal endothelial cell culture: density dependency of successful cultures in vitro. BMC Res. Notes 3, 176. Peh, G.S., Toh, K.P., Wu, F.Y., Tan, D.T., Mehta, J.S., 2011. Cultivation of human corneal endothelial cells isolated from paired donor corneas. PLoS One 6, e28310. Reca, R., Mastellos, D., Majka, M., Marquez, L., Ratajczak, J., Franchini, S., Glodek, A., Honczarenko, M., Spruce, L.A., Janowska-Wieczorek, A., Lambris, J.D., Ratajczak, M.Z., 2003. Functional receptor for C3a anaphylatoxin is expressed by normal hematopoietic stem/progenitor cells, and C3a enhances their homingrelated responses to SDF-1. Blood 101, 3784e3793. Sanie-Jahromi, F., Ahmadieh, H., Soheili, Z.S., Davari, M., Ghaderi, S., Kanavi, M.R., Samiei, S., Deezagi, A., Pakravesh, J., Bagheri, A., 2012. Enhanced generation of retinal progenitor cells from human retinal pigment epithelial cells induced by amniotic fluid. BMC Res. Notes 5, 182. Siebert, J.W., Burd, A.R., McCarthy, J.G., Weinzweig, J., Ehrlich, H.P., 1990. Fetal wound healing: a biochemical study of scarless healing. Plast. Reconstr. Surg. 85, 495e502. Teo, B.K., Goh, K.J., Ng, Z.J., Koo, S., Yim, E.K., 2012. Functional reconstruction of corneal endothelium using nanotopography for tissue-engineering applications. Acta. Biomater. 8, 2941e2952. Tripathi, R.C., Borisuth, N.S., Tripathi, B.J., Fang, V.S., 1991. Analysis of human aqueous humor for epidermal growth factor. Exp. Eye. Res. 53, 407e409. Underwood, M.A., Gilbert, W.M., Sherman, M.P., 2005. Amniotic fluid: not just fetal urine anymore. J. Perinatol. 25, 341e348. Wilkinson, D.I., Theeuwes, M.J., Farber, E.M., 1994. Nerve growth factor increases the mitogenicity of certain growth factors for cultured human keratinocytes: a comparison with epidermal growth factor. Exp. Dermatol. 3, 239e245. Yokoo, S., Yamagami, S., Yanagi, Y., Uchida, S., Mimura, T., Usui, T., Amano, S., 2005. Human corneal endothelial cell precursors isolated by sphere-forming assay. Investig. Ophthalmol. Vis. Sci. 46, 1626e1631. Zhu, C., Joyce, N.C., 2004. Proliferative response of corneal endothelial cells from young and older donors. Investig. Ophthalmol. Vis. Sci. 45, 1743e1751.
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Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer
Effect of amniotic fluid on the in vitro culture of human corneal endothelial cells Sepehr Feizi a, *, Zahra-Soheila Soheili b, Abouzar Bagheri a, Sahar Balagholi a, Azam Mohammadian b, Mozhgan Rezaei-Kanavi a, Hamid Ahmadieh a, Shahram Samiei c, Kambiz Negahban d a
Ophthalmic Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran National Institute of Genetic Engineering and Biotechnology, Tehran, Iran Iranian Blood Transfusion Organization Research Center, Tehran, Iran d Department of Ophthalmology, Boston University School of Medicine, Boston, USA b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 September 2013 Accepted in revised form 1 April 2014 Available online 12 April 2014
The present study was designed to evaluate the effects of human amniotic fluid (HAF) on the growth of human corneal endothelial cells (HCECs) and to establish an in vitro method for expanding HCECs. HCECs were cultured in DMEM-F12 supplemented with 20% fetal bovine serum (FBS). Confluent monolayer cultures were trypsinized and passaged using either FBS- or HAF-containing media. Cell proliferation and cell death ELISA assays were performed to determine the effect of HAF on cell growth and viability. The identity of the cells cultured in 20% HAF was determined using immunocytochemistry (ICC) and realtime reverse transcription polymerase chain reaction (RT-PCR) techniques to evaluate the expression of factors that are characteristic of HCECs, including Ki-67, Vimentin, Naþ/Kþ-ATPase and ZO-1. HCEC primary cultures were successfully established using 20% HAF-containing medium, and these cultures demonstrated rapid cell proliferation according to the cell proliferation and death ELISA assay results. The ICC and real time RT-PCR results indicated that there was a higher expression of Naþ/Kþ-ATPase and ZO-1 in the 20% HAF cell cultures compared with the control (20% FBS) (P < 0.05). The 20% HAFcontaining medium exhibited a greater stimulatory effect on HCEC growth and could represent a potential enriched supplement for HCEC regeneration studies. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: human corneal endothelial cells human amniotic fluid in vitro culture
1. Introduction Human corneal endothelial cells (HCECs) help to maintain corneal clarity by serving the following two functions: a pump function that drives water from the corneal stroma to the aqueous humor and a barrier function that involves focal tight junctions. Therefore, these cells regulate the corneal stromal hydration state, which is critical for corneal transparency (Dikstein and Maurice, 1972). The density of HCECs decreases with age and in various diseases, such as bullous keratopathy and Fuchs’ endothelial dystrophy. This cell decrease is predominantly compensated by cell migration and enlargement, rather than by cell division, to replace dead cells (Joyce, 2004).
* Corresponding author. Ophthalmic Research Center, Department of Ophthalmology, Labbafinejad Medical Center, Boostan 9 St., Pasdaran Ave., Tehran 16666, Iran. Tel.: þ98 21 2258 4733/2259 1616; fax: þ98 21 2256 2138. E-mail address: [email protected] (S. Feizi). http://dx.doi.org/10.1016/j.exer.2014.04.002 0014-4835/Ó 2014 Elsevier Ltd. All rights reserved.
Corneal transplantations for endothelial dysfunction have evolved over the last decade from using penetrating keratoplasty to posterior lamellar keratoplasty techniques (Patel, 2007). The recently introduced posterior lamellar keratoplasty techniques, such as Descemet stripping endothelial keratoplasty (DSEK), are an effective therapeutic approach for these conditions. These techniques, however, are dependent on the availability of a good quality donor corneal tissue for transplantation, which can be scarce in some regions of the world because of a lack of donors. Tissue-engineered corneal endothelium is a novel approach that addresses this issue (Joyce and Zhu, 2004; Lai et al., 2006). HCECs do not proliferate in vivo because the cell cycle is arrested in the G1 phase (Chen et al., 2003; Kim et al., 2001; Paull and Whikehart, 2005). HCECs, however, retain the ability to proliferate in vitro under the proper culture conditions that include growth factors and eliminate suppressing factors from the culture medium (Ishino et al., 2004; Li et al., 2007; Zhu and Joyce, 2004; Engelmann and Friedl, 1989; Nakahara et al., 2013).
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Previously, several techniques and culture media have been developed and optimized using numerous agents and growth factors, including epidermal growth factor (EGF), fibroblast growth factor (FGF), nerve growth factor (NGF), and bovine pituitary extract (BPE) (Ishino et al., 2004; Li et al., 2007; Zhu and Joyce, 2004; Engelmann and Friedl, 1989; Joyce, 2004). Human amniotic fluid (HAF) is enriched with a variety of growth factors and nutrients, and different reports have shown that it is necessary for embryonic cell proliferation, differentiation and dedifferentiation (Hirai et al., 2002). In addition, HAF has also been found to be an effective stimulator of retinal progenitor cell development from the retinal pigment epithelium (Ghaderi et al., 2011; Sanie-Jahromi et al., 2012). The purpose of this study was to evaluate the effect of adding HAF to a culture system for the isolation and cultivation of HCECs. 2. Materials and methods 2.1. Tissue preparation Ninety-six human corneas, of which 95.8% were cultured successfully, were obtained from 75 donors between the ages of 5 and 51 years old (mean age, 35 6 years). All of the donor human corneas were obtained from the Central Eye Bank of Iran, preserved at 4 C (Optisol-GS preservative; Chiron Vision, Irvine, CA, USA) and used within 1 week of collection. All of the corneas had an endothelial cell count >2500 cells/mm2 but were deemed unsuitable for clinical use. The study was approved by the ethics committee of the Ophthalmic Research Center. The corneas were transferred to a plastic dish with the epithelium facing down. Then, under an operating microscope and sterile conditions, a 27-gauge needle attached to a 5-cc syringe (bevel facing up) was inserted into the stroma beneath Descemet’s membrane (DM) and up to the center of the cornea. Balanced salt solution was gently injected to detach DM and the endothelial cells from the stroma to Schwalbe’s line at 360 when possible. The samples were transferred to the laboratory in phosphate buffered saline (PBS) solution. 2.2. Human amniotic fluid preparation Ten ml HAF samples were collected from pregnant women who underwent amniocentesis to assess for genetic deficiencies during the first trimester of gestation. The amniotic fluid cells were collected for karyotype analysis. The supernatants from the samples showing no evidence of chromosomal abnormalities were pooled and used in our experimental procedures. The procedure used to collect these samples was approved by the ethics committees of the National Institute of Genetic Engineering and Biotechnology (NIGEB) and the Ophthalmic Research Center of Iran. All of the donors signed the informed consent document after the investigators explained the purpose of the study. The HAF samples were centrifuged at 300 g for 5 min at 4 C, and the resulting supernatants were then sterilized using a 0.2 mm membrane filter (Orange Scientific, Belgium) and stored at 70 C until further analysis. 2.3. Ex vivo cell culture The separated DM and endothelial cells were incubated in 3.5 mg/mL collagenase A (Roche, USA) for 50 min and then cultured in DMEM:F12 at a 1:1 (v/v) ratio (Sigma, Germany) and supplemented with 20% fetal bovine serum (FBS), 120 mg/ml penicillin (Sigma, Germany), and 220 mg/ml streptomycin (Sigma, Germany) on 6-well plates coated with 20 mg/ml of type IV collagen (Sigma,
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USA). All cultures were carried out in an incubator at 37 C with a humidified atmosphere of 5% CO2. The culture medium was first changed with DMEM:F12 1:1 supplemented with three different concentrations (10%, 20% and 30%) of HAF on the seventh day and subsequently replaced every 6 days. To potentially preserve any secreted growth factors, the culture medium was only partially replaced. Upon reaching 80% confluency, the cells were subcultured by treating with trypsin/EDTA. The trypsinized cells were gently centrifuged (5 min at 300 g), the supernatant was discarded and the residual precipitates were re-suspended in complete medium supplemented with 10%, 20% or 30% HAF and seeded on new plates. Seeding density was approximately 1 104 cells/cm2. Control samples were exposed to the same procedure and incubation conditions, however, the culture medium was supplemented with 20% FBS instead of HAF. 2.4. Purification of endothelial cells The presence of spindle shaped cells in the primary culture was indicative of stromal fibroblast contamination, and the colony of endothelial cells was transferred to a new 6-well plate using a cell scraper. This procedure was repeated as necessary until no fibroblasts were observed in any of the culture dishes. 2.5. Cell identification The characteristics of the cultured HCECs were determined and verified based on the morphology and the expression of molecular markers. In terms of morphology, the appearance of hexagons or “cobblestone”, which is characteristic of HCECs, was used to differentiate these cells from human corneal stromal fibroblasts, which have an elongated and spindle-shaped appearance (phase-contrast microscope, Olympus IX71). Cell morphology was quantitatively evaluated using morphometric analyses to compare cell size, coefficient of variance, as well as cell circularity between the different groups. For this purpose, morphometric data of the area and perimeter of randomly selected cells from phase contrast images of each culture medium (when cells became confluent) was manually outlined by pointto-point tracing of the cell borders using ImageJ software (Teo et al., 2012; Peh et al., 2013). When cultures reached a sufficient confluency, the cells were trypsinized and cultured in a 24-well plate. The expression level of the molecular markers was detected using immunocytochemistry (ICC). Furthermore, the RNA was extracted and reverse transcribed using a cDNA synthesis kit and subjected to amplification by realtime reverse transcription polymerase chain reaction (RT-PCR). Detailed procedures for the ICC and real time RT-PCR were provided by the product manufacturers. 2.6. Cell proliferation and death ELISA assays HCECs were plated on 96-well plates at 5000 cells/well in 200 ml complete medium. Next, the medium was replaced with 100 ml of fresh serum-free medium plus the indicated concentration of HAF. To determine whether the HAF altered cell proliferation, bromodeoxyuridine (BrdU) was added after 6 days of treatment, and the proliferation assay was performed according to the manufacturer’s instructions (Cell Proliferation assay kit; Roche, USA). The cytotoxicity was evaluated after 6 days of treatment using the Cell Death assay kit (including positive control; DNA-histone complex) according to the manufacturer’s instructions (Roche, USA).
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2.7. Immunocytochemistry (ICC)
3. Results
Cultured cells from passage 3 in 24-well plates were first fixed by incubating in 10 C methanol for 10 min. The cells were permeablized using Triton X-100 (0.25%) and then blocked in 1% bovine serum albumin (BSA) in PBS for 1 h at room temperature. Specific primary rabbit anti-human polyclonal antibodies were used to detect the Ki-67 (Rabbit anti-Ki67 H-300, Santa Cruz, USA, sc-15402, 60 min, 1:400), Vimentin (Rabbit anti-Vimentin H-84, Santa Cruz, USA, sc-5565, 60 min, 1:800), Naþ/Kþ-ATPase (Rabbit anti-Naþ/Kþ-ATPase H-300, Santa Cruz, USA, sc-28800, 90 min, 1:500) and ZO-1 (Rabbit anti-ZO-1 H-300, Santa Cruz, USA, sc10804, 90 min, 1:400) protein levels. Secondary fluorescein isothiocyanate (FITC) conjugated antibody (Goat anti-rabbit IgG-FITC, Santa Cruz, USA, sc-2012) against the primary antibodies were diluted (1:200) and used to detect the immunoreactivity of the cultures to the primary antibodies. All of the antibodies were diluted in PBS. After the final wash, the slides were incubated in mounting medium containing 4,6-diamidino-2-phenyindole dihydrochloride (DAPI) (1.5 mg/mL, Santa Cruz, USA, sc-24941) for 10 min to counterstain the nuclear DNA. The slides were examined using a fluorescence microscope (Olympus IX71) equipped with a 460 nm filter for the DAPI dye and a 520 nm filter for the FITC conjugated antibodies. To determine the specificity of our antibodies, we used a negative control for each marker that underwent the same procedure and used all of the same materials except for the primary antibody.
3.1. Endothelial cell morphology and purification Compared with the cells grown in 10% HAF, 30% HAF and 20% FBS, the cells grown in 20% HAF demonstrated a more uniform distinctive corneal endothelial morphology, which was very similar to the hexagonal mosaic appearance of corneal endothelial cells in vivo, produced a higher density in culture and reached complete confluency earlier (Fig. 1). Morphologic assessment demonstrated that HCECs cultured in 20% HAF were distinctly the most compact (2599.91 528.54 mm2), most homogeneous (coefficient of variation: 0.20) and hexagonal in shape as suggested by their cellular circularity index of 0.88 0.04 (Table 1). As shown in Fig. 1E, the 20% HAF-supplemented medium yielded a cell density that was 7-, 3-, and 1.5-times greater than the cell density achieved using 20% FBS, 10% HAF, and 30% HAF, respectively (P < 0.05 for all comparisons). Although the cells cultured in the 20% HAF-treated medium retained the morphology of endothelial cells, some vesicles began to grow and the HCECs became wider and lost their hexagonal shape after passage 4 and died after several weeks (Fig. 2A). In the beginning, the rate of fibroblast fusiform cell contamination in the cultures was 5%. Using a cell scraper to isolate the HCECs from the stromal fibroblasts (mainly performed at the first passage), no spindle shaped cells were observed after two passages (Fig. 2B). 3.2. Cell proliferation and death ELISA assays
2.8. Real-time reverse transcription polymerase chain reaction The total RNA was extracted from passage 3 cultured cells using QIAzolLysis Reagent (Qiagen, Germany). The concentration and purity of the isolated RNA were determined using a NanoDrop, and the integrity of the RNA was verified using agarose gel electrophoresis and ethidium bromide staining. Reverse transcription was performed using oligodT primers and the SuperScript reverse transcriptase kit (Invitrogen, USA). Real time RT-PCR was then performed using the QuantiFast SYBR Green PCR Kit (QIAGEN, Germany, 204054). To perform PCR, specific primers for Ki-67 (Qiagen, Germany, QT00014203), Vimentin (Qiagen, Germany, QT00095795), Naþ/Kþ-ATPase (Qiagen, Germany, QT00059808), ZO-1 (Qiagen, Germany, QT00077308) and GAPDH (QIAGEN, Germany, QT01192646), as a housekeeping gene control, were used. The PCR parameters were an initial denaturation (one cycle at 95 C for 10 min); denaturation and annealing/amplification at 95 C for 10 s and 60 C for 30 s, respectively, for 40 cycles; and a melting curve, 72 C, with the temperature gradually increasing (0.5 C) to 95 C.
2.9. Statistical analysis Real time RT-PCR, ICC and cell counts were performed in triplicate and ELISA tests were performed in duplicate. The morphometric parameters and cell density resulting from specific culture media as well as cell proliferation and death (determined by ELISA assay) were quantitatively compared between the control and experimental conditions (various concentrations of HAF) using a one-way ANOVA test. Differences in the expression levels of the different markers (determined by ICC and RT-PCR) were quantitatively evaluated between the control and the 20% HAF-treated cells using Student’s t-test (SPSS 16.0; IBM Corp., USA). A P < 0.05 was considered statistically significant.
The ELISA test results showed that the concentrations of HAF and FBS used to supplement the media had no cytotoxic effects on the HCECs compared with the positive control sample included with the kit (DNA-histone complex) (Fig. 3A). The HAF at all of the concentrations tested increased the cell proliferation rate (1.5 to two fold) compared with the control cultures grown in FBScontaining medium (P < 0.05). The greatest effect was observed in the cells cultured in 20% HAF-containing medium compared with the 20% FBS-, 10% HAF-, and 30% HAF-containing media (P < 0.05 for all comparisons) (Fig. 3B). 3.3. Demonstrative markers for characteristics specific to corneal endothelial cells 3.3.1. Immunocytochemistry The ICC results revealed that there were Vimentin, Ki-67, Naþ/ Kþ-ATPase and ZO-1 positive cells in the cultures (Fig. 4). The expression of the Naþ/Kþ-ATPase and ZO-1 proteins confirmed the identity of the isolated cells as corneal endothelial cells. For the HCECs cultured in 20% HAF, more than 98% of the cells expressed Vimentin and more than 95% of the HCECs expressed ZO-1. Furthermore, Naþ/Kþ-ATPase expression was detected in nearly 100% of the HCECs. Taken together, these results indicate that the HCECs retained the original characteristics of corneal endothelial cells. Additionally, more than 70% of the HCECs expressed the nuclear Ki-67 cell proliferation index, which indicates that the isolated cultivated cells also retained their proliferative ability in vitro. The negative controls (ICC test without primary antibody) did not exhibit any relative FITC signal. Compared with the control, the HCECs cultured in 20% HAFcontaining medium demonstrated a 3.5-fold, fivefold and threefold increase in the number of Naþ/Kþ-ATPase, Vimentin and ZO-1 positive cells, respectively (P < 0.05 for all comparisons) (Fig. 5). The number of cells positive for Ki-67, however, did not differ
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Fig. 1. Human corneal endothelial cell (HCEC) cultures in control serum (20% fetal bovine serum; A), 10% human amniotic fluid (HAF; B), and 10% HAF (C). The HCECs grown in 20% HAF (D) demonstrate a more uniform distinctive corneal endothelial morphology and produce a higher density, (phase-contrast microscope, Olympus IX71, 200). (E) HCEC numbers after 4 weeks of treatment with 10%, 20%, 30% HAF and 20% FBS as a control. The 20% HAF-supplemented medium yielded a cell density that was 7-, 3-, and 1.5-times greater than the cell density achieved using 20% FBS, 10% HAF, and 30% HAF, respectively (*P < 0.05 for all comparisons). Table 1 Cell size, coefficient of variation and cellular circularity of corneal endothelial cells cultured in different media. Culture medium Cell size (mm2) 20% FBS 10% HAF 20% HAF 30% HAF P value
6484.70 4025.22 2599.91 3372.88 0.05). Compared with the control, the number of cells positive for Naþ/K þ -ATPase (B), Vimentin (C) and ZO-1 (D) was significantly higher in the 20% HAF-treated HCECs (3.5-fold, fivefold and threefold increase, respectively, *P < 0.05 for all comparisons).
for the ex vivo expansion of RPE cells and the trans-differentiation of these cells into differentiated neurons (retinal ganglion cells and rod photoreceptors) (Ghaderi et al., 2011; Sanie-Jahromi et al., 2012). Currently, the collective profile of AF proteins, the precise interactions between these proteins and their potential beneficial effects remain to be determined. Furthermore, potential variability can exist between samples obtained from different donors and at different gestational ages (Michaels et al., 2007). More than 98% of HAF is composed of water. Electrolytes, proteins, peptides, carbohydrates, lipids, hormones and various growth factors, including epidermal growth factor (EGF), fibroblast growth factor (FGF), nerve growth factor (NGF), hepatocyte growth factor (HGF), transforming growth factor alpha (TGF-a), transforming growth factor beta (TGF-b1), insulin-like growth factor-I (IGF-I), insulinlike growth factor-II (IGF-II), erythropoietin (EPO), granulocyte colony stimulating factor (G-CSF), and macrophage colony stimulating factor (M-CSF), make up the remaining portion (Hirai et al., 2002; Colombo et al., 1993; Marx et al., 1999; Kimble et al., 1999). Cho et al. (2007) identified the 15 most abundant proteins in HAF at the gestational ages between 16 and 18 weeks, which include albumin, fibronectin, serotransferrin, complement C3, and ceruloplasmin. Complement C3 is a factor in HAF that has been found to be responsible for the regeneration of damaged tissue (Kimura et al., 2003; Reca et al., 2003). Plasminogen is a factor that is also involved in cell proliferation and wound healing. Ceruloplasmin, a1 microglobulin, serotransferrin, apolipoprotein A and
albumin are other HAF proteins that are essential for cell homeostasis and transport. The hyaluronic acid in HAF fosters an extracellular environment that is permissive for cell motility and proliferation (Longaker et al., 1989; Siebert et al., 1990). These factors may work in a synergistic manner and may play a more important role in tissue development when acting in combination (Bry and Hallman, 1992; Wilkinson et al., 1994). The results of this study suggest that HAF is a valuable compound because it contains all of these aforementioned factors, and therefore, it should be considered a potential substantial supplemental medium. Based on the morphometric analysis, HCECs demonstrated more organized alignment and higher density in the 20% HAF culture media. After passage 4, however, the HCECs cultured in 20% HAF demonstrated some vesicles and became wider suggestive of senescence. A higher concentration of HAF (30%) repressed further cell proliferation. It is possible that the proliferative effects of the growth factors are overshadowed by the inhibitory factors at this higher concentration. For example, TGF-b, an ingredient in HAF, has been found to suppress S-phase entry in cultured corneal endothelial cells (Chen et al., 1999). To prevent stromal fibroblast contamination, we attempted to strip only the endothelial cells along with DM. Some posterior stroma (between 6 and 20 microns), however, inevitably remain attached to the underlying DM, no matter how precisely DM is removed (Jafarinasab et al., 2010). Therefore, the explant in vitro will normally give rise to a mixed cell population that contains stromal fibroblasts and endothelial cells. Because stromal
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Fig. 6. Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) analysis of the Ki-67, Naþ/K þ -ATPase, Vimentin and ZO-1 gene expression in cultured cells treated with 20% human amniotic fluid (HAF). The mRNA levels were normalized to GAPDH and are presented as a percentage of the control value. Compared with the control, the HAF treatment increased the Naþ/K þ -ATPase and ZO-1 gene expression by 400-fold and 600-fold, respectively (*P < 0.05). There appeared to be no significant difference in the expression levels of Ki-67 and Vimentin between the HAF-treated cells and the control (P > 0.05).
fibroblasts have a faster growth rate, they can outnumber the endothelial cells after 10 days of culture (Hyldahl, 1984). To eliminate the contaminating fibroblasts, a cell scraper was used on the primary culture as soon as a few fibroblasts were visible to transfer only the endothelium to a new culture vessel. The results of the present study demonstrate that the method of DM removal from the donor corneas and the use of a cell scraper to remove the fibroblasts at a very early passage of cell culture is an effective method for eliminating irrelevant cellular contamination. Some investigators have used a selective medium, containing D-valine instead of L-valine, in which fibroblastic cells are unable to grow (Engelmann et al., 1988) to eliminate unrelated cellular contamination. Alternatively, contaminating stromal fibroblasts can effectively be depleted from HCEC cultures through a negative celleselection strategy by magnetic affinity cell separation using antifibroblast magnetic microbeads. Using this technique, Peh et al. (2012) reported a separation efficacy of 96.88%. Additionally, a positive selection strategy recruiting two monoclonal antibodies (anti-GPC4 and anti-CD200) has been adopted to isolate HCECs from a mixed population of HCECs and stromal fibroblasts by fluorescence-activated cell sorting (Cheong et al., 2013). In conclusion, to the best of our knowledge, this study is the first to report that 20% HAF significantly promotes the proliferation of and maintains the morphology of HCECs. The data presented in this study suggest that HAF possesses an invaluable stimulatory effect on HCEC growth and should be considered as a potential novel enriched supplement that can be used in serum-free media for the in vitro propagation of these cells.
Financial propriety There is no financial or propriety interest in any of the materials used in this manuscript.
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