Current Eye Research, 2015; 40(1): 66–71 ! Informa Healthcare USA, Inc. ISSN: 0271-3683 print / 1460-2202 online DOI: 10.3109/02713683.2014.917189
ORIGINAL ARTICLE
Cellular Toxicity of Calf Blood Extract on Human Corneal Epithelial Cells In Vitro Young Min Park1, Su Jin Kim2, Young Sang Han3 and Jong Soo Lee1
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1
Department of Ophthalmology, School of Medicine, Pusan National University & Medical Research Institute, Pusan National University Hospital, Pusan, South Korea, 2Department of Ophthalmology, Maryknoll Hospital, Pusan, Korea, and 3Department of Ophthalmology, St. Mary’s Medical Center, Pusan, Korea
ABSTRACT Purpose: To investigate the biologic effects of the calf blood extract on corneal epithelial cells in vitro. Materials and methods: The effects on corneal epithelial cells were evaluated after 1, 4, 12, and 24 h of exposure to various concentrations of calf blood extract (3, 5, 8 and 16%). The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5diphenyl tetrazolium bromide) assay was performed to measure levels of cellular metabolic activity. The lactate dehydrogenase (LDH) assay was performed to determine the extent of cellular damage. Cellular morphology was examined using phase-contrast microscopy. The scratch wound assay was performed to quantify the migration of corneal epithelial cells. Results: At the 3 and 5% concentrations of calf blood extract, MTT values were similar to those observed in the control group. However, at a concentration of 8 and 16%, cellular metabolic activity was significantly decreased after 4 h of exposure to calf blood extract. After 12 h of exposure to 8 and 16% concentrations of calf blood extract, LDH activity and cellular morphological damage to the corneal epithelial cells were significantly increased. There was no evidence of cellular migration after 12 h exposure to 5% or higher concentration of calf blood extract because of cellular toxicity. Conclusions: Compared with normal corneal epithelial cells, the cellular activity was decreased, and toxicity was increased after over 12 h of exposure to more than 5% concentration of calf blood extract. Further clinical studies will be necessary to determine the optimal concentration and exposure time for the topical application of eye drops containing calf blood extract. Keywords: Calf blood extract, corneal epithelium, scratch wound assay, Solcoseryl, toxicity
INTRODUCTION
However, the main disadvantages of ongoing treatment with serum drops are the need for repeated blood collection, which excludes patients who cannot donate blood; it also involves a risk of secondary infection.8 SolcoserylÕ is a protein-free, standardized dialysate/ultrafiltrate derived from calf blood, which has been used to enhance wound healing in both experimental and animal studies, including man in vitro.11–14 Preliminary studies showed that higher concentrations of calf blood extract were associated with decreased cellular metabolic activity and
Autologous serum contains substances such as epidermal growth factor (EGF), vitamin A, transforming growth factor beta (TGF-b), fibronectin, insulin-like growth factor 1, substance P, nerve growth factor and other cytokines that are essential for the proliferation, differentiation and maturation of the normal corneal and conjunctival epithelium.1–6 Thus, autologous serum eye drops have been applied as a novel approach to treat severe dry eye syndrome and as a supportive measure in corneal wound healing.7–10
Received 25 November 2013; revised 19 March 2014; accepted 17 April 2014; published online 14 May 2014 Correspondence: Jong-Soo Lee, MD, PhD, Department of Ophthalmology, School of Medicine, Pusan National University & Medical Research Institute, Pusan National University Hospital, Pusan, Republic of Korea. Tel: 82-51-240-7321. Fax: 82-51-242-7341. E-mail: [email protected]; [email protected]
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Calf Blood Extract on Corneal Epithelial Cells increased cellular toxicity.15 To date, the effects of SolcoserylÕ on human corneal epithelial cells are not well understood, especially regarding drug concentration. This study is designed to investigate the efficacy and cytotoxicity of calf blood extract (SolcoserylÕ ) when applied to corneal epithelial cells, with special attention to drug concentration and exposure time.
METHODS
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Cell Culture and Preparation This study was in accordance with the tenets of the Declaration of Helsinki. Human corneal epithelial cell primary cultures were obtained using left over human donor peripheral corneas after transplantation.16 Under a tissue culture hood, Descemet’s membrane, endothelium and posterior stroma were removed using forceps under a dissecting microscope. The anterior cornea with intact epithelium was covered with 1.2 U/mL of Dispase II (Boehringer Mannheim, Mannheim, Germany) in calcium and magnesiumfree phosphate-buffered saline (PBS) and incubated at 37 C in a humidified 5% CO2 incubator for 1 h. Epithelial cells were then centrifuged at 1000 revolutions per minute (rpm) for 5 min. Primary cultures of corneal epithelial cells were prepared in 35 mm Petri tissue-culture dishes (Corning Incorporated, Corning, NY), containing Dulbecco’s modified Eagle’s medium (DMEM; Gibco BRL, Rockville, NY) supplemented with 10% fetal bovine serum (Gibco BRL), 20 ng/mL EGF (Gibco BRL), 100 U/mL penicillin (Gibco BRL) and 100 mg/mg streptomycin (Gibco BRL). Cells were incubated at 37 C in a humidified atmosphere of 95% air and 5% CO2. Culture medium was changed every two or three days. Corneal epithelial cells usually grew after 4–7 d and reached confluence within 21–28 d. The cells were then enzymatically detached with 0.25% trypsin and 0.002% EDTA (Irvine Scientific, Santa Ana, CA) at 37 C for 10 min after washing once with Dulbecco’s PBS (D-PBS) (Gibco BRL). The suspended epithelial cells were then centrifuged at 400 rpm for 10 min. The supernatant was removed and fresh medium was added. The cell suspension was counted in a hemocytometer, and 5 103 cells/well were plated using 96-well tissue culture plates. Second-passage human corneal epithelial cells were used in all experiments. These cells were incubated in 1 mL of culture media at 37 C (5% CO2, 95% air) and were allowed to attach to the bottom of the well for 24 h before calf blood extract was added. The effect of the drug on corneal epithelial cells can be underestimated when cells are too dense, so the cells were cultured for approximately 4–5 d to ensure 80–90% confluence. !
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MTT Assay for Cellular Metabolic Activity To determine the proliferation rate of human corneal epithelial cells, the colorimetric tetrazolium salt MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide; Sigma, St. Louis, MO) test was performed.17 The MTT assay is based on the production of purple formazan from a methyl tetrazolium salt by the mitochondrial enzymes of viable cells. Cultured cells were seeded in 96-well culture plates at a concentration of 4000 cells/well and allowed to form a monolayer for 24 h. The cells were then exposed to 150 mL of DMEM medium containing 3, 5, 8 and 16% calf blood extract for 1, 4, 12 and 24 h. After the exposure, cells were washed twice with PBS and they were incubated in culture media for 24 h, and then MTT assay was performed. The balanced salt solution treated group was used as a control. At the end of the incubation period, the MTT solution was carefully aspirated, with care taken not to disturb the purple formazan crystal at the bottom of each well. The formazan reaction product was dissolved by adding 150 mL dimethyl sulfoxide (Sigma), and the optical density of each well was measured using an automatic plate reader (Molecular Devices, Sunnyvale, CA) with a 570-nm test wavelength and a 690-nm reference wavelength. In each experiment, eight wells were used for each concentration. This procedure was repeated in triplicate.17 Cellular metabolic activity was calculated with the mean of the absorption rates at each exposure time and concentration. Cellular metabolic activity was demonstrated by the following calculation: cellular metabolic activity (%) = absorption rate of each well/ absorption rate of control group 100. Data were analyzed using Wilcoxon signed rank test and were considered statistically significant at p50.05.
Lactate Dehydrogenase Assay for Cellular Toxicity The lactate dehydrogenase (LDH) assay measures leakage of the cytoplasmic enzyme LDH into the extracellular medium. The presence of LDH in the cell culture medium represents cell membrane damage. For this assay, 4.0 103 corneal epithelial cells/mL were seeded per well of 96-microtiter plates. Twentyfour hours after cell seeding, cells were exposed to each concentration of calf blood extracts. The LDH titer was assessed at 1, 4, 12 and 24 h after addition of the agent. The supernatant was collected from each well. LDH activity was measured in the supernatant using CytoTox 96, a nonradioactive cytotoxicity assay kit (Promega, Madison, WI), in accordance with the manufacturer’s instructions. Absorbance was determined at 490 nm with one 96-well plate enzymelinked immunosorbent assay reader. LDH activity,
68 Y. M. Park et al. which is proportional to color intensity, was expressed as optical density. The effect of the balanced salt solution-treated group was used as a control. To evaluate the significance of the differences of cell toxicity, the data were analyzed by ANOVA test and were considered statistically significant at p50.05.
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Cellular Morphology Analysis with Phase-Contrast Microscopy The cellular morphological changes were investigated using a phase-contrast microscope (Nikon, Tokyo, Japan). The cells (5 103 cell/mL/well) that were grown to confluence in 24-well plates were seeded at 500 mL/well in six-well plates, and then these cells were subsequently incubated in DMEM in 5% CO2 and 95% air at 37 C. After exposure to 3, 5, 8 and 16% calf blood extracts for 1, 4, 12 and 24 h, cells were incubated at 37 C for 24 h after being rinsed with D-PBS. The morphologic evaluation of the cytotoxic drug effects was performed using phase-contrast microscopy by a well-trained technician.
Scratch-Wound Assay for Cellular Migration A scratch-wound assay with human corneal epithelial cells was used to determine whether calf blood extract could promote wound closure. Human corneal epithelial cells (5 103 cell/mL/well), which were grown to confluence on 24-well plates, were then seeded at 500 mL/well on six-well plates, and then they were subsequently incubated in DMEM in 5% CO2 and 95% air at 37 C. The human corneal epithelial cells were wounded by scratching the surface of the culture with a 100 mL pipette tip. The scratched human corneal epithelial cells were washed with fresh medium to
remove the detached cells, and then the cells were incubated in the medium in the presence of 3, 5, 8 or 16% calf blood extract for 1, 12 and 24 h. The migration of cells was measured using an inverted phase-contrast light microscope. The balanced salt solution treated group was used as a control. To ensure that wounds of similar area were compared, multiple positioning marks were made at the center of the denuded surface with a small needle. Eighteen hours after wounding, the monolayers were fixed and stained, and the migrated cells’ edges were imaged.
RESULTS Effects of Calf Blood Extract on Cellular Metabolic Activity The MTT assay is used as a marker for cell viability or metabolic activity. In this study, DMEM culture medium was used as a control. After incubation with calf blood extract for 1, 4, 12 and 24 h, MTT values were significantly decreased in a time- and dose-dependent manner compared to control, especially with 8 and 16% concentrations of calf blood extract. For 8% concentrations of calf blood extract, MTT values showed significant decrease after 4 (p = 0.032), 12 (p = 0.012) and 24 h (p = 0.014) of exposure. In addition, for 16% concentrations of calf blood extract, MTT values showed significant decrease after 4 (p = 0.021), 12 (p = 0.010) and 24 h (p = 0.009) of exposure. For 3 and 5% concentrations of calf blood extract, MTT values were similar to those of controls (p40.05). However, at concentrations of 8 and 16%, cellular metabolic activity was significantly decreased after 4-h exposure to calf blood extract (Figure 1).
FIGURE 1 The absorption rate of the water-insoluble formazan dye in corneal epithelial cells exposed to calf blood extract, as determined by a scanning spectrometer (ELISA reader). The metabolic activity of corneal epithelial cells decreased at 8 and 16% concentration and longer exposure durations. Current Eye Research
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Calf Blood Extract on Corneal Epithelial Cells
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FIGURE 2 Lactate dehydrogenase (LDH) titers of cultured corneal epithelial cells exposed to calf blood extract. The LDH titers exhibit a dose- and time-dependent response relationship. LDH activity and cellular damage to corneal epithelial cells were most evident after exposure to 8 and 16% calf blood extract.
Effects of Calf Blood Extract on Cellular Toxicity Within 24 h, the LDH titer of cultured human corneal epithelial cells in calf blood extract exhibited a timeand dose-dependent relationship. At 8 and 16% concentrations of calf blood extract, LDH titers increased significantly up to 12 h of exposure (p = 0.012 and 0.009) and then decreased (after 24 h of exposure, p = 0.018 and 0.012). In addition, for 8 and 16% concentrations of calf blood extract, the LDH titer increased greater than 1.7-fold, when compared to the 3 and 5% concentrations of calf blood extract (50.8 at 4, 12 and 24 h) (Figure 2).
Effects of Calf Blood Extract on Cellular Morphology Using phase-contrast microscopy, control human corneal epithelial cells were observed to be densely distributed throughout the culture media. At the 3 and 5% concentrations of calf blood extract, cellular density was similar to control after 4, 12 and 24 h of exposure. However, as the concentration of calf blood extract and/or exposure time increased, more corneal epithelial cells swelled and detached from the dishes (Figure 3).
Effect of Calf Blood Extract on Cellular Migration Twenty-four hours after wounding, 3% concentration of calf blood extract promoted cell migration, with significantly increased wound closure as compared to higher concentrations of calf blood extract. At the 5% concentrations of calf blood extract, human !
2015 Informa Healthcare USA, Inc.
corneal epithelial cell migration decreased significantly after 24 h exposure. Furthermore, prominent cellular damage was found after 12 h exposure to a concentration of 8 and 16% calf blood extract (Figure 4).
DISCUSSION In 1984, Fox et al.18 first reported beneficial effects of autologous serum eye drops for the treatment of dry eye in Sjogren’s syndrome. Thereafter, autologous serum eye drops have been reported to be effective for the treatment of severe dry eye-related ocular surface disorders such as Sjogren’s syndrome, graft-versushost disease, Stevens–Johnson syndrome, superior limbic keratitis, exposure keratitis, persistent epithelial defects, recurrent corneal erosion and neurotrophic keratitis.3,4,7,18 However, there are some disadvantages of treatment with autologous serums drops. Repeated blood collection is required for ongoing treatment with fresh serum, and the preparation of serum eye drops requires a well-equipped laboratory and trained personnel. The use of autologous serum is contraindicated in patients with severe systemic disease, those with a fear of extracting blood, those who are infants or extremely elderly and those with chronic illness. There is also the risk of secondary infection due to drop contamination. So topical allogenic serum eye drops have been proposed as an alternative therapeutic option for treating severe ocular surface diseases requiring serum treatment. Nonetheless, the risk of transmitting blood-borne diseases still remained, with one report of Human Immunodeficiency Virus transmission by a single droplet.19 Fetal calf blood extract is used extensively in the laboratory to promote cell growth in culture. Some studies have reported favorable effects on corneal
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FIGURE 3 Phase-contrast microscopic photographs of corneal epithelial cells after 1, 4, 12 and 24 h of exposure to (A) control, (B) 3% calf blood extract, (C) 5% calf blood extract, (D) 8% calf blood extract and (E) 16% calf blood extract. Corneal epithelial cell densities decreased after 4 h of exposure to 8 and 16% calf blood extract. Contrary to normal corneal epithelial cells showing hyper-reflective round edge with hypo-reflective center, damaged cells show hyper-reflective irregular morphology without hypo-reflective center.
FIGURE 4 Scratch assay of corneal epithelial cells immediately after exposure to (A) control, (B) 3% calf blood extract, (C) 5% calf blood extract, (D) 8% calf blood extract and (E) 16% calf blood extract after 1, 12 and 24 h of exposure. The effects of calf blood extract on wound closure were visualized after confluent human corneal epithelial cells were scratch wounded and incubated in the culture medium. At the 5% concentrations of calf blood extract, human corneal epithelial cell migration decreased significantly after 24 h exposure (C3). Furthermore, prominent cellular damage was found after 12-h exposure to a concentration of 8 and 16% calf blood extract (D2, D3, E2 and E3).
wound healing after the application of SolcoserylÕ , a protein-free hemodialysate derived from calf blood.11–14 Egger et al.12 showed that fetal calf blood extract accelerates the migration of corneal epithelial cells in vitro. Spessotto et al.11 showed that fetal calf blood extract increases the differentiation of human monocytes in vitro. But, on the contrary, preliminary studies showed decreased cellular metabolic activity and increased cellular toxicity after exposure to calf blood extract.15 Until now, the effect of calf blood extract concentration on human corneal epithelial cells has remained uncertain. This study was performed using lower concentrations of calf blood
extract to identify a formulation that is safe and effective for clinical use. In this study, the cellular metabolic activity of cultured human corneal epithelial cells in calf blood extract decreased in time- and dose-dependent manner, especially with 8 and 16% concentrations of calf blood extract. The cell death also showed a timeand dose-dependent response. Higher concentrations of drug caused far more damage to the corneal epithelial cells than did lower concentrations. In addition, there was no evidence of cellular migration after 12 h exposure to 5% or higher concentration of calf blood extract. Current Eye Research
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Calf Blood Extract on Corneal Epithelial Cells The TGF-b concentration in human serum is around 50 ng/mL, five times higher than that in tears. TGF-b is known to have antiproliferative effects, and high concentrations of this molecule may suppress wound healing of the ocular surface epithelium. This is one of the reasons for diluting serum to 20%.8,10 Furthermore, there is a report suggesting dilution of serum from 12.5 to 25%, which growth factors might reach their optimal range.20 Although, the level of TGF-b nor growth factors was not measured in this study, the higher concentration of TGF-b is expected in the higher concentration of calf blood extract. As no study has proven the pharmacokinetics and the cytokines of calf blood extract, further study might be necessary to support our hypothesis. In this study, the metabolic activity and the migration of corneal epithelial cell were inhibited more strongly at higher concentrations of calf blood extract. Although, clinically calf blood extracts are not used in the settings of this study, prolongation of exposure time with higher concentration of calf blood extract needs precaution. This study demonstrated that a solution of less than 8% (3 and 5%) concentration of calf blood extract caused less damage to corneal epithelial cells than did higher concentrations. Therefore, lower concentrations of calf blood extract should be safe for clinical use. In case of prescribing high concentration of calf blood extract, shortening the drug exposure time by prolonging the instillation term seems necessary. Although the results of the in vitro test may not always be applicable in vivo, it is believed that it will be a valuable guide for determining the optimal drug concentration for use in clinical situations. Further clinical studies concerned with the concentration and exposure duration to be used when treating patients with topical formulations of calf blood extract will be necessary.
DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This study was supported by Biomedical Research Institute Grant (2010-4), Pusan National University Hospital, and a grant from the Korean Health technology R&D Project, Ministry of Health and Welfare (A070001), Republic of Korea.
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REFERENCES 20. 1. Noble BA, Loh RS, MacLennan S, Pesudovs K, Reynolds A, Bridges LR, et al. Comparison of autologous serum eye drops with conventional therapy in a randomized
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controlled crossover trial for ocular surface disease. Br J Ophthalmol 2004;88:647–652. Liu L, Hartwig D, Harloff S, Herminghaus P, Wedel T, Geerling G. An optimized protocol for the production of autologous serum eyedrops. Graefes Arch Clin Exp Ophthalmol 2005;243:706–714. Poon AC, Geerling G, Dart JK, Fraenkel GE, Daniels JT. Autologous serum eye drops for dry eyes an epithelial cell defects: clinical and in vitro toxicity studies. Br J Ophthalmol 2001;85:1188–1197. Matsumoto Y, Dogru M, Goto E, Ohashi Y, Kojima T, Ishida R, et al. Autologous serum application in the treatment of neurotrophic keratopathy. Ophthalmology 2004;111:1115–2220. Phan TM, Foster CS, Boruchoff SA, Zagachin LM, Colvin RB. Topical fibronectin in the treatment of persistent corneal epithelial defects and trophic ulcers. Am J Ophthalmol 1987;104:494–501. Fredj-Reygrobellet D, Plouet J, Delayre T, Baudouin C, Bourret F, Lapalus P. Effects of aFGF and bFGF on wound healing in rabbit cornea. Current Eye Res 1987;6:1205–2309. Tsubota K, Goto E, Fujita H, Ono M, Inoue H, Saito I, et al. Treatment of dry eye by autologous serum application in Sjogren’s syndrome. Br J Ophthalmol 1999;83:390–395. Kojima T, Higuchi A, Goto E, Matsumoto Y, Dogru M, Tsubota K. Autologous serum eye drops for the treatment of dry eye disease. Cornea 2008;27:25–30. Lee GA, Chen SX. Autologous serum in the management of recalcitrant dry eye syndrome. Clin Experiment Ophthalmol 2008;36:119–122. Tsubota K, Goto E, Shimmura S, Shimazaki J. Treatment of persistent epithelial defect by autologous serum application. Ophthalmology 1999;106:1984–1989. Spessotto P, Dri P, Baschong W, Mittenzwei H, Patriarca P. Effect of protein-free dialysate from calf blood on human monocyte differentiation in vitro. Arzeimittelforschung 1993;43:747–751. Egger SF, Huber-Spitzy V, Alzner E, Scholda C, Vecsei VP. Corneal wound healing after superficial foreign body injury: vitamin A and dexpanthenol versus a calf blood extract. Ophthalmologica 1999;213:246–249. Studer O. A comparative clinical study of SolcoserylÕ eye gel and cystein eye gel 2.4% in the treatment of foreign body injuries of the cornea. Ophthalmic Res 1984;16: 179–184. Kuniaka T, Senga Y, Senga H, Weiner M. Nature of enhanced mitochondrial oxidative metabolism by a calf blood extract. J Cell Physiol 1991;146:148–155. Lee JS, Kim EH, Kim SJ. Effects of calf serum on human corneal epithelial cells in vitro. J Korean Ophthalmol Soc 2012;52:852–858. Gipson IK, Grill SM. A technique for obtaining sheets of intact rabbit corneal epithelium. Invest Opthahlmol Vis Sci 1982;23:269–273. Mosmann TJ. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxic assays. J Immunol Methods 1983;65:55–63. Fox RI, Chan R, Michelson JB, Belmont JB, Michelson PE. Beneficial effect of artificail tears made with autologous serum in patients with keratoconjunctivitis sicca. Arthritis Rheum 1984;27:459–461. Eberle J, Habermann J, Gurtler LG. HIV-1 infection transmitted by serum droplets into the eye: a case report. AIDS 2000;14:206–207. Liu L, Hartwig D, Harloff S, Herminghaus P, Wedel T, Geerling G. An optimised protocol for the production of autologous serum eyedrops. Graefes Arch Clin Exp Ophthalmol 2005;243:706–714.
ORIGINAL ARTICLE
Cellular Toxicity of Calf Blood Extract on Human Corneal Epithelial Cells In Vitro Young Min Park1, Su Jin Kim2, Young Sang Han3 and Jong Soo Lee1
Curr Eye Res 2015.40:66-71. Downloaded from informahealthcare.com by Library Univ. of Texas on 12/27/14. For personal use only.
1
Department of Ophthalmology, School of Medicine, Pusan National University & Medical Research Institute, Pusan National University Hospital, Pusan, South Korea, 2Department of Ophthalmology, Maryknoll Hospital, Pusan, Korea, and 3Department of Ophthalmology, St. Mary’s Medical Center, Pusan, Korea
ABSTRACT Purpose: To investigate the biologic effects of the calf blood extract on corneal epithelial cells in vitro. Materials and methods: The effects on corneal epithelial cells were evaluated after 1, 4, 12, and 24 h of exposure to various concentrations of calf blood extract (3, 5, 8 and 16%). The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5diphenyl tetrazolium bromide) assay was performed to measure levels of cellular metabolic activity. The lactate dehydrogenase (LDH) assay was performed to determine the extent of cellular damage. Cellular morphology was examined using phase-contrast microscopy. The scratch wound assay was performed to quantify the migration of corneal epithelial cells. Results: At the 3 and 5% concentrations of calf blood extract, MTT values were similar to those observed in the control group. However, at a concentration of 8 and 16%, cellular metabolic activity was significantly decreased after 4 h of exposure to calf blood extract. After 12 h of exposure to 8 and 16% concentrations of calf blood extract, LDH activity and cellular morphological damage to the corneal epithelial cells were significantly increased. There was no evidence of cellular migration after 12 h exposure to 5% or higher concentration of calf blood extract because of cellular toxicity. Conclusions: Compared with normal corneal epithelial cells, the cellular activity was decreased, and toxicity was increased after over 12 h of exposure to more than 5% concentration of calf blood extract. Further clinical studies will be necessary to determine the optimal concentration and exposure time for the topical application of eye drops containing calf blood extract. Keywords: Calf blood extract, corneal epithelium, scratch wound assay, Solcoseryl, toxicity
INTRODUCTION
However, the main disadvantages of ongoing treatment with serum drops are the need for repeated blood collection, which excludes patients who cannot donate blood; it also involves a risk of secondary infection.8 SolcoserylÕ is a protein-free, standardized dialysate/ultrafiltrate derived from calf blood, which has been used to enhance wound healing in both experimental and animal studies, including man in vitro.11–14 Preliminary studies showed that higher concentrations of calf blood extract were associated with decreased cellular metabolic activity and
Autologous serum contains substances such as epidermal growth factor (EGF), vitamin A, transforming growth factor beta (TGF-b), fibronectin, insulin-like growth factor 1, substance P, nerve growth factor and other cytokines that are essential for the proliferation, differentiation and maturation of the normal corneal and conjunctival epithelium.1–6 Thus, autologous serum eye drops have been applied as a novel approach to treat severe dry eye syndrome and as a supportive measure in corneal wound healing.7–10
Received 25 November 2013; revised 19 March 2014; accepted 17 April 2014; published online 14 May 2014 Correspondence: Jong-Soo Lee, MD, PhD, Department of Ophthalmology, School of Medicine, Pusan National University & Medical Research Institute, Pusan National University Hospital, Pusan, Republic of Korea. Tel: 82-51-240-7321. Fax: 82-51-242-7341. E-mail: [email protected]; [email protected]
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Calf Blood Extract on Corneal Epithelial Cells increased cellular toxicity.15 To date, the effects of SolcoserylÕ on human corneal epithelial cells are not well understood, especially regarding drug concentration. This study is designed to investigate the efficacy and cytotoxicity of calf blood extract (SolcoserylÕ ) when applied to corneal epithelial cells, with special attention to drug concentration and exposure time.
METHODS
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Cell Culture and Preparation This study was in accordance with the tenets of the Declaration of Helsinki. Human corneal epithelial cell primary cultures were obtained using left over human donor peripheral corneas after transplantation.16 Under a tissue culture hood, Descemet’s membrane, endothelium and posterior stroma were removed using forceps under a dissecting microscope. The anterior cornea with intact epithelium was covered with 1.2 U/mL of Dispase II (Boehringer Mannheim, Mannheim, Germany) in calcium and magnesiumfree phosphate-buffered saline (PBS) and incubated at 37 C in a humidified 5% CO2 incubator for 1 h. Epithelial cells were then centrifuged at 1000 revolutions per minute (rpm) for 5 min. Primary cultures of corneal epithelial cells were prepared in 35 mm Petri tissue-culture dishes (Corning Incorporated, Corning, NY), containing Dulbecco’s modified Eagle’s medium (DMEM; Gibco BRL, Rockville, NY) supplemented with 10% fetal bovine serum (Gibco BRL), 20 ng/mL EGF (Gibco BRL), 100 U/mL penicillin (Gibco BRL) and 100 mg/mg streptomycin (Gibco BRL). Cells were incubated at 37 C in a humidified atmosphere of 95% air and 5% CO2. Culture medium was changed every two or three days. Corneal epithelial cells usually grew after 4–7 d and reached confluence within 21–28 d. The cells were then enzymatically detached with 0.25% trypsin and 0.002% EDTA (Irvine Scientific, Santa Ana, CA) at 37 C for 10 min after washing once with Dulbecco’s PBS (D-PBS) (Gibco BRL). The suspended epithelial cells were then centrifuged at 400 rpm for 10 min. The supernatant was removed and fresh medium was added. The cell suspension was counted in a hemocytometer, and 5 103 cells/well were plated using 96-well tissue culture plates. Second-passage human corneal epithelial cells were used in all experiments. These cells were incubated in 1 mL of culture media at 37 C (5% CO2, 95% air) and were allowed to attach to the bottom of the well for 24 h before calf blood extract was added. The effect of the drug on corneal epithelial cells can be underestimated when cells are too dense, so the cells were cultured for approximately 4–5 d to ensure 80–90% confluence. !
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MTT Assay for Cellular Metabolic Activity To determine the proliferation rate of human corneal epithelial cells, the colorimetric tetrazolium salt MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide; Sigma, St. Louis, MO) test was performed.17 The MTT assay is based on the production of purple formazan from a methyl tetrazolium salt by the mitochondrial enzymes of viable cells. Cultured cells were seeded in 96-well culture plates at a concentration of 4000 cells/well and allowed to form a monolayer for 24 h. The cells were then exposed to 150 mL of DMEM medium containing 3, 5, 8 and 16% calf blood extract for 1, 4, 12 and 24 h. After the exposure, cells were washed twice with PBS and they were incubated in culture media for 24 h, and then MTT assay was performed. The balanced salt solution treated group was used as a control. At the end of the incubation period, the MTT solution was carefully aspirated, with care taken not to disturb the purple formazan crystal at the bottom of each well. The formazan reaction product was dissolved by adding 150 mL dimethyl sulfoxide (Sigma), and the optical density of each well was measured using an automatic plate reader (Molecular Devices, Sunnyvale, CA) with a 570-nm test wavelength and a 690-nm reference wavelength. In each experiment, eight wells were used for each concentration. This procedure was repeated in triplicate.17 Cellular metabolic activity was calculated with the mean of the absorption rates at each exposure time and concentration. Cellular metabolic activity was demonstrated by the following calculation: cellular metabolic activity (%) = absorption rate of each well/ absorption rate of control group 100. Data were analyzed using Wilcoxon signed rank test and were considered statistically significant at p50.05.
Lactate Dehydrogenase Assay for Cellular Toxicity The lactate dehydrogenase (LDH) assay measures leakage of the cytoplasmic enzyme LDH into the extracellular medium. The presence of LDH in the cell culture medium represents cell membrane damage. For this assay, 4.0 103 corneal epithelial cells/mL were seeded per well of 96-microtiter plates. Twentyfour hours after cell seeding, cells were exposed to each concentration of calf blood extracts. The LDH titer was assessed at 1, 4, 12 and 24 h after addition of the agent. The supernatant was collected from each well. LDH activity was measured in the supernatant using CytoTox 96, a nonradioactive cytotoxicity assay kit (Promega, Madison, WI), in accordance with the manufacturer’s instructions. Absorbance was determined at 490 nm with one 96-well plate enzymelinked immunosorbent assay reader. LDH activity,
68 Y. M. Park et al. which is proportional to color intensity, was expressed as optical density. The effect of the balanced salt solution-treated group was used as a control. To evaluate the significance of the differences of cell toxicity, the data were analyzed by ANOVA test and were considered statistically significant at p50.05.
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Cellular Morphology Analysis with Phase-Contrast Microscopy The cellular morphological changes were investigated using a phase-contrast microscope (Nikon, Tokyo, Japan). The cells (5 103 cell/mL/well) that were grown to confluence in 24-well plates were seeded at 500 mL/well in six-well plates, and then these cells were subsequently incubated in DMEM in 5% CO2 and 95% air at 37 C. After exposure to 3, 5, 8 and 16% calf blood extracts for 1, 4, 12 and 24 h, cells were incubated at 37 C for 24 h after being rinsed with D-PBS. The morphologic evaluation of the cytotoxic drug effects was performed using phase-contrast microscopy by a well-trained technician.
Scratch-Wound Assay for Cellular Migration A scratch-wound assay with human corneal epithelial cells was used to determine whether calf blood extract could promote wound closure. Human corneal epithelial cells (5 103 cell/mL/well), which were grown to confluence on 24-well plates, were then seeded at 500 mL/well on six-well plates, and then they were subsequently incubated in DMEM in 5% CO2 and 95% air at 37 C. The human corneal epithelial cells were wounded by scratching the surface of the culture with a 100 mL pipette tip. The scratched human corneal epithelial cells were washed with fresh medium to
remove the detached cells, and then the cells were incubated in the medium in the presence of 3, 5, 8 or 16% calf blood extract for 1, 12 and 24 h. The migration of cells was measured using an inverted phase-contrast light microscope. The balanced salt solution treated group was used as a control. To ensure that wounds of similar area were compared, multiple positioning marks were made at the center of the denuded surface with a small needle. Eighteen hours after wounding, the monolayers were fixed and stained, and the migrated cells’ edges were imaged.
RESULTS Effects of Calf Blood Extract on Cellular Metabolic Activity The MTT assay is used as a marker for cell viability or metabolic activity. In this study, DMEM culture medium was used as a control. After incubation with calf blood extract for 1, 4, 12 and 24 h, MTT values were significantly decreased in a time- and dose-dependent manner compared to control, especially with 8 and 16% concentrations of calf blood extract. For 8% concentrations of calf blood extract, MTT values showed significant decrease after 4 (p = 0.032), 12 (p = 0.012) and 24 h (p = 0.014) of exposure. In addition, for 16% concentrations of calf blood extract, MTT values showed significant decrease after 4 (p = 0.021), 12 (p = 0.010) and 24 h (p = 0.009) of exposure. For 3 and 5% concentrations of calf blood extract, MTT values were similar to those of controls (p40.05). However, at concentrations of 8 and 16%, cellular metabolic activity was significantly decreased after 4-h exposure to calf blood extract (Figure 1).
FIGURE 1 The absorption rate of the water-insoluble formazan dye in corneal epithelial cells exposed to calf blood extract, as determined by a scanning spectrometer (ELISA reader). The metabolic activity of corneal epithelial cells decreased at 8 and 16% concentration and longer exposure durations. Current Eye Research
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FIGURE 2 Lactate dehydrogenase (LDH) titers of cultured corneal epithelial cells exposed to calf blood extract. The LDH titers exhibit a dose- and time-dependent response relationship. LDH activity and cellular damage to corneal epithelial cells were most evident after exposure to 8 and 16% calf blood extract.
Effects of Calf Blood Extract on Cellular Toxicity Within 24 h, the LDH titer of cultured human corneal epithelial cells in calf blood extract exhibited a timeand dose-dependent relationship. At 8 and 16% concentrations of calf blood extract, LDH titers increased significantly up to 12 h of exposure (p = 0.012 and 0.009) and then decreased (after 24 h of exposure, p = 0.018 and 0.012). In addition, for 8 and 16% concentrations of calf blood extract, the LDH titer increased greater than 1.7-fold, when compared to the 3 and 5% concentrations of calf blood extract (50.8 at 4, 12 and 24 h) (Figure 2).
Effects of Calf Blood Extract on Cellular Morphology Using phase-contrast microscopy, control human corneal epithelial cells were observed to be densely distributed throughout the culture media. At the 3 and 5% concentrations of calf blood extract, cellular density was similar to control after 4, 12 and 24 h of exposure. However, as the concentration of calf blood extract and/or exposure time increased, more corneal epithelial cells swelled and detached from the dishes (Figure 3).
Effect of Calf Blood Extract on Cellular Migration Twenty-four hours after wounding, 3% concentration of calf blood extract promoted cell migration, with significantly increased wound closure as compared to higher concentrations of calf blood extract. At the 5% concentrations of calf blood extract, human !
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corneal epithelial cell migration decreased significantly after 24 h exposure. Furthermore, prominent cellular damage was found after 12 h exposure to a concentration of 8 and 16% calf blood extract (Figure 4).
DISCUSSION In 1984, Fox et al.18 first reported beneficial effects of autologous serum eye drops for the treatment of dry eye in Sjogren’s syndrome. Thereafter, autologous serum eye drops have been reported to be effective for the treatment of severe dry eye-related ocular surface disorders such as Sjogren’s syndrome, graft-versushost disease, Stevens–Johnson syndrome, superior limbic keratitis, exposure keratitis, persistent epithelial defects, recurrent corneal erosion and neurotrophic keratitis.3,4,7,18 However, there are some disadvantages of treatment with autologous serums drops. Repeated blood collection is required for ongoing treatment with fresh serum, and the preparation of serum eye drops requires a well-equipped laboratory and trained personnel. The use of autologous serum is contraindicated in patients with severe systemic disease, those with a fear of extracting blood, those who are infants or extremely elderly and those with chronic illness. There is also the risk of secondary infection due to drop contamination. So topical allogenic serum eye drops have been proposed as an alternative therapeutic option for treating severe ocular surface diseases requiring serum treatment. Nonetheless, the risk of transmitting blood-borne diseases still remained, with one report of Human Immunodeficiency Virus transmission by a single droplet.19 Fetal calf blood extract is used extensively in the laboratory to promote cell growth in culture. Some studies have reported favorable effects on corneal
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70 Y. M. Park et al.
FIGURE 3 Phase-contrast microscopic photographs of corneal epithelial cells after 1, 4, 12 and 24 h of exposure to (A) control, (B) 3% calf blood extract, (C) 5% calf blood extract, (D) 8% calf blood extract and (E) 16% calf blood extract. Corneal epithelial cell densities decreased after 4 h of exposure to 8 and 16% calf blood extract. Contrary to normal corneal epithelial cells showing hyper-reflective round edge with hypo-reflective center, damaged cells show hyper-reflective irregular morphology without hypo-reflective center.
FIGURE 4 Scratch assay of corneal epithelial cells immediately after exposure to (A) control, (B) 3% calf blood extract, (C) 5% calf blood extract, (D) 8% calf blood extract and (E) 16% calf blood extract after 1, 12 and 24 h of exposure. The effects of calf blood extract on wound closure were visualized after confluent human corneal epithelial cells were scratch wounded and incubated in the culture medium. At the 5% concentrations of calf blood extract, human corneal epithelial cell migration decreased significantly after 24 h exposure (C3). Furthermore, prominent cellular damage was found after 12-h exposure to a concentration of 8 and 16% calf blood extract (D2, D3, E2 and E3).
wound healing after the application of SolcoserylÕ , a protein-free hemodialysate derived from calf blood.11–14 Egger et al.12 showed that fetal calf blood extract accelerates the migration of corneal epithelial cells in vitro. Spessotto et al.11 showed that fetal calf blood extract increases the differentiation of human monocytes in vitro. But, on the contrary, preliminary studies showed decreased cellular metabolic activity and increased cellular toxicity after exposure to calf blood extract.15 Until now, the effect of calf blood extract concentration on human corneal epithelial cells has remained uncertain. This study was performed using lower concentrations of calf blood
extract to identify a formulation that is safe and effective for clinical use. In this study, the cellular metabolic activity of cultured human corneal epithelial cells in calf blood extract decreased in time- and dose-dependent manner, especially with 8 and 16% concentrations of calf blood extract. The cell death also showed a timeand dose-dependent response. Higher concentrations of drug caused far more damage to the corneal epithelial cells than did lower concentrations. In addition, there was no evidence of cellular migration after 12 h exposure to 5% or higher concentration of calf blood extract. Current Eye Research
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Calf Blood Extract on Corneal Epithelial Cells The TGF-b concentration in human serum is around 50 ng/mL, five times higher than that in tears. TGF-b is known to have antiproliferative effects, and high concentrations of this molecule may suppress wound healing of the ocular surface epithelium. This is one of the reasons for diluting serum to 20%.8,10 Furthermore, there is a report suggesting dilution of serum from 12.5 to 25%, which growth factors might reach their optimal range.20 Although, the level of TGF-b nor growth factors was not measured in this study, the higher concentration of TGF-b is expected in the higher concentration of calf blood extract. As no study has proven the pharmacokinetics and the cytokines of calf blood extract, further study might be necessary to support our hypothesis. In this study, the metabolic activity and the migration of corneal epithelial cell were inhibited more strongly at higher concentrations of calf blood extract. Although, clinically calf blood extracts are not used in the settings of this study, prolongation of exposure time with higher concentration of calf blood extract needs precaution. This study demonstrated that a solution of less than 8% (3 and 5%) concentration of calf blood extract caused less damage to corneal epithelial cells than did higher concentrations. Therefore, lower concentrations of calf blood extract should be safe for clinical use. In case of prescribing high concentration of calf blood extract, shortening the drug exposure time by prolonging the instillation term seems necessary. Although the results of the in vitro test may not always be applicable in vivo, it is believed that it will be a valuable guide for determining the optimal drug concentration for use in clinical situations. Further clinical studies concerned with the concentration and exposure duration to be used when treating patients with topical formulations of calf blood extract will be necessary.
DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This study was supported by Biomedical Research Institute Grant (2010-4), Pusan National University Hospital, and a grant from the Korean Health technology R&D Project, Ministry of Health and Welfare (A070001), Republic of Korea.
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