Quantifying Cell Confluency by Plasmonic Nanodot Arrays to Achieve

Article Cite This: ACS Sens. 2019, 4, 1816−1824

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Quantifying Cell Confluency by Plasmonic Nanodot Arrays to Achieve Cultivating Consistency Wen-Huei Chang,*,† Zi-Yi Yang,‡ Tak-Wang Chong,‡ Ya-Yu Liu,† Hung-Wei Pan,§ and Chun-Hung Lin*,‡ †

Department of Applied Chemistry, National Pingtung University, Pingtung 90003, Taiwan Department of Photonics, National Cheng Kung University, Tainan 70101, Taiwan § Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung 81362, Taiwan Downloaded via UNIV OF SOUTHERN INDIANA on July 28, 2019 at 09:20:14 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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S Supporting Information *

ABSTRACT: The determination of cell confluency and subculture timing for cell culture consistency is crucial in the field of cell-based research, but there is no universal standard concerning optimal confluence. In this study, gold nanodot arrays on glass substrates were used as culture substrates, and their spectral shifts of localized surface plasmon resonance (LSPR) were employed to monitor cell growth and quantify cell confluency. Experiments including cell counting, metabolic activity, focal adhesion, and cell cycle were also performed to confirm the cell growth monitoring accuracy of the LSPR signals. The LSPR signal exhibited the same trends like the increase of cell numbers and cell metabolic activity and reached the maximum as the cell growth achieved confluency, suggesting its great capability as an effective indicator to predict suitable subculture timing. The proposed sensing approach is a noninterventional, nondestructive, real-time, and useful tool to help biologists quantify the optimal subculture timing, achieve cell culture consistency, and obtain reproducible experimental results efficiently. KEYWORDS: surface plasmon, biosensor, cell culture, cell confluency, subculture timing ell culture has been widely applied to the field of biomedicine to produce various biotechnological products, including biomaterials, pharmaceutical proteins, growth factors, vaccines, and antibodies. Depending on their origin, animal cells grow either as an adherent monolayer or in a suspension. Most cells derived from solid tissues are adherent. Adherent cells are anchorage dependent and propagate as a monolayer attached to a cell culture vessel. This attachment is essential for proliferation, and a sufficient substrate surface is needed for cell growth. Confluency is a commonly used term in cell biology, referring to the proportion of the surface which is covered by adherent cells. When cell growth reaches about 80% confluency, cells must be subcultured or passaged. Failure to subculture nearly confluent cells leads to the reduced mitotic index and eventually cell death. By contrast, passaging cells too early prolongs the lag time. The gene expression and morphological characteristics of cultured cells can vary because of cell confluency.1−4 Some cell lines, such as normal lung epithelium, mammary gland epithelium, mammary gland ductal carcinoma, umbilical vein endothelium, and ovary fibroblasts, exhibit different morphological characteristics at low and high confluency.5,6 In terms of gene expression, endothelial cells secrete phosphotyrosine at low confluency, but this phenomenon does not appear at high confluency.4 To

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achieve optimal and consistent results, experiments are usually performed using cells at a particular confluence. However, ongoing cell culture experiments are dependent on human interventions, which are laborious and highly subjective, leading to large variations and inconsistent results, especially in the visual assessments of cell confluency to determine the appropriate time to subculture cells. Determining cell confluency accurately and finding the best subculture time remains challenging. Advances in image software have now introduced accuracy and standardization to confluency measurements for adherent cells.7,8 Cell segmentation is one of the analysis methods, which requires the customization of analysis parameters for each cell type. Each cell is then identified by the software, but the method is time-consuming and has complicated operation.9 Another method is to measure the confluence of cell colonies by the software, which is universal and applicable to a majority of cell types without customization; however, it cannot determine confluency when cells overlap.10−12 Received: March 15, 2019 Accepted: June 18, 2019 Published: June 18, 2019 1816

DOI: 10.1021/acssensors.9b00524 ACS Sens. 2019, 4, 1816−1824

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MO, USA). DMSO was purchased from Merck (Darmstadt, Germany). Actin cytoskeleton and focal adhesion staining kits were bought from Millipore (Billerica, MA, USA). Acetone and isopropanol were procured from JT Baker (Phillipsburg, NJ, USA). ARPE-19 was obtained from the Bioresource Collection and Research Center (Hsinchu, Taiwan). Cell Culture. ARPE-19 cells were cultured in DMEM and F-12, 10% FBS, and 100 units/mL penicillin−streptomycin. They were maintained at 37 °C in a 5% CO2 atmosphere in a humidified incubator. The culture medium was replaced every 2 days. For subculturing, the culture medium was removed and discarded. In brief, the cells were rinsed with PBS solution to remove all traces of serum containing trypsin inhibitor. Approximately 1 mL of trypsinEDTA was added to the solution in the culture dish, and the cells were observed under an inverted microscope until the cell layer was dispersed. Then, 5 mL of the complete growth medium was added, and the cells were aspirated by gently pipetting. The cell suspension was transferred to a centrifuge tube and spun at 1000 rpm for 3 min to remove the trypsin−EDTA solution. After centrifugation was completed, the supernatant was discarded, and the cells were resuspended in a fresh growth medium. The cell numbers were determined using a hemocytometer. Appropriate aliquots of the cell suspension were added to new culture vessels. The number of the cultured cells was counted from the optical images captured via an optical microscope to quantify cell growth by cell densities. Preparation of Silicon Master Molds and PFPE Working Molds. A flexible PFPE polymer mold, which exhibits advantages of flexibility and low surface energy, was used in our nanoimprint process to avoid the risk of damaging the expensive silicon mold during nanoimprinting. With flexibility, the mold could provide an ideal conformal contact with the imprinted substrate without the need for high pressure. With an extremely low surface energy (12 mN/ m),39 PFPE could be easily detached from the imprinted polymers during demolding. PFPE has a high elastic modulus (∼40.5 MPa at room temperature),40 so it is stiff enough to imprint nano features. PFPE working molds can be replicated easily from a silicon mold at a low cost. Silicon master molds and PFPE working molds were prepared in accordance with our previous work.40 Fabrication of GNDAs. The fabrication procedure of GNDAs is illustrated in Figure 1a. A bilayer resist scheme with a UV-curable resist as a top layer and PMMA as a bottom layer was used to create an undercut resist profile and to facilitate the metal lift-off process.

Localized surface plasmon resonance (LSPR) is the collective oscillations of conduction electrons at the interface of metallic nanostructures and environmental dielectric, resulting in remarkable localized field enhancement, which strengthens light−matter interactions close to a metal surface. The enhanced localized fields offer attractive biomedical applications, such as biological sensing,13−17 surface-enhanced Raman scattering,18,19 biological imaging,20,21 and photothermal therapy.22,23 LSPR refractive index sensing, which mostly depends on LSPR spectral shifts induced by environmental dielectric changes around the plasmonic nanostructures, provides an efficient and nondestructive way to detect and quantify a small quantity of chemical or biological species. This LSPR-based sensing technique, which is based on optical extinction measurement, exhibits low-cost, rapid, real-time, highly sensitive, and label-free advantages. With these advantages, studies on biosensors in accordance with the LSPR principle have been widely performed. Some of these studies have focused on sensing biomolecules, 13 proteins,14,24,25 microRNAs,16 cancer cells,26−28 protein−DNA interactions,29,30 and DNA/RNA hybridization.31−36 An LSPR-enhanced field is localized within a distance of several tens of nanometers to a metal surface.37 Therefore, the LSPR spectral shift is sensitive to a slight change in environmental refractive index adjacent to plasmonic nanostructures. Various shapes of plasmonic nanoparticles synthesized by chemical approaches are one of the major categories of LSPR sensors. Changes in colors can be detected by the naked eye in a few seconds when a reaction occurs.38 Aside from the synthesized plasmonic nanoparticles, ordered plasmonic nanostructures fabricated by self-assembly or top-down approaches are another majority. These nanostructures exhibit advantages of controllability and repeatability, providing an increased flexibility and precise control of sensor properties. Ordered patches are not aggregated within liquid media, making them suitable candidates for sensing during cell culture. In this study, nanoimprint lithography was applied to fabricate gold nanodot arrays (GNDAs) on a glass substrate as LSPR sensors. The GNDA sensors were then used as growth substrates for culturing human retinal pigmented epithelium cells (ARPE-19) and monitoring cell growth. The biocompatibility and performance of the GNDA sensors were evaluated. A series of experiments, including cell counting, (3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) (MTT) and cell counting kit-8 (CCK-8) assays, immunofluorescent staining of cell focal adhesions, and flow cytometric cell cycle analysis, was also performed to monitor the cell growth. The GNDA LSPR sensor was illustrated to be a powerful tool for the in situ monitoring of cell growth, quantifying of cell confluency, and predicting of the optimal subculture timing.

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MATERIALS AND METHODS

Reagents and Cell Line. Perfluoropolyether (PFPE) was purchased from Solvay Solexis Inc. (West Deptford, NJ, USA). Poly(methyl methacrylate) (PMMA) (Mw ≈ 35 K) was purchased from Acros Organics (Belgium). AMONIL, a UV-curable resist, was obtained from AMO GmbH (Austria). Dulbecco’s modified Eagle’s medium (DMEM), Ham’s F-12 nutrient mixture (F-12), trypsinEDTA, and fetal bovine serum (FBS) were obtained from Hyclone (South Logan, UT, USA). Phosphate buffered saline (PBS), propidium iodide (PI), RNase A, MTT, CCK-8, TritonX-100, formaldehyde, penicillin−streptomycin (5000 U/mL), and bovine serum albumin (BSA) were procured from Sigma-Aldrich (St. Louis,

Figure 1. Schematic of (a) the GNDA sensor and (b) the optical setup for cell growth monitoring. A GNDA sensor in a Petri dish was illuminated using a light source. The transmitted light was collected by a long working distance objective, and the extinction spectra were then obtained on a miniature UV−vis−NIR spectrometer. (This picture is not presented in relative proportions.). 1817


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Figure 2. GNDA fabrication and LSPR spectral results. (a−c) SEM images of (a) resist undercut profile, (b) GNDA after lift off, and (c) GNDA after annealing. (d) Size, (e) extinction spectra, and (f) LSPR peak wavelength and fwhm of nanodots under unannealed treatment and annealed treatment at 200 to 500 °C for 15 min. The fwhm was calculated by fitting the extinction curve to the Pseudo-Voigt function. (g) Extinction spectra of one GNDA sensor (sensor #2) immersed in water, acetone, and 2-propanol. (h) LSPR peak wavelengths of three GNDA sensors immersed in water (n = 1.33), acetone (n = 1.36), and 2-propanol (n = 1.38). The scale bars in a−c are 400 nm. The error bars in d represent one standard deviation of the measured data from five positions in each sample. with PBS, and 100 μL of MTT solution (5 mg/mL) was added to each well in the dark. The cells were incubated with MTT solution for another 4 h so that MTT could be metabolized by the cells. The MTT solution was discarded from the well, and the MTT crystals were dissolved using 1000 μL of DMSO. A total of 200 μL of the dissolved solution was aliquotted to a new 96-well microplate. The absorbance at 570 nm of the colored product was then determined using a microplate reader (Infinite M200, TECAN). For the CCK-8 assay, the 96-well microplate was used to culture cells directly. At the end of the experiment, 10 μL of CCK-8 solution was added directly to each well. The cells with CCK-8 were incubated for an additional 1 h. The absorbance at 470 nm of the solution was then measured. Immunofluorescent Staining of Actin Filaments in the Cytoskeleton, Focal Contacts, and Cell Nuclei. TRITCconjugated phalloidin, vinculin monoclonal antibody, and DAPI were obtained from the actin cytoskeleton and focal adhesion staining kits. ARPE-19 cells were cultured on microscope slides placed in a sixwell microplate for 1−4 days. Afterward, the cells were washed with prewarmed PBS twice, fixed in 4% formaldehyde solution at 4 °C for 10 min, permeabilized with 0.1% Triton X-100 in PBS at room temperature for 5 min, washed twice with 1× wash buffer (1× PBS containing 0.05% Tween-20), blocked with PBS containing 1% BSA for 30 min, incubated with antivinculin antibody at room temperature for 1 h, washed three times (5−10 min each) with 1× wash buffer, incubated with FITC-conjugated goat antimouse secondary antibody and TRITC-conjugated phalloidin simultaneously at room temperature and strictly in the dark for 1 h, and washed three times (5−10 min each) with 1× wash buffer. The nuclei were counterstained with DAPI, and the slides were sealed with an antifade mounting solution. Fluorescence images could be visualized with a fluorescence microscope. Cell Cycle Analysis through Flow Cytometry. ARPE-19 cells were cultured in a six-well microplate and incubated for 1−6 days. Afterward, they were detached by 0.25% trypsin-EDTA solution and centrifuged at 4000 rpm for 2 min. The supernatant was removed, and the cells were resuspended in 1 mL of PBS. Subsequently, drops of 70% cold ethanol (1 mL) were added to the samples while being shaken. The sample was transferred to a freezer at −20 °C and stored until measurement. Prior to flow cytometric analysis, the cells were centrifuged at 4000 rpm for 2 min. The supernatant was removed, and the pellets were washed twice with PBS. Then, 0.5 mL of PBS containing 10 μg/mL RNase A and 20 μg/mL PI stock solution was

First, the bottom PMMA layer was spin coated onto a glass substrate at a thickness of 150 nm and baked at 150 °C for 10 min. The AMONIL resist was then spin coated onto the bottom PMMA layer at 3000 rpm and baked at 100 °C for another 10 min. The PFPE mold and the glass substrate coated with bilayers were stacked together and placed into our home-built imprint chamber. A compressed air press with a pressure of 3 bar was applied to the mold at room temperature for 5 min, and the mode features were filled with an imprint resist. The resist was then hardened by illuminating with UV light (λ = 365 nm) for 10 min from the backside of the mold. After demolding was performed, the resist residual layer was removed in a reactive-ion etching (RIE) machine with a SF6/O2 gas mixture for 5.5 s. The features were then transferred to the underlying PMMA layer by oxygen plasma. An undercut resist profile was produced in this step to facilitate the subsequent lift-off process. A gold film with a thickness of 50 nm was deposited on the patterned resist through electron-beam evaporation. The GNDA was obtained after acetone lift-off and thermal annealing processes were completed. Cell Growth Monitoring by LSPR Signal. Changes in environmental refractive index over GNDAs can be determined by measuring the spectral shift of the LSPR wavelength. The optical setup is illustrated in Figure 1b. A GNDA sensor in a Petri dish was illuminated using a light source (tungsten halogen lamp, SL1FILTER, StellarNet Inc., USA) coupled with fiber optic cables and a collimating lens. The transmitted light was collected by a long working distance objective (20×, M Plan Apo, Mitutoyo, Japan), and the extinction spectra were obtained on a miniature UV−vis−NIR spectrometer (BLK-CSR-SR model, StellarNet Inc., USA). The bulk sensitivity of the GNDAs was determined by measuring the LSPR wavelength shift of samples immersed in various liquids, including water, acetone, and 2-propanol. The sensitivity of each sample was calculated from the LSPR spectral shift divided by the change in the refractive index. The same optical setup was used to monitor the succeeding cell growth. The integration of the miniature spectrometer with an inverted microscope was also possible so that the morphological characteristics of the cells could be observed and the spectrum could be obtained simultaneously in one setup. MTT and CCK-8 Assays. ARPE-19 cells were cultured in a 12-well or 96-well microplate and incubated at 37 °C and in a 5% CO2 atmosphere for 1−8 days. For the MTT assay, the 12-well microplate was used for placing glass substrates. At the end of the experiment, the culture medium was removed from the well, the cells were washed 1818


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Figure 3. Quantification of cell growth by (a) LSPR red shifts, (b) cell densities, and cell viability analyzed by (c) MTT and (d) CCK-8 assays. The initial cell densities were 123 (black), 57 (red), and 28 (green) cells/mm2. The error bars represent one standard deviation of the measured data from (a,b) four positions on the GNDAs and (c,d) three independent experiments.

its fwhm reduces more quickly, resulting in a larger figure of merit (FOM). Therefore, we chose the GNDAs annealed at 500 °C as the sensors for their better sensing capability. The sensitivity of each GNDA sensor was evaluated by measuring the LSPR spectral shift of the sensor immersed in various liquids, namely, water (n = 1.33), acetone (n = 1.36),41 and 2-propanol (n = 1.38).42 Figure 2g shows the extinction spectra of one GNDA sensor (sensor #2), and Figure 2h presents the LSPR peak wavelengths of the three sensors immersed in various liquids. The LSPR peaks red-shifted linearly as the refractive index increased. The sensor sensitivities calculated from the measured data were 300.0, 304.0, and 309.2 nm/RIU. Their sensing performance was similar. The slight differences in their LPPR peak wavelengths could be attributed to nanodot size variation in different sensors. However, this difference did not influence the sensing result because the sensing technique relied on the LSPR spectral shifts not on the absolute LSPR peak values. Cell Growth Monitoring by LSPR Sensing, Cell Density, MTT, and CCK-8 Assays. ARPE-19 cells were cultured on the GNDA sensors to ensure the biocompatibility and noncytotoxicity of the GNDAs. Microscopic observation revealed no evident differences in the morphological characteristics of the cells that grew on the GNDA, glass substrate, and culture dish (Figure S2, Supporting Information). The ARPE19 cells with initial densities of 123, 57, and 28 cells/mm2 were cultured on the GNDAs characterized in the Plasmonic GNDA Sensor and Its Sensitivity to determine the relationship between LSPR shift and cell growth. As the cells grew, their number increased, and they formed a monolayer cover on the GNDAs, possibly resulting in the change in the environmental refractive index over the GNDAs. Figure 3a shows the monitored LSPR peak shifts with respect to the cell culture time. The reference LSPR peak for determining the LSPR shifts was obtained from the extinction spectra of the GNDA sensor immersed in a fresh medium (DMEM/F-12) without the cultured cells. The individual influence of various culture

added to the resuspended cells and kept in the dark at room temperature for 30 min. The samples were transferred to Eppendorf tubes for flow cytometric analysis (BD FACSVerse). Cell cycle analysis was performed using Modfit software.

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RESULTS AND DISCUSSION Plasmonic GNDA Sensor and Its Sensitivity. Figure 2a shows the scanning electron microscopy (SEM) image of the undercut resist profile for patterning GNDA after nanoimprint patterning and RIE etching processes were completed. The deposited gold film did not fully cover the resist sidewall because of the undercut resist profile formed by the bilayer resist scheme. The gold film on the top of the resist was successfully lifted off, leaving the GDNA with an area of 5 mm × 5 mm on the glass substrate as shown in Figure 2b. The diameter and pitch of the nanodots were 236 and 400 nm, respectively. The GDNAs were annealed in a furnace at various temperatures from 200 to 500 °C for 15 min to further improve the sensing performance of the GDNAs. Figure 2c illustrates the SEM image of the annealed GNDA at 500 °C. The original rough surface of the nanodots became smooth, and the size decreased significantly. The diameter was approximately 184 nm. When the annealing temperature increased, the size of the annealed nanodots decreased as illustrated in Figure 2d. The annealed gold tended to cohere at a high temperature, resulting in a reduced size. The extinction spectra of the GNDAs were also measured to check their optical behavior as shown in Figure 2e. The LSPR peak wavelength and full width at half-maximum (fwhm) of the GNDAs annealed at various temperatures are illustrated in Figure 2f. The LSPR peak of the GNDA at a high annealing temperature blue-shifted because of its small size. The narrow spectral width of the GNDAs annealed at high temperatures could be attributed to the reduced nanodot size and the increased size uniformity. The size effect on the LSPR signal was also simulated to support the experimental results (Figure S1, Supporting Information). From the simulation, the sensitivity is slightly lower for a smaller nanodot. However, 1819


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Figure 4. Fluorescence images of cell nuclei (DAPI, blue), actin cytoskeleton (TRITC-conjugated phalloidin, red), and focal contacts (antivinculin, green) in ARPE-19 cells cultured for 1−96 h. The initial density of ARPE-19 was 123 cells/mm2. The right-most column shows the merged images of the three fluorescent stains.

mediums can be subtracted by this approach. Initially, the LSPR wavelength of the GNDAs continuously underwent a red shift with culture time, indicating an increase in the environmental refractive index because of an increase in the adherent cultured cells on the GNDA surfaces. The optical microscopic images of the cells at the time of LSPR monitoring were also captured for comparison. The corresponding cell densities were collected from the captured optical images, and the statistical data are illustrated in Figure 3b. Similar to the trends of the LSPR monitoring data, the cell densities of the cultured cells initially increased gradually. The cells with initial densities of 123, 57, and 28 cells/mm2 reached the same maximum LSPR red shift (approximately 40 nm) and the same maximum cell density (approximately 182 cells/mm2) at 72, 112, and 168 h, respectively. At the time that cell growth reached confluence, the GNDAs sensed the maximum environmental refractive index. The cell confluence accompanied the maximum red shift of the LSPR wavelength, indicating that the LSPR red shift could be a useful indicator of cell growth and confluence. After the cell reached confluency, the cells became unhealthy and underwent apoptosis, resulting in a decrease in cell densities. The monitoring LSPR peak also started to undergo a blue shift possibly as a result of the detachment of the apoptotic cells from the GNDA surfaces. The trends of the cell densities and LSPR signals with respect to the culture time were similar. The only major difference was that the initial

LSPR shifts were not significant regardless of the initial cell densities because the cells were not attached to the culture substrate at the initial culture stage. This difference should not influence the prediction ability of the subculture timing by LSPR monitoring. To make sure that the cell adhesion to the GNDA surface is necessary for the LSPR approach, another suspension cell line, THP-1,43 was tested. Figure S3 (Supporting Information) shows the monitored LSPR shifts and cell density with respect to the cell culture time of THP-1 cells. The initial density was 123 cells/mm2. Although the cell density increased as the cultured cells grew, there was no evident change in the LSPR shift. These data suggested that the use of GNDAs for cell growth sensing is specific for adherent cells. MTT and CCK-8 assays, which reflect the health status and cell metabolic activity of the cultured cells, were performed to confirm the results of LSPR monitoring and cell density data. MTT and CCK-8 assays are based on tetrazolium salt and commonly used as quantitative colorimetric methods for determining the number of viable cells in cell proliferation and cytotoxicity assays. These two tetrazolium salts are reduced by dehydrogenase activities in cells to produce a formazan dye. The amount of formazan dye, which is measured by a spectrometer and represented as absorbance, is directly proportional to the number of living cells and positively correlated with cell viability. An MTT assay is cheaper than a CCK-8 assay, but the former involves more steps and requires 1820


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4 shows the fluorescence images of the cell nuclei, actin cytoskeleton, and focal contacts in ARPE-19 cells cultured from 1 to 96 h. The initial density of ARPE-19 was 123 cells/ mm2. DAPI staining revealed that the cell nuclei increased as the cultured cells grew. The global outer shapes of the actin cytoskeleton and focal contact stains were similar, marking the shapes of the cultured cells. However, filamentous structures in the cytoskeleton stain were present, whereas many tiny dots in the focal contact stain were observed. These tiny dots likely came from the staining of the focal adhesions that anchored the cells to the cultured substrate. In the first hour, the cell cytoskeleton and the focal contacts formed small circles. The cells did not remarkably adhere to the cultured substrate at this stage, and this phenomenon could explain why the initial LSPR red shifts were not significant regardless of the initial cell densities (Figure 3a) as previously discussed in the Cell Growth Monitoring by LSPR Sensing, Cell Density, MTT, and CCK-8 Assays. The number of cells in the 24th hour was still similar to that in the first hour. The only major difference was that the size of the cells increased. The number of the tiny focal contact spots increased, indicating that the cells adhered to the culture substrate and grew stably. From the 24th hour to the 72nd hour, the shapes of the cells did not vary significantly, and the number of cells increased gradually. Additional focal contact spots appeared as the cells increased. The maximum confluence of the adhered cells and the maximum number of the focal contact spots were observed in the 72nd hour, at which the timing showed the greatest LSPR red shift and maximum cell density and metabolic activities (Figure 3). The increasing focal contact spots further enlarged the effective refractive index close to the GNDA surface, thereby causing a steady increase in the LSPR red shifts (Figure 4). The number of cells and focal contact spots decreased in the 96th hour compared with the results in the 72nd hour. The phenomena likely resulted in the slight LSPR blue shift (Figure 3). Using GNDA as part of the growth substrate for cell culture was a novel finding, and the LSPR red shift of GNDA was consistent with the increase in the number of cells, focal contact spots, and cell metabolic activities. Cell Cycle Analysis. This study aimed to develop a novel, quick, and label-free method to monitor cell growth and to suggest suitable subculture timing. As such, we analyzed the cell cycle, which includes an ordered, coordinated, and irreversible series of events that occur in a cell. Those events lead to the growth of cells and the replication and division of DNA. The cell cycle is divided into four phases in which a cell increases in size (gap 1 [G1] phase), replicates DNA (synthesis [S] phase), prepares to divide (gap 2 [G2] phase), and divides (mitosis [M] phase). The quantitation of DNA content through flow cytometry is a favorable method for cell cycle analysis. In this study, only the attached cells were included in the cell cycle analysis of our study because we believed that only the attached cells might influence the LSPR sensing of GNDA. Figure 5a shows the cellular DNA histogram of the cultured cells with an initial cell density of 123 cells/mm2 for 24−144 h. The left major peak represents G1 cells with a single chromosome set (2N), the right minor peak denotes G2/M cells containing a double chromosome set (4N), and the region between 2N and 4N corresponds to S cells. Figure 5b illustrates the corresponding statistical data. The histograms showed two clearly separated peaks, namely, G1 and G2/M peaks. The G1 peak increased gradually from

a longer time to generate data than the latter does. MTT is cytotoxic, and organic solvents such as DMSO are needed to dissolve formazan. Thus, using the cells after the assay is impossible. The CCK-8 assay is more efficient, reliable, sensitive, and time-saving than the MTT assay, but the former is much more expensive than the latter. Although formazan formed from the CCK-8 assay is water-soluble and can be directly measured using a spectrometer, it cannot be excluded from cell cultures and may affect the following experiments. Figure 3c,d show the results of MTT and CCK-8 assays. Both assays yielded the maximum absorbance at 72, 108, and 144 h with initial cell densities of 123, 57, and 28 cells/mm2, respectively, indicating the maximum number of viable cells present. The LSPR quantification method was strongly consistent with cell confluency, MTT, and CCK-8 assays. In all of the methods, the length of time in reaching the peak was approximately the same, and the cell growth curves were similar. Only the curves of the MTT and CCK-8 assays with an initial cell density of 28 cells/mm2 reached the maximum absorbance earlier than those of LSPR red shifts and cell densities. This result could be attributed to the low cell density at the beginning of cell culture. A slight variation in cell density at the beginning could lead to a large variation at the end, especially for a long culture time. This phenomenon further strengthened the need of cell growth monitoring to quantify subculture timing. The LSPR quantification method can be applied to monitor cell activities, and it is much faster than MTT and CCK-8 assays. MTT and CCK-8 assays require hours of waiting for the reactions to occur, whereas the LSPR quantification method requires only a few seconds to obtain the LSPR spectrum. The most important advantage of LSPR monitoring is its nonintervention effects on cell growth. Biologists, who conduct cell culture experiments, can use the LSPR monitoring method to predict the timing of cell confluency easily and efficiently. Immunofluorescent Staining of Cell Focal Adhesions. LSPR is sensitive to the environmental refractive index variation close to metal surfaces. As such, we focused on the adhesion behavior of the adherent cells during the culture period. Cells sense the environment by using their lamellipodia and filopodia at the early stage of cell−surface interactions.44−46 Lamellipodia and filopodia can also involve cell− cell interactions.47 As cells spread, they possess an adhesion trait at the budding stage. They form small dots known as focal complexes at the leading edge of the cell in lamellipodia. Focal complexes transform into focal adhesions, which are initially located at the cell periphery and then gradually move toward the cell center.47 The function of focal adhesions is structural, linking the external extracellular matrix to the internal actin cytoskeleton. These focal adhesions also serve as organizing centers to initiate signaling pathways in response to adhesion and transducing information from the outside of the cells into chemical and genetic messages that are interpreted within the cytoplasm and the nucleus. For investigation, the focal contacts of the cells were stained by antivinculin antibodies, which is a universal focal adhesion marker, and an FITC-conjugated secondary antibody in various culture periods from 1 to 96 h. The nuclei and the actin cytoskeletons of the cells were also marked with DAPI, which is a DNA binding dye, and TRITC-conjugated phalloidin, which localizes actin filaments, for positioning the cell nuclei and recognizing the cell shapes, respectively. Figure 1821


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confluency was consistent with the maximum LSPR red shift, indicating that the LSPR red shift could be a practical indicator of cell growth and confluency. Cell cycle analysis demonstrated that the timing of the maximum cell confluence reached the growth plateau, and the cells became unhealthy without further subculturing. In practice, with considering the cost of adding a GNDA layer on culture dishes, the GNDAs can be implemented only on the sampling points instead of on the entire area of the dish. The dish made of polystyrene with oxygen plasma treatment on the surface is the most widely used culture platform.49 The integration of an LSPR layer with the dishes could be possible using the nanotransfer printing technique.50,51 Assuming that the total area of the sampling region is 25 mm2 on a 100 mm culture dish, there is only 0.45% of the cells that grow on the GNDA when the cells reach confluency. We believe that the small ratio of cells on the GNDAs would not influence the gene expression of the entire culture system. This rapid, noninterventional, nondestructive, label-free, and real-time optical sensing approach could help researchers monitor cell growth, predict cell confluence, and standardize subculture timing by observing the same amount of LSPR shift.

Figure 5. (a) Cellular DNA content distribution and (b) cell cycle analysis of ARPE-19 cells cultured for 24 to 144 h through flow cytometry. The initial density was 123 cells/mm2. In a, a Savitzk− Golay filter was applied to visualize the data trend. The error bars in b represent one standard deviation of the measured data from three independent experiments.

the 24th hour to the 72nd hour and plateaued in the 96th hour and thereafter. Without subculturing the confluent cells after the 96th hour, the cell surface became rough and started floating in the medium. The decreased number of the attached cells might be the reason for the blue shift of the LSPR spectrum as shown in Figure 3a. For evaluating the growth potential of the cells after reaching confluency, some cells in the 72nd, 96th, 120th, and 144th hour were selected for subculturing to observe their morphological characteristics and further growth. The cells from the group of the 72nd hour could attach to and grow well on the culture dish. The cells from the group of the 144th hour could not attach to the culture dish anymore (Figure S4, Supporting Information). However, the cells from the groups of the 96th and 120th hour showed variable results, which could depend on the passage numbers of the cells.48 These data indicate that the cells could be no longer healthy after the confluence was reached and the maximum LSPR red shift occurred. Therefore, we would like to propose that the 72nd hour was a critical time to perform cell subculture.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssensors.9b00524.

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Simulated extinction spectra, fwhm, and FOM of the GNDAs; cell morphology of ARPE-19 on GNDA, glass, and culture dish substrates; monitored LSPR shifts and cell density with respect to the cell culture time of THP1 cells; and cell morphology of subcultured ARPE-19 after reaching confluency (PDF)

AUTHOR INFORMATION

Corresponding Authors

*Address: Department of Applied Chemistry, National Pingtung University, No. 1, Linsen Rd., Pingtung 90003, Taiwan. E-mail: [email protected]. *Address: Department of Photonics, National Cheng Kung University, No. 1, University Rd., Tainan 70101, Taiwan. Email: [email protected].

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CONCLUSIONS Cells were previously cultured by subjective observation and decision. However, nonstandardized culture conditions might alter growth patterns and likely change the gene expression of cellular functions. As such, cell biologists must consider many fundamental aspects at every stage of cell culture to ensure the quality and reproducibility of their experiments. The standardization of cell culture conditions enhances experimental success, ensuring that different batches of cells or samples are comparable. In this study, GNDA sensors patterned via a nanoimprint process on glass substrates were used as a part of cell culture substrates. Cell confluency could be quantified and the optimal subculture timing could be determined by monitoring the transmitted LSPR spectral shifts of the cultured cells on a GNDA sensor with a simple optical measuring apparatus to achieve cell culture consistency. Experiments on various cell growth aspects, including cell density, metabolic activity, focal adhesion, and growth cycle, were also performed to verify the monitoring accuracy of the proposed approach. The results of the LSPR red shift, cell density, and metabolic activity showed a similar trend and exhibited good correlation with one another. The immunofluorescence assay revealed that the LSPR red shift could be related to adhesion spots. Cell

ORCID

Chun-Hung Lin: 0000-0001-6400-9969 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the Ministry of Science and Technology (MOST) of Taiwan under grant nos. NSC 1012221-E-006-210-MY2 and MOST 103-2221-E-006-110. The authors thank Dr. Ching-Hsein Chen of Dept. of Microbiology, Immunology and Biopharmaceuticals, National Chiayi University; Dr. Jun-Jen Liu of School of Medical Laboratory Science and Biotechnology, Taipei Medical University; Dr. Chih-Chia Huang of Dept. of Photonics, National Cheng Kung University, Center for Micro/Nano Science and Technology of the National Cheng Kung University; and Taiwan Semiconductor Research Institute for providing the necessary equipment and technical support. 1822


Article

ACS Sensors

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Quantifying Cell Confluency by Plasmonic Nanodot Arrays to Achieve

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