Identification of Hydrogen Peroxide-Secreting Cells by Cytocompatible

Article pubs.acs.org/ac

Identification of Hydrogen Peroxide-Secreting Cells by Cytocompatible Coating with a Hydrogel Membrane Yang Liu, Shinji Sakai,* Shogo Kawa, and Masahito Taya* Division of Chemical Engineering, Department of Materials Engineering Science, Graduate School of Engineering Science, Osaka University, Toyonaka, Osaka 560-8531, Japan S Supporting Information *

ABSTRACT: A method for identifying each cell secreting reactive oxygen species (ROS) is highly desirable to advance the understanding of the physiological and pathological processes attributed to extracellular ROS. Here, we first report a method for realizing this. The individual cells secreting hydrogen peroxide (H2O2), a common ROS, could be coated by a hydrogel membrane through a horseradish peroxidasecatalyzed reaction consuming H2O2 secreted from the cells themselves. This hydrogel membrane coating was proved to be cytocompatible. In addition, the hydrogel membrane made from an alginate derivative could be removed on demand without causing damage to the enclosed cells. These results demonstrated the feasibility of the proposed method to be an effective tool in cellular ROS studies.

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studied as a possible route for obtaining hydrogels containing living cells because of its mild reaction conditions for cells.8−10 We expected the resultant hydrogel membrane would enable not only identification of the cells but also separation of the identified cells (Scheme 1). The material used for the hydrogel membrane was a sodium alginate derivative obtained by conjugation with tyramine for providing Ph moieties and 4aminofluorescein for providing a fluorescence signal (Fluor-AlgPh). Some polysaccharide-based hydrogels, such as alginate-, cellulose-, and amylopectin-based hydrogels, cross-linked through the HRP-catalyzed reaction are degradable on demand by the enzymes specific for the respective polysaccharides without inducing damage in mammalian cells.11−13 Thus, we expected that the enclosed cells would be enabled for release from the Fluor-Alg-Ph hydrogel membrane on demand with high viability.

eactive oxygen species (ROS), generated through metabolic pathways in living cells, play essential roles in both intracellular and extracellular events.1,2 For understanding the physiological or pathological processes attributed to ROS, a variety of methods have been developed for detecting ROSgenerating cells. A common method for identifying these cells is the use of fluorogenic probes, such as 2′,7′-dichlorodihydrofluorescein and dihydroethidium.3 These chemicals permeate into the cytoplasm from ambient solution and liberate florescence by reacting with ROS. They are useful for identifying the cells with intracellular ROS but may not correctly reflect the ROS secreted extracellularly because of the existence of ROSscavenging systems in cells, including superoxide dismutase, catalase, glutathione peroxidase, and others. In this study, we aimed to develop a method for identifying the cells secreting H2O2, an ROS. Recently, extracellular H2O2 has attracted increasing attention as one aspect of immunity or as a signal molecule of cell-to-cell communication, the same functions as those of other ROS.4,5 There are reagents for detecting H2O2 in the extracellular environment such as the medium,6,7 but the values obtained using the conventional method only reflect the content of H2O2 in the whole measured bulk liquid; i.e., it is impossible to identify the cells secreting H2O2. To the best of our knowledge, there is no method for identifying the cells secreting ROS, including H2O2. For identifying H2O2-secreting cells, our strategy is to apply an enzymatic reaction, in which H2O2 released from the target cells is utilized for the progress of the reaction. We tried to form a thin hydrogel membrane on the surface of these individual cells, as a label for identification, through a horseradish peroxidase (HRP)-catalyzed reaction. In this reaction, phenolic hydroxyl (Ph) moieties were cross-linked via consumption of H2O2. Recently, this enzymatic reaction has been intensively © XXXX American Chemical Society

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EXPERIMENTAL SECTION Reagents. Sodium alginate (MW 70 000, Kimica I-1G) was obtained from Kimica (Japan). Alginate lyase (from Flavobacterium multivorum), amylopectin (from maize), gelatin (types A and B), and 4-aminofluorescein were purchased from SigmaAldrich (United States). Sodium hyaluronate (MW 1000000− 1200000) was obtained from JNC Corp. (Japan). HRP (200 units/mL), N-hydroxysuccinimide (NHS), N,N′-carbonyldiimidazole (CDI), and dimethyl sulfoxide (DMSO) were obtained from Wako Pure Chemical Industries (Japan). Tyramine hydrochloride was purchased from Tokyo Chemical Industry

Received: June 6, 2014 Accepted: October 31, 2014

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dx.doi.org/10.1021/ac503342k | Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry

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ethanol aqueous solution sequentially until an ultraviolet− visible spectrum peak at 275 nm was undetectable in the spent ethanol. The synthesis of amylopectin and gelatin (type A) derivatives possessing Ph moieties (AP-Ph and gelatin-Ph, respectively) followed that described in previous papers.12−14 To prepare rhodamine-labeled gelatin-Ph (Rhod-gelatin-Ph), NHS−rhodamine was dissolved in DMSO at 10 g/L in advance and then added to phosphate-buffered saline (PBS; pH 7.4) containing 80 g/L gelatin-Ph. The final concentration of NHS− rhodamine was 0.1 g/L. The mixture was stirred for 2 h at 37 °C, then dialyzed against deionized water, and lyophilized to collect the polymer derivatives. Cell Culture. Mouse embryo cell line 10T1/2 cells, human hepatocellular carcinoma cell line HepG2 cells, and human pancreatic carcinoma cell line Panc1 cells (Riken Cell Bank, Japan) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Nissui, Japan) containing 10% fetal bovine serum (FBS; Gibco, United States). Rat adipose stem cells (ADSCs) were isolated from the subcutaneous fat of a female rat (12 weeks old, Crl:CD (SD), Charles River, Japan) and were cultured in DMEM containing 15% FBS. Animal experiments complied with the recommendations of the Osaka University manual entitled “Guide for the Care and Use of Laboratory Animals” under the permission of our institutional committee (no. 22-3). The isolation process followed that described in a previous paper.15 More than 90% of the resultant ADSCs expressed CD44 and CD90, and less than 5% of the cells expressed CD45 and CD31. All the cells were incubated at 37 °C in humidified air containing 5% CO2. Flow Cytometric Analysis. Flow cytometric detection of cells coated with Fluor-Alg-Ph membrane was performed using a BD Accuri C6 flow cytometer. FCS Express 4 software (De Novo, United States) was used for data analysis. Identification of Suspended H2O2-Secreting Cells. For investigating various cell lines, 10T1/2 cells were dispersed at 4 × 105 cells/mL in PBS containing Fluor-Alg-Ph (1−2%, w/v) and HRP (10−100 units/mL) for 30−60 min at room temperature. Panc1 cells or ADSCs were soaked in PBS containing 2% (w/v) Fluor-Alg-Ph and 100 units/mL HRP at 1 × 106 cells/mL for 30 min at room temperature. All cell lines were subsequently washed with PBS before fluorescence microscopic observation (BIOREVE BZ-9000, Keyence, Japan) or flow cytometric analysis. For investigating various hydrogel materials, ADSCs were dispersed at 1 × 106 cells/mL in PBS containing 10% (w/v) AP-Ph, 5% (w/v) gelatin-Ph, or 1% (w/v) Fluor-HA-Ph and 100 units/mL HRP for 30 min at room temperature and were subsequently washed with PBS before fluorescence microscopic observation. Identification of Adhered H2O2-Secreting Cells. To confirm the possibility of identification of adhered H2O2secreting cells, PBS containing 1% (w/v) Fluor-Alg-Ph and 100 units/mL HRP was poured into a culture dish having adhered 10T1/2 cells, and the dish was allowed to stand for 30 min at room temperature. The culture dish was washed with PBS before observation. Model System. To confirm the contribution of H2O2 secreted from the H2O2-secreting cells to hydrogel membrane formation, HepG2 cells immobilizing glucose oxidase (GOx; BBI Solutions, United Kingdom) were used as a model of H2O2secreting cells (GOx−HepG2), because GOx oxidizes glucose to produce H2O2. HepG2 cells were chosen because of their undetectable H2O2-producing ability (Figure S4a, Supporting Information) confirmed with a commercially available fluoro-

Scheme 1. Illustration of (a) Selective Coating of H2O2Secreting Cells with a Hydrogel Membrane via HRPCatalyzed Reaction, (b) Cell Detection and Separation Based on Hindrance of Adhesion by the Hydrogel Membrane, and (c) Degradation of the Hydrogel Membrane Using a Specific Enzyme

(Japan). 2-Morpholinoethanesulfonic acid (MES) and watersoluble carbodiimide hydrochloride (WSCD) were obtained from Dojindo (Japan) and the Peptide Institute (Japan), respectively. NHS−rhodamine was obtained from Thermo Fisher Scientific Inc. (United States). Synthesis of Fluor-Alg-Ph and Other Polymers Possessing Ph Moieties. For synthesizing Fluor-Alg-Ph (see the Supporting Information, Figure S1), sodium alginate was first dissolved at a concentration of 10 g/L in 50 mmol/L MES buffer (pH 6.0). Tyramine hydrochloride, NHS, and WSCD were added to the sodium alginate solution at 7.0, 1.2, and 3.9 g/ L, respectively. 4-Aminofluorescein was dissolved in DMSO in advance at 50 g/L and then added to the above-mentioned solution to give a concentration of 0.25 g/L. The mixtures were stirred overnight at room temperature, then precipitated, and washed with 80% ethanol aqueous solution sequentially until an ultraviolet−visible spectrum peak at 275 nm attributed to the remaining tyramine and 4-aminofluorescein was undetectable in the spent ethanol. 1H nuclear magnetic resonance (NMR) spectra of the resultant Fluor-Alg-Ph and sodium alginate (dissolved in D2O at 1% (w/v)) were recorded at 30 °C on a JNM-ECS400 spectrometer (JEOL RESONANCE, Japan). The δ scale is relative to (trimethylsilyl)propanoic acid (TMSP) at δ = 0. The NMR data confirmed that the aromatic moieties from tyramine hydrochloride and 4-aminofluorescein were conjugated with the side chains of sodium alginate (Figure S2, Supporting Information). For the synthesis of a fluorescent hyaluronic acid derivative (Fluor-HA-Ph; Figure S3, Supporting Information), sodium hyaluronate was dissolved at a concentration of 7.5 g/L in 50 mmol/L MES buffer (pH 6.0). Tyramine hydrochloride, NHS, and WSCD were added to the solution at 3.3, 1.1, and 1.8 g/L, respectively. 4-Aminofluorescein was dissolved in DMSO in advance at 50 g/L and then added to the above-mentioned solution to give a concentration of 0.81 g/L. The mixtures were stirred overnight at room temperature, then precipitated, and washed with 80% B



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Figure 1. (a) Overlaid micrographs of transmitted light and fluorescence images of 10T1/2 cells after treatment with 1% (w/v) Fluor-Alg-Ph and 100 units/mL HRP for 30 min and with 1% (w/v) Fluor-Alg-Ph alone for 30 min. Scale bars = 100 μm. (b) Fluorescent signal from the hydrogel membrane covering 10T1/2 cells after treatment with 1% (w/v) Fluor-Alg-Ph and 100 units/mL HRP in the presence or absence of 4.5 × 104 units/ mL catalase. Effects of the concentrations of (c) Fluor-Alg-Ph and (d) HRP, and (e) the soaking time in polymeric solutions on the content of cells coated with the hydrogel membrane. The contents were compared in every experimental set and are represented as a relative coated cell ratio. Vertical bars: standard deviation (n = 3).

separated 10T1/2 cells, recovered after treatment with 0.02 mg/ mL alginate lyase for 10 min, were seeded on 96-well dishes at 2.0 × 103 cells/well. At 4, 20, 33, and 48 h of culture, the spent media in the wells were exchanged with media (200 μL/well) containing 10% (v/v) kit reagent. After 50 min of standing in an incubator, the absorbance at 450 nm of the resultant solutions was measured by a microplate reader.

genic probe (BES-H2O2-Ac, Wako Pure Chemical Industries, Japan), according to the supplier’s protocol.16 The GOx− HepG2 cells were prepared by soaking HepG2 cells in PBS containing a conjugate of GOx and biocompatible PEG anchors for cell membrane insertion (BAM; SUNBRIGHT OE-080CS, NOF, Japan). The conjugation was prepared on the basis of that described in previous papers.17,18 The GOx−HepG2 cells were soaked at 1.56 × 106 cells/mL in PBS containing 1% (w/v) Fluor-Alg-Ph, 100 units/mL HRP, and 0, 0.156, 0.311, 0.622, or 1.24 μmol/mL glucose. The mixtures were allowed to stand at room temperature for 6 min to exhaust glucose and subsequently washed with PBS before fluorescence microscopic observation. The exhaustion of glucose was confirmed from no increase in fluorescence intensities even at an extended standing time of 10 min under each condition. The amounts of H2O2 produced by each GOx−HepG2 cell in the solutions containing 0, 0.156, 0.311, 0.622, or 1.24 μmol/mL glucose were stoichiometrically calculated as 0, 0.1, 0.2, 0.4, and 0.8 pmol of H2O2/cell, respectively. Detection of Intracellular and Extracellular H2O2. 10T1/2 cells were stained with BES-H2O2-Ac probe according to the supplier’s protocol. Then the cells were dispersed at 5 × 105 cells/mL in PBS containing 4.6% (w/v) Rhod-gelatin-Ph and 100 units/mL HRP for 30 min at room temperature and subsequently washed with PBS before fluorescence microscopic observation. The Rhod-gelatin-Ph was used for avoiding the overlapping of the fluorescence from the BES-H2O2-Ac probe. Separation of Identified Cells. 10T1/2 cells, after treatment for identification with the Flour-Alg-Ph hydrogel membrane, were suspended in cell culture medium, poured into a gelatin (type B)-coated polystyrene culture dish, and then incubated for 1 h at 37 °C in 5% CO2/95% air. The suspension was collected for further analysis. Growth Profile Measurement. The cell growth profile was determined by using Cell Counting Kit-8 (Dojindo, Japan). The

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RESULTS AND DISCUSSION To reveal the feasibility of our idea, first, we dispersed mouse embryo 10T1/2 cells in PBS containing 1% (w/v) Fluor-Alg-Ph and 100 units/mL HRP for 30 min at room temperature. The cells were abundant in intracellular H2O2, as detected by the commercially available fluorogenic probe (Figure S4b, Supporting Information). Fluorescence microscopic observation after washing with PBS confirmed the existence of cells coated with the Fluor-Alg-Ph hydrogel membrane. In contrast, no fluorescent signal arising from the Fluor-Alg-Ph membrane was found when the cells were soaked in the solution containing Fluor-Alg-Ph alone (Figure 1a). Formation of the membrane on individual cells was also confirmed by the flow cytometer (Figure 1b). Two peaks indicating the existence of two groups of cells with (higher intensity peak) and without (lower intensity peak) the hydrogel membrane were observed in the histogram of the fluorescence intensity attributed to Fluor-Alg-Ph. The induction of Fluor-Alg-Ph hydrogel membrane formation by H2O2 secreted from the cells was also confirmed from the decrease in the content of the cells coated with the membrane in the solution containing 1% (w/v) Fluor-Alg-Ph, 100 units/mL HRP, and additional 4.5 × 104 units/mL catalase. Catalase catalyzes the decomposition of H2O2 to water and oxygen. For the above-mentioned results, one may wonder why both cells coated with the hydrogel membrane and noncoated cells exist despite the use of the same lot of the same cell line. However, recently, the heterogeneity of individual cells even in the same C



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lot of the same cell line has been well recognized.19,20 The ROS secretion ability also might be different among individual cells. To confirm the possibility of controlling the sensitivity of H2O2-secreting cell identification, we investigated the effects of the concentrations of Fluor-Alg-Ph and HRP and the soaking time in polymeric solution on hydrogel membrane formation. The results were that the content of the cells coated with FluorAlg-Ph membrane increased 1.3-fold with increasing Fluor-AlgPh concentration from 1% to 2% (w/v) at 100 units/mL HRP for 30 min of soaking in the solution (Figure 1c). This result demonstrates that the cells with a slower rate of H2O2 secretion are detectable by increasing the Fluor-Alg-Ph concentration. The HRP concentration (Figure 1d) and soaking time in polymeric solution (Figure 1e) also showed significant effects: the increases of HRP concentration from 10 to 100 units/mL (1% (w/v) Fluor-Alg-Ph, 30 min of soaking time) and soaking time from 30 to 60 min (1% (w/v) Fluor-Alg-Ph, 100 units/mL HRP) induced 2.5-fold and 2.8-fold increases in the content of the identified cells, respectively. These results were all in line with the general principle of enzyme kinetics and suggested the possibility of controlling the sensitivity of H2O2-secreting cell detection. To ascertain the contribution of H2O2 secreted from cells to coating of the cells with a hydrogel membrane, we studied Fluor-Alg-Ph hydrogel membrane formation using GOx− HepG2 cells in solutions containing different amounts of glucose to produce different amounts of H2O2 on the surface of individual cells. We measured the mean fluorescence intensity (MFI) arising from the hydrogel membrane by the flow cytometer to correlate hydrogel membrane formation with H2O2 production per cell (Figure 2). When the amounts of

On the basis of this formula, the average amount of H2O2 secreted by 10T1/2 cells coated with a hydrogel membrane, shown in Figure 1b (black line, higher intensity peak), was calculated as around 0.46 pmol of H2O2/cell during 30 min of soaking in the solution containing 1% (w/v) Fluor-Alg-Ph and 100 units/mL HRP. In addition, we applied the identification method of H2O2secreting cells to 10T1/2 cells adhering on a culture surface. A portion of them were covered with a Fluor-Alg-Ph membrane while keeping the cell shape extended (Figure 3). The

Figure 3. Overlaid micrographs of transmitted light and fluorescence images of adhered 10T1/2 cells after treatment with 1% (w/v) FluorAlg-Ph and 100 units/mL HRP for 30 min. Scale bar = 50 μm.

cytocompatible identification of H2O2-secreting cells, while maintaining adhesion of both the identified and surrounding cells, would be useful for studying the effect of secreted H2O2 on the surrounding cells. The versatility of this method was confirmed by tests using other cells and polymers. Rat adipose stem cells and human pancreatic cancer cell line Panc1 cells, possible ROS producers,21,22 could also be coated by a Fluor-Alg-Ph membrane (Figure 4a,b). Cell identification was also possible using other polymers possessing Ph moieties, such as AP-Ph, gelatin-Ph, and Fluor-HA-Ph (Figure 4c−e). An attractive point of the method developed in this study is that there are a variety of materials applicable as the membrane. A required condition for the membrane materials is possession of moieties crosslinkable through the HRP-catalyzed reaction consuming H2O2. This means the potential for fabricating hydrogel membranes having preferable properties for individual applications. For example, the AP-Ph membrane is degradable simply by being soaked in a medium containing serum because of the existing amylase, which can hydrolyze amylopectin (Figure S5, Supporting Information). In addition, the hydrogel membranes obtained from the materials, which can regulate cellular behaviors, e.g., hyaluronic acid or gelatin derivatives, may be useful for selectively providing stimuli to H2O2-secreting cells. An interesting result was obtained from the concurrent treatment of 10T1/2 cells with the fluorogenic probe for detecting intracellular H2O2 and Rhod-gelatin-Ph for forming a hydrogel membrane around the cells via HRP reaction with extracellular H2O2. As shown in Figure 5, we found that some cells were poorly coated with the hydrogel membrane despite showing a relatively strong signal from the probe for detecting intracellular H2O2. This result clearly demonstrates that the

Figure 2. MFI values from hydrogel membranes formed on the surfaces of GOx−HepG2 cells with different amounts of H2O2 produced. Vertical bars: standard deviation (n = 3).

H2O2 produced from each cell were 0 and 0.1 pmol/cell, the MFI value of the majority group of these cells was around 0.45 × 105, and no gelated cells were found. When H2O2 production was 0.2 pmol/cell, the MFI reached 13.6 × 105. Furthermore, the MFI increased as the H2O2 production increased from 0.2 to 0.8 pmol. The results confirmed the relationship between the amount of hydrogel membrane formed and that of H2O2 produced by the cells and gave a referable calibration of the H2O2 product of each cell and fluorescence intensity arising from the hydrogel membrane prepared by 1% Fluor-Alg-Ph and 100 unit/mL HRP: MFI = {64.3 × (amount of H2O2 produced) + 0.59} × 105 (in the range of 0.2 and 0.8 pmol of H2O2/cell). D



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Figure 4. Overlaid micrographs of transmitted light and fluorescence images of (a) Panc1 cells and (b) ADSCs after treatment with 2% (w/v) FluorAlg-Ph and 100 units/mL HRP for 30 min. Transmitted light images of ADSCs after treatment with (c) 10% (w/v) AP-Ph or (d) 5% (w/v) gelatin-Ph and 100 units/mL HRP for 30 min. The arrows in panel d mark the identified cells with a gelatin-Ph hydrogel membrane. (e) Overlaid micrograph of transmitted light and fluorescence images of ADSCs after treatment with 1% (w/v) Fluor-HA-Ph and 100 units/mL HRP for 30 min. Scale bars = 100 μm.

Figure 5. Micrographs of fluorescence images of 10T1/2 cells after concurrent treatment with 10 μmol/L BES-H2O2-Ac for detecting intracellular H2O2 (green) and 4.6% (w/v) Rhod-gelatin-Ph and 100 units/mL HRP for forming a hydrogel membrane (red). Scale bars = 100 μm.

the hydrogel membrane. The viability of the cells remained at 94.3 ± 3.3% (n = 3) after treatment with alginate lyase. In addition, cells recovered from the membrane coating proliferated in a fashion similar to that of the cells seeded through a conventional subculture protocol without any treatments for cell identification and separation (Figure 6b). The adhered cells experiencing no membrane coating in the separation method also similarly proliferated (Figure 6c, noncoated cells). These results demonstrate the cytocompatibility of the method developed in this study.

intracellular H2O2 level detected by the conventional method does not always reflect H2O2 secreted from the individual cells. In some studies using the identified H2O2-secreting cells, separation of these cells would be required. It is intuitive that the cells coated with a Fluor-Alg-Ph membrane showing a different peak compared with noncoated cells in the measurement using a flow cytometer (Figure 1b) can be separated using a fluorescence-activated cell sorter (FACS). As an alternative method instead of use of an FACS, we investigated a fluorescence-independent separation method for adherent cells. We put the medium suspending the cells obtained right after the treatment of 10T1/2 cells for identifying H2O2secreting cells (Figure 6a, red line) in a gelatin (type B)-coated polystyrene culture dish. After 1 h of standing in an incubator, the majority of the cells without the membrane adhered to the dish bottom. Meanwhile, the cells coated with the hydrogel membrane continued to float in the medium (Figure 6a, black line). The Fluor-Alg-Ph membrane of the coated cells disappeared after 10 min of treatment with 0.2 mg/mL alginate lyase. In fact, only the fluorescence peak from noncoated cells was detected (Figure 6a, blue line). HRP-catalyzed hydrogelation has been reported as a cell-friendly route for enclosing cells in hydrogels.8−13 However, this is the first attempt at applying the system to enclosing individual cells, and thus, we evaluated the viability of the coated cells purified by the separation method on the basis of the hindrance of adhesion by

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CONCLUSION In this study, we have presented the novel method for identifying cells secreting H2O2 at the single-cell level. This was realized by applying HRP-catalyzed reaction consuming H2O2. H2O2 secreted from cells was immediately consumed by the enzymatic reaction in the solution containing a polymer possessing Ph moieties and resulted in hydrogel membrane formation on the surface of the individual cells. The sensitivity of detection of H2O2-secreting cells could be controlled by the concentrations of the polymer or HRP or the soaking time in the polymeric solution containing HRP. Moreover, the method was cytocompatible, and the hydrogel membrane could be obtained from a variety of polymers possessing Ph moieties. The hydrogel membranes obtained from AP-Ph and Fluor-Alg-Ph were removable on demand simply by being soaked in media E



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Figure 6. (a) Fluorescence signal from the hydrogel membrane of cells before (red line) and after (black line) treatment to separate coated cells and after degradation of the hydrogel membrane coating on the surface of the separated cells with alginate lyase (blue line). (b) Growth profiles of separated cells after removal of the hydrogel membranes with alginate lyase (coated cells) and cells experiencing the normal subculture process (control). The data were standardized against the values at 4 h. Vertical bars: standard deviation (n = 4). (c) Micrographs of coated cells and noncoated cells separated on the basis of hindrance of adhesion by the hydrogel membrane and control cells. The coated cells were treated with alginate lyase and then reseeded on culture dishes. Scale bars = 200 μm. (5) Oshikawa, J.; Urao, N.; Kim, H. W.; Kaplan, N.; Razvi, M.; McKinney, R.; Poole, L. B.; Fukai, T.; Ushio-Fukai, M. PLoS One 2010, 5, e10189. (6) Ruch, W.; Cooper, P. H.; Baggiolini, M. J. Immunol. Methods 1983, 63, 347−357. (7) Zhou, M.; Diwu, Z.; Panchuk-Voloshina, N.; Haugland, R. P. Anal. Biochem. 1997, 253, 162−168. (8) Wang, L. S.; Lee, F.; Lim, J.; Du, C.; Wan, A. C.; Lee, S. S.; Kurisawa, M. Acta Biomater. 2014, 10, 2539−2550. (9) Tran, N. Q.; Joung, Y. K.; Lih, E.; Park, K. M.; Park, K. D. Biomacromolecules 2010, 11, 617−625. (10) Jin, R.; Moreira Teixeira, L. S.; Dijkstra, P. J.; Zhong, Z.; van Blitterswijk, C. A.; Karperien, M.; Feijen, J. Tissue Eng., Part A 2010, 16, 2429−2440. (11) Sakai, S.; Kawakami, K. J. Biomed. Mater. Res., Part A 2008, 85, 345−351. (12) Sakai, S.; Hirose, K.; Taguchi, K.; Ogushi, Y.; Kawakami, K. Biomaterials 2009, 30, 3371−3377. (13) Sakai, S.; Liu, Y.; Matsuyama, T.; Kawakami, K.; Taya, M. J. Mater. Chem. 2012, 22, 1944−1949. (14) Liu, Y.; Sakai, S.; Taya, M. Acta Biomater. 2013, 9, 6616−6623. (15) Matsumoto, T.; Kano, K.; Kondo, D.; Fukuda, N.; Iribe, Y.; Tanaka, N.; Matsubara, Y.; Sakuma, T.; Satomi, A.; Otaki, M.; Ryu, J.; Mugishima, H. J. Cell Physiol. 2008, 215, 210−222. (16) Maeda, H.; Fukuyasu, Y.; Yoshida, S.; Fukuda, M.; Saeki, K.; Matsuno, H.; Yamauchi, Y.; Yoshida, K.; Hirata, K.; Miyamoto, K. Angew. Chem., Int. Ed. 2004, 43, 2389−2391. (17) Kato, K.; Itoh, C.; Yasukouchi, T.; Nagamune, T. Biotechnol. Prog. 2004, 20, 897−904. (18) Sakai, S.; Taya, M. ACS Macro Lett. 2014, 3, 972−975. (19) Lugovtsev, V. Y.; Melnyk, D.; Weir, J. P. PLoS One 2013, 8, e75014. (20) Qi, W.; Zhao, C.; Zhao, L.; Liu, N.; Li, X.; Yu, W.; Wei, L. Cancer Cell Int. 2014, 14, 3. (21) Carrière, A.; Ebrahimian, T. G.; Dehez, S.; Augé, N.; Joffre, C.; André, M.; Arnal, S.; Duriez, M.; Barreau, C.; Arnaud, E.; Fernandez, Y.; Planat-Benard, V.; Lévy, B.; Pénicaud, L.; Silvestre, J.-S.; Casteilla, L. Arterioscler., Thromb., Vasc. Biol. 2009, 29, 1093−1099.

containing serum (normally containing amylase) and alginate lyase, respectively. Considering these promising results and the possibility of applying other materials as the membrane, we anticipate that this method would greatly contribute to research related to cellular ROS.

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ASSOCIATED CONTENT

S Supporting Information *

Additional figures as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: (+81) 6-68506252. Fax: (+81) 6-6850-6254. *E-mail: [email protected]. Phone: (+81) 6-68506251. Fax: (+81) 6-6850-6254. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study has no applicable funding. REFERENCES

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Identification of Hydrogen Peroxide-Secreting Cells by Cytocompatible

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