Impedance-Based Cell Culture Platform To Assess Light-Induced

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Impedance-based Cell Culture Platform to Assess Light-Induced Stress Changes with Antagonist Drugs using Retinal Cells Devasier Bennet, and Sanghyo Kim Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac303068t • Publication Date (Web): 19 Apr 2013 Downloaded from http://pubs.acs.org on May 3, 2013

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Analytical Chemistry

Impedance-based Cell Culture Platform to Assess Light-Induced Stress Changes with Antagonist Drugs using Retinal Cells Devasier Bennet and Sanghyo Kim* Department of Bionanotechnology, Gachon University, San 65, Bokjeong-Dong, Sujeong-Gu, Seongnam-Si, Gyeonggi- Do 461-701, Republic of Korea

ACS Paragon Plus Environment

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ABSTRACT: This paper describes an unprecedented, simple and real-time in vitro analytical tool to measure the luminous effect on the time responses function of retinal ganglion cells (RGC-5) by electric cell substrate impedance sensing (ECIS) system. The ECIS system was used for the continuous measurement of different color light-induced effects on the response of cells that exposed to protective drugs. The measurement suggests that the association of photooxidative stress was mediated by reactive oxygen species (ROS); which plays a critical role that leads to cell stress, damages and retinopathy, resulting in eye degenerative diseases. Continuous light-radiation caused time dependent decline of RGC-5 response and resulted in photo-damage within 10-hours due to adenosine 5′-triphosphate depletion and increased ROS level, which is similar to in vivo photo-damage. The ECIS results were correlated with standard cell viability assay. ECIS is very helpful to determine the protective effects of analyzed drugs such as βcarotene, quercetin, agmatine, and glutathione in RGC-5 cells, and the maximum drug activity of non-toxic safer drug concentrations was found to be 0.25, 0.25, 0.25 and 1.0 mM respectively. All drugs show protection against light radiation toxicity in a dose-dependent manner, the most effective drug was found to be glutathione. The proposed system identifies the photo-toxic effects in RGC-5 and provides high throughput drug screening for photo-oxidative stress during early stages of drug discovery. This study is convenient and potential enough for the direct measurements of the photo-protective effect in vitro and would be of broad interest in the field of therapeutics.


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INTRODUCTION The rate of oxygen consumption in eye cells decreases during the light adaptation process, and this condition favors the production of reactive oxygen species (ROS).1-3 Production of ROS is an initiator to oxidative stress. Oxidative stress is a general initiator of several neurodegenerative diseases, photo-damage and retinal disorders.4 The retina is mostly sensitive to oxidative stress due to a high intake of oxygen, high photo-sensitizes and pigments, and electromagnetic visiblelight radiation.2, 4 Numerous studies5-7 have demonstrated the damaging effects of light exposure to retinas. The first sign of degeneration has been observed by electron microscopy.8 The severity of eye cell damage is based on the duration of light exposure, wavelength, and type. In particular, the time point evaluation of 1000 and 4000 lux light exposed to RGC-5 cells had minimum cell damage in 24 hours and had a greater cell damage in 48 hours, which shows that increase light exposure cause decreased activity in cell viability assays.9 The various forms of light-exposure can induce different types of photo-damage, ocular retinopathies, and degenerative disease or disorders.10 However, the potential role of these disorders is neither well understood nor explored. Several studies have been conducted in order to obtain qualitative results of light-induced stress-related changes.11-13 Toxicity, cell viability, cell proliferation and apoptosis analysis methods are frequently used in high-throughput screening,9,10,14-19 though are not suitable for continuous or real-time analysis. Real-time quantitative assays on various phenotypic cells have been examined by the electric cell substrate impedance sensing (ECIS) system; that involved in the measurements of cellular behavior such as ultramotion, structural integrity, morphological changes, barrier integrity and toxicity studies.20-25 There is an essential need for determination of the toxic potential of light radiation. ECIS systems have the potentiality to study cellular responses of


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photo-toxicity, with applications in drug discovery and screening. Visible light electromagnetic radiations and their toxicities were examined in this study: white light shows 380 to 780 nm broad regions, which is comprised of blue, green and red light of the narrow region; 460 nm, 530 nm, and 625 nm, respectively. Light is known to induce stress damage and apoptosis in photoreceptor cells,12 though the specific risks of specific forms of electromagnetic radiation on cells needs to be analyzed more deeply. In order to better understand light-induced processes in cells, the ECIS system (impedance-based cell study) can predict which levels of illuminations can cause damage to the cells. In the present study, a simple and convenient tool; ECIS was used for continuous, realtime assessment of light-induced stresses of antagonistic drugs on photo-toxicity responses of rat retinal ganglion cells (RGC-5). RGC-5 is the only available robust retinal ganglion cell line with many resemblances to RGCs.26, 27 Therefore it is well suited as an in vitro photo-toxic testing model to establish real-time methodology for our study which could be beneficial to high throughput drug screening and development programs. As a test of this, with the aim of identifying compounds that might protect RGCs from light toxicity, we decided to focus on the following families of protective agents: ﬂavonoids,28 carotenoids,29 neuroprotective agents30 and antioxidants31 (quercetin, β -carotene, agmatine and glutathione respectively), and to determine their photo-protective effects on cells. The toxicity assays provide the 50% inhibition concentration in a real time manner. In parallel, cellular responses of RGC-5 cells were studied for photo-toxicity using MTT assay (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). An additional investigation was carried out using biochemical assays for adenosine 5′triphosphate (ATP), and ROS level before and after treatments. All experiments were preformed


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with triplicate; corresponding control experiments were carried out for all the studies and the cell behaviors were confirmed by fluorescence microscopy before and after treatments. EXPERIMENTAL SECTION Materials

and

reagents.

Agmatine, quercetin, β-carotene, L-glutathione and 2′,7′-

dichlorofluorescin diacetate were purchased from Sigma Aldrich, Co., (St. Louis, MO). Dulbecco's modified eagle medium standard (DMEM without phenol red), fetal bovine serum (FBS), trypsin-EDTA solution, and penicillin-streptomycin were purchased from Gibco Laboratories

(Grand

Island,

NY).

Alexa

fluor

488,

4,

6-diamidine-2-phenylindole

dihydrochloride (DAPI), rhodamine phalloidin (PHDR1), ATP bioluminescent kit and phosphate-buffered saline (PBS) were purchased from Invitrogen. Trypan blue dye was purchased from Alfa Aesar. The other chemicals used were of pharmaceutical grade. Milli-Q water (18.2 MΩ) was used throughout the experiment. Design of LED light setup on the microelectrode system. For the continuous real-time study of the photo-damaging effect on RGC cells, a novel and custom-made light-emitting diode (LED-RGB) strip light system was demonstrated in an ECIS system. The designed method was used for the monitoring of photoreceptor cell activities under controlled light exposure. The detailed design in the ECIS to assess light-induced behavioral changes has been described previously.24 In brief; the light was built just above the ECIS culture well (8 wells were focused with 8 round LED lights that was construct into a single strip, and make sure that all wells received the same lighting). The visible region of the electromagnetic spectrum (400–700 nm) was used in this study and was measured by a spectroradiometer (CS-1000, Minolta). The visible regions of the electromagnetic spectrum of each color light spectral characteristics are shown in


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Figure S-1. Also the typical experiments of detailed cell culture protocol and impedance measurement procedure are given in Supporting Information, Section S1. Real-time monitoring of the cellular response with white-light. Software, electrode arrays, and a lock-in amplifier were obtained from Applied Biophysics (Troy, NY, USA). Before starting the experiments, all equipments were sterilized with 70 % ethanol for 15 minutes, dried and irradiated with ultraviolet radiation for 15 minutes. The electrodes were rinsed with serumfree culture medium before initiating the experiment. Standard DMEM medium (250 µl) was then added to the ECIS 8W10E electrode arrays, then the arrays was plugged into a lock-in amplifier and incubated at 37°C for 20 minutes to record the background impedance value (Z0). Simultaneously, RGC-5 cells prepared with exact numbers (5 × 105cells), were added to each well, and the total volume of each electrode well was adjusted to 400 μL. The seeded cells were found to be adhered to the surface of the electrode after 8 to 10 hours incubation, and the LED white light was then switched on over the cultured well. Light exposures on a monolayer of each cultured well were maintained under normal culture environmental conditions (95% air-5% CO2 at 37°C). Control wells were kept under the same condition to normal cell growth, but were covered with a black cap to preventing light exposure. The cell activity changes resulting from the change of dark to light effect were reflected as impedance signals registered by the ECIS. The photo-toxicity experiment was performed for 10 hours continuously in the ECIS system. Impedance was measured every 1 minute, and data acquisition and processing was carried out with the software. The impedance-based cell study allowed for the measurement of light-induced responses in cell behavior, and receiving relevant physical information by evaluating the electrical impedance developed from cells, which are cultured under in vitro on a microelectrode system. The outcome data were normalized as Zx/Z0 to decrease the result of background


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impedance. The RGC-5 cells were inoculated in the micro-electrode chambers of the system for the photo-toxicity study, and the impedance was calculated from the average signal of the entire cells. In parallel, MTT assays were performed to determine the time-dependent toxicity of light radiation, and provide a comparison of the impedance data with a standard MTT measurement for correlation analysis. The light exposed RGC-5 cell viability was determined after various time points. In detail, 5 × 104 cells mL-1 concentrations of 100 µL RGC-5 cell suspensions ware seeded in each well of a 96-well culture plate and were incubated under same environment as previously described. After 10-hours of incubation the attached cells were exposed to light for up to 10-hours. After the initiation of light exposure, cell viability was quantified every hour using MTT reagent. Simultaneously cell viability was assessed using Live/Dead Staining dye kit (Biovision), 1 μL each of dye mixtures A (Live-Dye, a cell-permeable green fluorescent dye and B (propidium iodide (PI), a cell nonpermeable red fluorescent dye were diluted in 1000 μL of dilution buffer (followed the manufacturer’s protocol). On every two hours of light exposed culture, medium was removed and the previously prepared staining solution was added to the wells and incubated for 10 minutes at 37 °C. The stained sample wells were examined under fluorescence microscope. Similar times and conditions were set for control experiments. All experiments were carried out in triplicate. In addition, the independent confirmation of RGC-5 cell microtubule disassembly responses before and after light exposure was analyzed by fluorescence microscopy for comparison purposes. The levels of ATP and ROS production32 were simultaneously measured in the cells, and these data were compared with those of control cultures in detailed experimental protocol as provided in the Supporting Information, Section S2.


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Cytotoxicity screening for antagonist drugs. ECIS can be used to measure the changes occurring during the real-time recording of cell adhesion and spreading, as previously described by Keese and Giaever.33 Cell attachment and spreading over the surface of the electrodes impedes current flow and impedance in hence increased in a quantifiable manner. The loss of cell activity causes cells to detach from the electrode surface, restoring impedance.34–36 In this experiment, RGC-5 cells were used for cytotoxicity screening. Appropriate amounts of cells were seeded on the 8 well ECIS chip and they were incubated, and the impedance values were then measured. This system allows for the measurement of changes in cell behavior and measures the average electrical impedance signal developed by cells, which are cultured in vitro on a microelectrode system. After 10 to 15 hours incubation, a cell monolayer was obtained on the ECIS electrodes, the cell monolayers were exposed to five different concentrations of each antagonist drug by replacing the initial cell culture medium. The various concentrations of drugs (0.5 – 5.0 mM) were prepared using the standard fresh medium without phenol red, and solutions were filtered through a 0.22 µm membrane prior to use. Impedance was measured in all wells every second up to 10 hours, with identical times and conditions being applied to the control groups. Cell responses from each electrode were studied for different concentration effects and the effect of the different concentrations of drugs over time on the RGC-5 cells was determined. When cells are exposed to high concentrations of some drugs, the impedance values decrease due to the total cell mortality. The concentration of each compound required to achieve 50% inhibition was determined based on observations of the ECIS signal (ECIS 50). The inhibition concentration (IC) for treatment with various drugs at different concentrations after 10 hours was calculated using the equation (1). The optimum non-toxic concentrations for biological use were examined to show that maximum cell activity concentration is safe and nontoxic for the cells.


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(1) Zdrugs refers to cellular impedance at 10 hours after drugs treatment, while ZC refers to the cellular impedance of the control. Real-time measurements of drugs antagonistic effects on white-light toxicity. The suitable concentration of protective drugs of light radiation, glutathione, β-carotene, agmatine and quercetin were dissolved separately in standard DMEM medium. Exact numbers of RGC-5 cells were added to each well, and the total volume of each electrode well was adjusted to 400 μL using culture medium. After a RGC-5 cell monolayer was obtained, the cultured well was exposed to light under normal culture environmental conditions. The fresh medium containing drugs were then added, replacing the initial cell culture medium. The cell activity changes resulting from movement from a dark environment to light exposed stress were reflected as impedance signals registered by the ECIS. Control wells were also maintained; positive control well impedance was maintained in culture medium without any treatment, and continuous whitelight exposure throughout the experiment served as a negative control. The levels of ATP and ROS production were simultaneously measured in the RGC-5 cells, before and after applications of white-light toxicity in the ECIS system, and these data were compared with those of control cultures. In order to determine the antagonistic effect of white light-induced toxicity, ATP and ROS were determined before and after the addition of drugs to the cell suspension of each drug, the used concentration was most effective at preventing cell damage. The effect of antioxidant or free radical compounds on DCFH-DA can be measured against the fluorescence of the provided DCF standard. Correspondingly control experiments were conducted. All experiments were carried out in triplicate.


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Antagonistic effects on different color light toxicity. The present study we compared different color light effect and the protective compound of RGC-5 cells. The antagonistic effects of different wavelengths of light radiation (red, green, and blue) exposures under a normal culture environment were studied. This system included different colors of lights to assess their cellular effects. Cellular impedance effects of three different antioxidants (β-carotene, quercetin and glutathione) were used for each color light radiation-induced RGC-5 cell damages. Pre-treated and un-treated cells were used on the same chip; three well cells were pre-treated with suitable concentrations of glutathione, β-carotene, and quercetin before light exposure and three well untreated cells were used to compare cell behavior. In the eight wells of ECIS array, three wells were seeded with pre-treated RGC-5 cells, three wells with normal RGC-5 cells (un-treated), and two wells with control experiments. In control experiments, one well acts as a negative control and the other acts as a positive control, and the ECIS data was compared to a control culture and are presented in this paper. Cells were exposed to different color light radiation (cell stressed, approximately 150 – 180 minutes), then treated using different drugs, and the procedure was carried out thereafter as detailed above. Protection of RGC-5 cells from different color light induced toxicity by different drugs was determined and the impedance data of pre-treated and un-treated cells effects were compared. RESULTS AND DISCUSSION System with light setup for real-time model. Our previous work24 proved that the impedancebased cell study is well suited for light-induced cell morphological study, and it is an unprecedented model to establish real-time methodology to assess light-induced stress changes with antagonist drugs in RGC-5 cell models. Figure 1 shows a schematic diagram of the ECIS light setup device, which is an in-house made light - emitting diode (LED) light on/off setup, and


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was established in an ECIS system to conduct a light-induced cell behavior study with in vitro drug screening model for optometric intervention. In addition, the input sensors included a thermocouple which was used to monitor the temperature. Light applications were ventilated with a fan to avoid heat build-up throughout the experiments. An LED strip light was inverted above the ECIS cultured chip to attain uniform exposure, and each LED light was designed to focus on each well of an eight well chip. Responses of RGC-5 to light radiation. In order to characterize the effects of white-light radiation on the RGC-5 cells, we used this system setup to monitor light-induced damage in RGC-5 cells by measuring cellular impendence every minute over an experiment period. We used LED with white, red, green, and blue light, which was applied to RGC-5 cells, and monitored the severity of light-induced toxicity in cells. In the initial experiment, attached RGC5 cells were exposed to white-light radiation continuously for 10 hours. The normalized impedance curve is shown in Figure 2. As the RGC-5 cells attached to the detecting electrode, the resistance of the well increased very rapidly and then reached a steady-state within 10 to 15 hours from the initial cell seed. After reach their steady-state (stationary phase) the experiments was started and ended before decline phase. All the experiment was done within the stationary phase. After 16 hours later, the resistance of the control well impedance started to change in to accelerated death phase; we therefore limited our total cell culture time to 32 hours. The cell-free data could not make any disturbance in impedance value, and the cell covered electrode with control experiment having no light exposure shows no changes in impedance value; which means there are no influences on cell growth. Test wells were exposed to white-light radiation continuously for 10-hours are presented in Figure 2a. It shows a substantial decline in impedance value from initial light exposure. Simultaneously, these measurements were confirmed by


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fluorescent microscopic imaging (Figure S-2), and time-dependent effects on cell morphology with different time intervals as shown in the Supporting Information, Section S3. In addition, the test cell covered electrode, during light exposure showed a substantial decline in impedance value from initial light exposure. In the continuous light exposure, rapid impedance reductions occur; which means that the rapid cell growth impedes the over exposure of white-light radiation. Cellular electrical impedance sensing shows growth reduction in cellular impedance, and subsequently a significant decrease in cellular impedance with increased exposure time. Increased duration of light exposure had a greater effect on cell damage. The physical developments of RGC-5 cells were impaired, and their strength was hindered. The test well peak shows a subsequent decline in impedance compared to the control well peak. In brief, electrical cell impedance increased with time initially due to the cell-substrate, attachment, spreading, and mitosis of cells on the electrode. After the development of a monolayer the cells were exposed to light radiation and the impedance showed a small reduction initially, which indicated a reduction in cell function and strength. Followed by prolonged exposure of light, the cell function decreased, indicating the reducing cell activity; absence or undergoing cell apoptosis or cell viability. Comparison of the time course of effects on normalized resistance drops were elicited from untreated (control) and light-treated RGC-5 cells. Figure 2c showed the light-induced toxicity of time-dependent impedance drops in RGC-5 cells. The impedance curve shows a great reduction after prolonged exposure times, suggesting that the onset and severity of the photo-toxic effects increased with increasing light exposure. The initial ECIS data shows minor reduction in initial cellular impedance, and increased light exposure shows the greater effect on cell damage; which was impaired, and their strength was hindered. The initial reduction in cellular function might be related to the increased levels of some stress factors. As a result of


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light irradiation, retinal photoreceptors trigger apoptotic cell death.37 The light is a risk factor in glaucoma and the mitochondrial optic neuropathies.38 The light-toxicity evolves by chemical reaction in the forms of ionizing radiation39, it enter into the cells then it lead to severe damage by induces some noxious factors, which in turn trigger the mitochondria-mediated apoptotic activity, and apoptotic death. The other causes are elevation of mitochondrial oxidative damage and massive levels of DNA damage, leading to increased mitochondrial ROS production and decreased mitochondrial ATP synthesis.39,40 The presented therapeutic agents can protect the cells by prevent the initial elevation of noxious factors. In contrast to the additional time point assessments, Figure 2d shows cellular MTT assay to find the time-dependent and radiation-dependent toxicity of light. Survival was confirmed by the MTT assay. The viability of RGC-5 cells exposed to continuous white-light radiation was determined after various durations of exposure. Figure 2d shows the MTT values of RGC-5 cells exposed to continuous white-light radiation for up to 10 hours, which shows that the continuous light radiation cause decrease in cell viability. It is clearly shown that the quantified MTT values did not decline immediately after initial light exposure, but after four hours the MTT values were decreased and continued to 10-hours light exposure. Comparison of the impedance results with a standard MTT measurement is shown in Figure 2e, and the MTT results were correlated with the cellular impedance drop as shown in Figure 2c. During dark exposure did not affect the impedance, but after light exposure the increased impedance has started to decline. It was indicating that the initial light exposure also change the impedance value, due to elevation of stress on the cells started to shrink, and the actin filaments started to detach from the electrode surface because of the loss of cell strength. However, MTT values did not decline immediately after initial light exposures, but after two-hours light exposures the cell activities were decreased.


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Live/dead staining test helps to optically visualizing the viability of RGC-5 cells (Figure 2f). Green and red fluorescence corresponding to live and dead cells respectively, the cell viability decreased with increased time of light exposure. In comparison, cellular electrical impedance sensing shows initial minor reduction in cellular impedance and subsequently significant decreases in cellular impedance on increased light exposure. Increased time of light exposure levels had a greater effect on cell damage. The retarded RGC-5 cells were impaired, and their strength was hindered followed by cell damage. Consistent with decrease in cell viability, there was an increase in the percentage of cell death as detected with Live/dead staining. Qualitatively determined results coincided well with quantitative cell viability assay results. The initial response was related to the light-induce stress activity. Impedance-based cell study can therefore immediately monitor some of the onset cell changes as they begin to occur. Correlation analysis between the impedance data and MTT assays. Correlation analysis between MTT assays and cellular impedance are depicted in Figure 2d, e. The comparison of time-dependent photo-toxicity and impedance values was normalized at zero by establishing the control value (without light exposure). Correlations suggested a close agreement between the MTT value and ECIS data. This time-dependent photo-toxicity comparison, the correlation efficiency (linearity, R = 0.988) shows that close agreement has achieved between the MTT value and the impedance data. A decline in impedance proves that ECIS systems can evaluate light-induced damage to the cell membrane at early stages of the photo-toxic process. The realtime measurement can give a better outcome for toxicity studies with minimum numbers of cells because the end point measurements could miss the onset actions and intermittent measurements. Light-induced RGC-5 cellular activities


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Biochemical analysis. In the study, the cellular pathology of oxidative stresses and cell damage are a common initiator of many different neurodegenerative diseases, including retinal disorders.41 Cellular function is particularly susceptible to oxidative damage, leading to decreased ATP synthesis and increased ROS production, which in turn leads to cell damage followed by necrosis or apoptosis.42 The ATP and ROS levels were measured during the light exposure; in order to determine whether the RGC-5 cells responded similarly. Here, the RGC-5 cells responded in the same manner; these same changes are seen in RGC-5 cells exposed to light radiation in vitro. As shown in Figure S-3a (see in the Supporting Information), the initial exposure time of the ROS and ATP levels does not show a big difference, but after increasing the duration of exposure the ROS and ATP levels were changed. ATP levels fell by nearly 25 % compared to the control value, within five hours of light exposure to the cells, and levels continued to decline up to the end of experiment. The ROS production responded dissimilarly; the ROS levels increased to nearly twice those of the control values within the first 4 hours of light radiation exposure to the cells, and continued to increases thereafter. We further confirmed with fluorescence image (Figure S-3b (see in the Supporting Information)), the cells showed that increased light radiation triggers releasing ROS as detected by an increase in fluorescence and ATP cellular activity level shows decrease in fluorescence vice versa, this optically visualizing the fluorescence image is coincided with the quantitative assay. ECIS profiling for the cytotoxicity of drugs. Several drugs were used as protective compounds against light-induced photoreceptor death in vivo.43,44 In the present study, we used β-carotene, quercetin, agmatine,45 and glutathione to investigate their effects on the light-induced RGC-5 cell damage in an in vitro model, see in the See supporting information, Section S4. The RGC-5 cell monolayers were exposed to different concentrations of drug and the responses ware


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monitored. The continuous cellular response was monitored in real time using ECIS, which provides a continuous readout of the electrical resistance of RGC-5 monolayers. The normalized impedance profile of RGC-5 cell monolayer exposed to different concentrations of drugs is shown in Figure S-4. The high concentrations of β-carotene, quercetin, agmatine, and glutathione (3, 3, 1.25 and 5 mM respectively) showed a decline in impedance (Figure S-4- left side See Supporting Information), where the impedance value of the cellular response f (C, t) was significantly lower than its control well immediately after drug administration, which is thought to be as a result of membrane damage. In parallel, the cellular response functions f (ZC, t) of all well data were measured. Using above equation, the inhibition concentrations (ECIS 50) of drugs were evaluated by ECIS data from the inhibition curve. Figure S-4-right side (Supporting Information) showed the inhibition curves of β-carotene, quercetin, agmatine, and glutathione for 12 hours. Their ECIS50 values were then determined to be 2.0, 2.0, 1.0 and 3.5 mM, respectively. Concentrations above these were in effect a lethal dose of β-carotene, quercetin, agmatine, and glutathione. Figure S-4 in the Supporting Information clearly shows that β-carotene and agmatine developed toxicity at concentrations ＞0.50 mM, quercetin developed toxicity at ＞1 mM concentration, and glutathione developed toxicity at ＞2 mM concentration. Less than the above respective concentration shows maximum drug activity, which provides the establishment of non-toxic safer drug concentrations. The effective safe doses less than toxic concentrations of drugs are well tolerated and were used for further study. Evaluation of toxicity protective agents by the ECIS system. We tested the ability of the general protectors of light-induced stress and stress-induced cell death to prevent toxicity. The drug concentrations selection and administration in to the electrode as provided in details in the


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Supporting Information, Section S5. Positive control wells showed no changes in impedance growth, which indicating that there was no influence upon the cell growth (Figure 3a). Negative control wells show a continuous impedance drop, which indicating that cells were loosened their strength, and followed by loss of cell activity. Additionally, the test cell covered electrodes shows significant changes during the light exposure and following antagonistic effects. During light exposure the entire well showed a decline in impedance except the control one, after four hours of light exposure four different drugs were added in four wells. Figure 3a shows that the cells were stressed at initial light exposure and that impedance were decreased. Cells were then treated with drugs; all the drugs provided significant levels of protection from cell damage, which is clearly shown by the impedance recovery. The maximum cell protection was shown after glutathione treatment, which recovered the impedance and maintained maximum cell activity. None of the test cells endured any secondary damage after the administration of drugs. Figure 3b shows the decrement in impedance levels during light exposure; RGC-5 cells were treated with white light for up to 4 hours, and were then switched to drugs containing media for cell recovery. The resistive portion of impedance was normalized to its initial value, the value of relative changes in cellular resistance were presented. The average value of light exposure induces a persistent reduction in resistance with a peak decline initiating within few minutes of exposure. Continuous light exposure showed the maximum reduction in cell function. The % of reduction in cellular resistance induced by light radiation was significantly greater than control. Similarly, the % of recovery in cellular resistance treated by drugs was comparatively greater than control; Figure 3b shows that all of these compounds were protected from the light-induced toxicity. The most effective drug compound for protection was determined to be glutathione, followed by quercetin and β-carotene. The least protection was shown by agmatine, as compared


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to other drugs. Simultaneously we confirmed the light-induced fibrillary reductions and recovery (using different drugs) on single RGC-5 cells by fluorescence microscopy (Figure 3c). The reduction in cellular function may be associated to the reduced degrees of actin fibers, which increase stress. Most notably, this light radiation was found to cleave actin, hindered the functional and structural integrity. After treatments cells were extended their periphery (cell border, microtubules) to renew cell contact (which increased in impedance value, restore cell activity), they initiate cell-cell contacts and restore their own structure. We further confirmed the above effects on cell morphology by fluorescence microscopy images, and showed identical results in detailed explanation in the Supporting Information, Section S6 (Figure S-5; See Supporting Information), which coincided with cellular physical signals. Furthermore, we checked the capability of drugs to reduce the loss of ATP level and increase the ROS level; these alterations are possible contributing factors to the photo-toxicity of wide range white-light radiation. Figure S-6 in the Supporting Information shows the level of ROS and ATP present in RGC-5 cells before and after treatment (see the Supporting Information, Section S7). White light radiation-induced RGC-5 cells also lead to a deprivation of ATP, and this reduction can be prevented by glutathione and agmatine treatment. Quercetin and β-carotene showed a minimum effect ATP levels but significantly reduced ROS levels compared to the control. Table S1 shows the intracellular ROS and ATP levels after each drug treatment to the experiments. Glutathione gives the most significant results followed by agmatine, quercetin and β-carotene. However, all the drugs were comparatively efficient at treating oxidative stress. Protective effect of drugs against different light–induced cell damage. The effect of narrow wavelength lights on cellular responses with three different antioxidant drugs (β-carotene, quercetin and glutathione) were studied, and the normalized variance ratio was used to quantify


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the changes (Figure 4). After cells became attached, they were stressed with different color lights followed by treatment with different antioxidant drugs, and the cellular responses were monitored in real time. Simultaneously, we compared the effect of pre-treated cells and untreated cells. In comparison, the pre-treated cell shows more lively activity and rapid mobility during and after light exposure in all the experiments. Figure 4 shows that impedance declines rapidly after red and blue-light exposure and that green light shows the minimum impedance drop. Blue color light resulted in large reductions in impedance; almost double that of red color light, but green color light showed the least effect of all. The different narrow wavelength light (red, green, and blue) effect on the cell were studied and quantified and shown in Figure S-7. The red and blue lights were nearly identical but green light showed comparatively less effects, and pre-treated cell shows less effect compared to un-treated cells. It might be the presence of chromophore content in the mitochiondria, absorbing energy, and photodynamic production of free radicals in cells (details see in the Supporting Information, Section S8).46-48 In general the shorter wavelengths of light produce harm to cells because of high energy, 49 the blue light with the lowest wavelength also reaching the retina,50 has been shown to induce retinal damage, particularly to the photoreceptors,51-53 by the production of reactive oxygen intermediates. It depends on the biologic chromophore present in the cells and elevated oxygen content present in the environment as well as the color and intensity of the light exposure. There was no cell death observed for different color light exposure for 180 minutes (data not shown here), however, cellular resistance was decreased due to the lack of cell function, lack of cell strength and increased cell stress. The idea that initial light exposure can cause stress to the cells, because the stimulation of ROS production and play a crucial role in the activation of intracellular death pathways.54 Now it is believed that stimulation of ROS are central to the death


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pathways. The presented therapeutic agents prevent elevation of noxious factors. All the antioxidant drugs prevented secondary damage by scavenging free radicals produced from light radiation. In particular, Figure 4a showed blue color light-induced response with the antagonistic effect of drugs on cells. Complete protection against blue color light-induced damage was observed in pre-treated cells with 0.50 mM glutathione, 0.25 mM β-carotene and 0.25 mM quercetin, which showed that the cellular impedances were completely recovered to normal cell growth by comparing with the control cell. In the case of un-treated cells, they showed a rapid decrease in impedance with no subsequent recovery, but the cells are re-strengthened on the surface attachment. Figure 4b shows the green color light-induced response with the antagonistic effect of drugs on cells. Figure 4b pre-treated cells showed identical results of Figure 4a; which showed that cellular impedance was completely recovered. Pre-treated cells showed some fluctuations in impedance after exposure to green color light, and minimum cellular photodamage. Figure 4c showed red color light-induced response with the antagonistic effect of drugs on cells which gives identical results of Figure 4a. Complete protection against red color lightinduced damage was observed in pre-treated cell culture wells. In parallel, cell morphological changes were studied for different color light exposure and drug treated responses using fluorescence microscopy (Figure S-8; See Supporting Information), which shows all color light exposure causes fibrillary reduction in RGC-5 cells. The images were agreed with the physical information of ECIS impedance.

As shown in Table S-2 in the Supporting Information,

glutathione and quercetin provide a high level of protection from photo-damage in pre-treated cells. Under conditions of green-light exposure there is less of a reduction in impedance and high impedance recovery compared to red or blue light. This indicates that glutathione and quercetin were well able to scavenge cellular free radicals to prevent cell damage, compared to β-carotene.


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Our results suggested that glutathione with agmatine treatment protected RGC-5 from lightinduced cellular damage, light-induced ATP loss, and light-induced oxidative stress. This is very reliable approach to discover the onset of early stage cell response towards high throughput drug screening and developments. This unprecedented setup was established using a bioimpedance system to study the photo damaging effect in a real-time manner. Other existing methods to monitor toxicant induced stress and cells undergoing changes were established by radioactive labeling, which is assessed by microscopy and biochemical assays. These activities of toxicant-induced damage related to enzymes and DNA fragmentations can be detected by electrophoresis or enzyme-linked immunoassay techniques.15 The major goal of this study was to attain and develop a new bioanalytical method capable of continuous real-time monitoring without any reagent addition. The present model was used to reveal an unconventional method for monitoring photo-damaging effects in retinal cells by assessing light responses in vitro. This method revealed one of the most sensitive morphological onset changes in cell-cell and cell-substrate behavior, which was well suited for continuous monitoring of lightinduced cell behavioral responses. In the present study, we established that an impedance-based system can be used effectively for in vitro photo-toxicity testing, and evaluation of toxicity protective agents in an in vitro RGC-5 cell model test. The impedance-based cell study with light setup is allowed for direct, continuous real-time evaluation of cellular status; it has been proven to be a useful tool for the study of photo-toxicity and light-induced stress study as well as for the evaluation of photo-toxic effects of some chemical agents; which may retard or cure diseases. However, further study is necessary to investigate the expression and reaction of cellular pigments and drug-bind protein in the RGC-5 cell line, as well as to achieve efficacy for the treatment of light-induced retinopathy by providing suitable optometric dosage formulation.


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CONCLUSION In order to assess the light-induced changes with antagonist drugs, we have developed an ECIS system for in vitro cellular assay of retinal cells model. A light setup for artificial photodamaging and drug design and drug screening for retard or cure cell damage, which will provide a platform for optometry interventions. We found that the ECIS results are reproducible with the other standards, by comparison of impedance signals with MTT values. These measurements proposed that ECIS could be used to independently evaluate the photo-toxicity study in vitro. In addition, ECIS data of drugs protection from different color light-induced damage exposed different RGC-5 cell behaviors, providing insight into the various concentration dependence activities of these drugs. Moreover, different color light shows variable impedance drops for red and blue color light whereas a minimum impedance drop occurred under green light exposure. All drugs showed some protection against light radiation toxicity. In specific, pre-treated cells showed better efficacy compared to non-treated cells. Our results provided that specific agmatine and quercetin could be more beneficial for the treatment of retinal photo-damage. This system is capable of high-throughput screening uses, and explores the hypotheses of photo-toxicity induced disease and its pathophysiological mechanisms by monitoring the cellular behavior without specific labeling. The in vitro cell-based ECIS system allows for the study of lightinduced behavior, and thus provides reproducible and statistically significant information, which is collected in real-time. So, this impedance-based cell study can find more use in applications such as light-induced retinopathy and photo-damage testing.


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ASSOCIATED CONTENT Supporting Information. Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author. E-mail: [email protected]. Phone: +82-31-750-8554. Fax: +8231-750-8819; ACKNOWLEDGMENT This research was supported by the Gachon University Research Fund in 2012 (GCU 2012-R177)


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FIGURES

Figure 1. Schematic drawing of the cell monitoring system under controlled light environment in an in vitro cell model. The experimental setup includes electric cell–substrate impedance sensing under custom-design and fabricated light setup with ventilated device. Schematic shows that custom-made system for real-time monitoring of cell behavior in vitro cells in a microelectrode environment under lights which are controlled from outside the incubator.


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Figure 2. The effects of light-induced RGC-5 cell behavior under the ECIS system with continuous light exposure responses. (a and b), The control showed no decrease in growth even after 24 hours, but test cells showed a continuous growth decline. (c), Time dependant impedance drop of light-exposed cells. (d), RGC-5 cells time dependence of photo-toxicity. The cell viability was determined after various exposure time points for the continuous white-light radiation effect using MTT. (e), Correlation analysis between MTT assays and cellular impedance. The correlation data shows linearity, indicates a close agreement between the MTT value and the impedance data. (f), Fluorescence micrographs of live/dead staining of RGC-5 cells grown on electrodes, the images of cells on circular detecting electrodes of ECIS wells, in which the images were taken after various exposure time points. Dead cells were stained in red and are denoted with white arrows. All sets of experiments were carried out in triplicate, error bars show the SD (n = 3 for all cases). Scale bars, 50 μm.


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Figure 3. Effects of protective agents on light-induced RGC-5 cell damage under ECIS system. (a), ECIS data of photo-protective effect with different magnification. The continuous light exposures causes decrease cellular responses, and then followed by different drugs treatment the cellular impedance signals were recovered. (b), Effects of light-induced cellular resistance (decline) and drugs treated cellular resistance (recovery). Light exposure induces a persistent reduction in resistance indicating a reduction in cellular function and the following administration of various drugs the cellular resistance has retrieved. (c) Fluorescence micrographs of light-induced stress fibrillary reduction on single RGC-5 cells and recovery using different drugs. The control cells showed clear fibrillary network and the light exposed cells shows hindered functional and structural integrity, after drug treatments restore the cell activity.


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Actin filament detachments were denoted with white arrows. All sets of experiments were carried out in triplicate, error bars show the SD (n = 3 for all cases). Scale bars, 10 μm.


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Figure 4. ECIS real-time impedance data of cellular responses under different color light (red, green and blue) exposures followed by treatment with different drugs. (a) The effects of antioxidants on blue light-induced toxicity of retinal cells. (b) The effects of antioxidants on green light-induced toxicity of retinal cells. (c) The effects of antioxidants on red light-induced toxicity of retinal cells. All sets of experiments were carried out in triplicate. Corresponding morphological changes were noticed in different color light exposure and drug treated responses using fluorescence microscopy (Figure S-6; See Supporting Information).


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For TOC only


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Impedance-Based Cell Culture Platform To Assess Light-Induced

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