Noninvasive Measurement of Alkaline Phosphatase Activity in

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Noninvasive Measurement of Alkaline Phosphatase Activity in Embryoid Bodies and Coculture Spheroids with Scanning Electrochemical Microscopy Toshiharu Arai,† Taku Nishijo,† Yoshiharu Matsumae,† Yuanshu Zhou,† Kosuke Ino,† Hitoshi Shiku,†,* and Tomokazu Matsue*,†,‡ †

Graduate School of Environmental Studies, Tohoku University, Sendai 980-8579, Japan WPI-Advanced Institute for Materials Research, Tohoku University, 2-1-1 Katahira, Aoba, Sendai 980-8577, Japan

‡

S Supporting Information *

ABSTRACT: Alkaline phosphatase (ALP) is an enzyme commonly used as an undifferentiated marker of embryonic stem cells (ESCs). Although noninvasive ALP detection has long been desired for stem cell research and in cell transplantation therapy, little progress has been made in developing such techniques. In this study, we propose a noninvasive evaluation method for detecting ALP activity in mouse embryoid bodies (mEBs) using scanning electrochemical microscopy (SECM). SECM has several advantages, including being noninvasive, nonlabeled, quantitative, and highly sensitive. First, we found that SECM-based ALP evaluation permits the comparison of ALP activity among mEBs of different sizes by monitoring the p-aminophenol (PAP) production rate in aqueous solution containing p-aminophenylphosphate (PAPP) normal to the surface area of each sample. Second, coculture spheroids, consisting of mEB and MCF-7 cells for the core and the concentric outer layer, respectively, were prepared as model samples showing heterogeneous ALP activities. The overall PAP production rate dramatically declined in the presence of the MCF-7 cell outer layer, which blocked the mass transfer of PAPP to inner mEB. This result indicated that the SECM response mainly originated from ALP located at the surface of the cellular aggregate, including mEBs and coculture spheroids. Third, taking advantage of the noninvasive nature of SECM, we examined the relevance of ALP activity and cardiomyocyte differentiation. Collectively, these results suggested that noninvasive SECM-based ALP activity normalized by the sample surface enables the selection of EBs with a higher potential to differentiate into cardiomyocytes, which can contribute toward various types of stem cell research.

A

enable the reuse of cellular samples whose ALP activity was precisely measured, is highly desired in order to detect the presence of accidental undifferentiated cells causing tumor formation within pluripotent cell-derived somatic tissues in transplantation medicine. The research target of the present study was the mouse embryoid body (mEB), a three-dimensional aggregate of ESCs. By forming EBs, various types of somatic cells were derived in vitro for the investigation of the cell differentiation potentials. Various factors are involved in determining the fate of ESCs differentiation,9 including the two- or three-dimensional culture, the size of EBs, soluble factors, the extracellular matrix, and the oxygen concentration, among others. In this study, we evaluated the ALP activity of individual mEBs that formed under various experimental conditions, including variations in the mEB size and cultivation period.

lkaline phosphatase (ALP) is an enzyme that hydrolyzes a phosphate ester compound under alkaline conditions. ALP has been widely used as a biomarker of bone or liver diseases, labeling for staining, ELISA, electrochemical immunoassays,1−3 and an undifferentiated marker since it is expressed at high levels in various types of stem cells, such as embryonic germ cells,4 embryonic stem cells (ESCs),5 and induced pluripotent stem cells (iPSCs).6 Recently, the measurement of ALP activity has been used not only to investigate the undifferentiated state of cultured ESCs and iPSCs but also to confirm the acquisition of the undifferentiated state under practical situations for regenerative medicine, including the initial screening stage of successful reprogramming to iPSCs7 and in studies of cancer stem cells.8 However, in general, ALP activity is evaluated under invasive methods such as dye staining or absorbance detection using appropriate substrates. Because of the invasiveness of the methods, it is currently difficult to reuse the same samples used for ALP assays in subsequent experiments or recultivation procedures. Therefore, development of noninvasive ALP detection procedures, which © 2013 American Chemical Society

Received: June 20, 2013 Accepted: September 22, 2013 Published: September 23, 2013 9647

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Cell Tracker Green CMFDA (Invitrogen, CA) and Cell Tracker Orange CMRA (Invitrogen), respectively, for fluorescence detection. After staining, monoculture mEB and MCF-7 cell spheroids were independently formed using the hanging drop method in each medium for 1 or 3 days. Day 1 or day 3 fusion was defined as the condition under which monoculture spheroids with day 1 or day 3 MCF-7 cells and ESCs were transferred within a single droplet. To establish coculture spheroids with concentric layers, ESC (initial concentrations of 250 cells/20 μL drop) and MCF-7 cell (initial concentrations of 250, 500, 1000, or 2000 cells/20 μL drop) monoculture spheroids were prepared. Subsequently, each monoculture day 1 spheroid was pipetted and transferred to the 20 μL droplet of the ESC differentiation medium. Three days later (day 4 overall), coculture spheroids were formed, showing four patterns of MCF-7 cell layers, and were used for ALP activity measurement. Day 1 fusion was determined when the ESCs were completely covered with a layer of MCF-7 cells. Day 3 spheroids were used to establish Janus spheroids. Day 3 fusion was determined when neither spheroid was completely covered. ESC (initial concentrations of 250 cells/20 μL drop) and MCF-7 cell (initial concentrations of 2000 cells/20 μL drop) monoculture spheroids were fused on day 3 in the same manner as described above. The next day (day 4 overall), the two spheroids aggregated together to form a single Janus spheroid with a small area of ESCs and a large area of MCF-7 cells, which were used for ALP activity imaging. Alkaline Phosphatase Activity Measurement. Absorbance Detection and ALP Staining. To detect ALP activity of whole cells that constituted mEBs, we performed absorbance detection after mEBs were dissociated by soaking and pipetting in Accutase solution (Millipore). In an optical conventional method for ALP detection, 4.0 mM p-nitrophenyl phosphate (PNPP) was used as a substrate, and the enzymatic product, pnitrophenol (PNP), was monitored by measuring the rate of increase in absorbance at 405 nm by using a microplate reader (model 680, Bio-Rad Laboratories, Inc., CA). ALP staining was conducted using a StemTAG alkaline phosphatase staining kit (Cell Biolabs, Inc., San Diego, CA). Stained cells were observed under an optical microscope (Olympus IX-71). Further detail was written in the Supporting Information. Estimation of the PAP Production Rate from Single mEB Using SECM. A mEB was transferred into a cell composed of six cone-shaped microwells with a radius and depth of 2.0 mm each (Research Institute for the Functional Peptides, Yamagata, Japan). Electrochemical ALP activity was detected in a HEPESbased saline solution (10 mM HEPES, 150 mM NaCl, 4.2 mM KCl, 2.7 mM MgCl2, 1.0 mM NaHPO4, and 11.2 mM glucose; pH 9.5) containing 4.7 mM PAPP. PAP oxidation currents were monitored at +0.3 V versus Ag/AgCl using a 10 μm radius platinum disk microelectrode probe, and it was set manually at the closest side of the mEB under a microscopic viewing. The electrode probe was scanned vertically from the side of the sample up to 320 μm automatically, and this movement was repeated up and down 6 times using a commercial SECM system containing an XYZ stage and potentiostat (HV405; Hokuto Denko). The PAP production rate (FPAP (mol/s)) was obtained by multiplying the difference in PAP concentration between the bulk and surface of the spheroid (ΔCPAP (mol/ cm3)) and sample radius (rs (cm)) according to the spherical diffusion equation:11,18

Here, we describe a noninvasive protocol for the measurement of ALP activity in mEBs by using scanning electrochemical microscopy (SECM). SECM, a scanning probe microscopy (SPM) technique, is commonly used to measure local electrochemical behavior and to capture images of redox species. Since SECM allows highly quantitative and noninvasive measurements of various cellular functions, this tool has been widely applied in evaluations of various mammalian embryos or cellular aggregates.10−14 With respect to the electrochemical detection of ALP activity, p-aminophenylphosphate (PAPP) is generally used as a substrate. The enzymatic activity of ALP catalytically hydrolyzes PAPP, yielding p-aminophenol (PAP). Therefore, ALP activity can be evaluated by monitoring the PAP oxidation current at a suitable tip potential where PAPP is not oxidized.15 However, kinetics of the localized enzymes in three-dimensional samples will become complicated due to several causes such as heterogeneous location of enzymes and/ or mass transport hiding of enzymatic substrate and product.16 Therefore, analysis from the viewpoint of mass transfer should contribute to precisely understanding the origin of the electrochemical responses. In the present study, an SECM-based ALP evaluation was carried out to monitor the decrease in ALP activity during the mEB culture process under a wide range of EB sizes and cultivation periods. Moreover, coculture spheroids, containing ESCs and human breast cancer cells (MCF-7 cells, which show very low ALP activity), were prepared as a model cellular aggregate with partially different ALP activities. First, coculture spheroids of ESCs and MCF-7 cells were prepared for the core and the concentric outer layer, respectively. By using these biological samples, we demonstrated experiments relevant for mass transfer. Electrochemical responses of ALP from inside and at the surface of ESCs were evaluated in detail by changing the thickness of the outer layer of MCF-7 cells. Second, Janus spheroids were prepared as a model sample for SECM imaging of heterogeneous ALP activity. Janus spheroids are spheroids composed of two groups of cells that are nonconcentrically juxtaposed to one another, so that each group of cells essentially forms a hemisphere.17 We demonstrated Janus spheroid patterning of MCF-7 cells with ESCs and continuous imaging of respiratory activity and enzyme activity for the identical Janus spheroid. We further examined the relevance of ALP activity and cardiomyocyte differentiation to demonstrate the significance and noninvasiveness of the SECM technique. The results suggest that noninvasive SECM-based ALP evaluation enables suitable selection of EBs with a high potential to differentiate into cardiomyocytes, which can contribute toward various types of stem cell research.

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MATERIALS AND METHODS Cell Culture, EB Formation, and Differentiation. The mouse ES cells (strain 129/SVE, purchased from DS Pharma Biomedical Co., Ltd.) were cultured and mEBs were formed by the hanging drop method (200−10 000 cells/20 μL) as described in the Supporting Information. To evaluate their differentiation potential toward cardiomyocytes, the mEBs collected were transferred onto a gelatin-coated 24-well plate and further cultured in a differentiation medium; thereafter, morphological observations were performed to judge whether the cell aggregates were beating or not beating. Formation of Coculture Spheroids. Before formation of coculture spheroids, ESCs and MCF-7 cells were stained with 9648

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Figure 1. (A) Total ALP activity (absorbance at 405 nm) of dispersed mEBs that were formed using 200−10 000 cells per drop on days 2, 5, and 8. (B) ALP activity (absorbance at 405 nm) per volume of individual mEBs. (C) ALP activity (absorbance at 405 nm) versus radius of mEBs. (A−C) show the same data from different perspectives.

Figure 2. (A) ALP dye staining of individual mEBs that were formed using 200−5000 cells per drop on days 2, 5, and 8. Scale bars: 200 μm. (B) ALP activity (FPAP) obtained by SECM vs mEB radius on days 2, 5, and 8. Initial cell numbers of 200−5000 are shown. (C) The data of (A) are organized with respect to each initial cell number. (D) ALP activity (FPAP) per surface area of individual mEBs. (B−D) show the same data from different perspectives.

⎛ 1 ⎞ ⎟(1 + FPAP = D × 2π ⎜1 − ⎝ 2⎠

2 )rsΔC PAP

probe was accomplished using a motor-driven XYZ stage (K701−20RMS; Suruga Seiki) and a stage controller (D70; Suruga Seiki). The tip scan rate was 10 μm/s, the pixel resolution was 20 × 20 μm, and the scan area was 700 × 700 μm. A single image was acquired in 42 min. The distance between the electrode tip and the top of the cellular sample was set at approximately 20 μm, based on the approach curve to the bottom of the culture dish. From the position that negative feedback was observed, the tip was raised 470 and 20 μm for imaging of the Janus spheroid and the outgrowth region, respectively (see Figure 5A). We first detected respiration activity (oxygen reduction) at −0.5 V versus Ag/AgCl, and we sequentially detected ALP activity (PAP oxidization) at +0.3V versus Ag/AgCl. We captured an optical micrograph of the SECM imaging area, including the SECM tip located at the

(1)

The diffusion coefficient (D) of PAP was 7.1 × 10−6 cm2/s .18 The mean radius (rs) of the individual mEB was determined according to the equation: rs = 1/2(ab)1/2, where a and b represent the two orthogonal diameters of the mEB.19 Further details on the estimation of the PAP production rate was written in Supporting Information. SECM Imaging of Respiration Activity and ALP Activity. For constant height mode SECM imaging, a different instrument was used from that used for the FPAP estimation . Details of the SECM imaging system were reported previously.20,21 The current was amplified with a current amplifier (428, Keithley). Movement of the microelectrode 9649

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For the electrochemical evaluation, we first determined the PAP oxidation current value at the surface of mEBs on day 2 (not scanning). The tip was located at a distance of 30 μm from the sample surface (Supporting Information, Figure S1). Next, we executed tip scanning and obtained the PAP production rate of individual mEBs (FPAP) by SECM measurement as a function of the mEB radius (Figure 2B). SECM measurements were performed on days 2, 5, and 8. The FPAP value was obtained as a function of rs and ΔCPAP from eq 1. The procedure for estimating ΔCPAP from the PAP oxidation current profile of SECM was conducted as previously described.18 For mEB samples with rs values ranging from 50 to 180 μm, the FPAP value was largest on day 2 and decreased proportionally to the cultivation period. Therefore, the differentiation status of individual mEBs of identical sample size could be easily compared on the basis of the FPAP value. For instance, our group developed electrode array devices for predicting the differentiation potential of individual EBs using their electrochemical responses.22−25 However, because the FPAP was affected by several parameters, including the number of cells in the mEB and the ALP activity per cell, comparison of the ALP activity in mEBs of different sizes was complicated. Additionally, the results strongly suggest that the PAP oxidation current monitored by SECM preferentially originated from the ALP that was localized at the surface of the mEB rather than inside the mEB because FPAP was proportional to the power of 2.24, 1.41, and 1.46 of the mEB radius for mEBs on days 2, 5, and 8, respectively (Figure 2B). These power numbers were remarkably small relative to the third power of the sample radius when the ALP activity was measured at the cell suspension levels shown in Figure 1C. Figure 2C shows the FPAP values for mEBs formed using various initial cell numbers per drop. FPAP clearly increased with increasing initial cell numbers. By contrast, in the case of mEBs formed with initial cell numbers of 250 and 500, FPAP did not decrease significantly in accordance with the cultivation period. This was due to the fact that cellular proliferation in mEBs was canceled out by the decrease in the ALP activity per cell due to differentiation. For mEBs formed with initial cell numbers ranging from 1000 to 10 00, the decrease in ALP activity of individual mEBs was sufficient. This is because the decline in ALP activity per cell was preferential to the total ALP increase due to the proliferation in mEBs. Therefore, the FPAP value obtained by SECM was found to be more sensitive to the differentiation state of individual cells than to the cellular proliferation. Consequently, FPAP obtained by SECM (Figure 2C) did not match the total ALP activity in mEBs measured by absorbance detection (Figure 1B). These results demonstrate the need to clarify the origin of FPAP values that are measured using SECM. Normalization of the SECM response by the sample surface (S = S sample = 4πrs2) allowed us to conduct a quantitative comparison of ALP activity in mEBs of different sample sizes. For comparison, we also calculated FPAP per volume (FPAP/V) and FPAP per surface area (FPAP/S) values on day 2 as a function of the mEB radius (Supporting Information, Figure S2). This figure clearly demonstrates that normalization of the SECM response by the sample surface is preferential to normalization by the sample volume, since FPAP/S values on day 2 were independent of the mEB radius, whereas FPAP/V values decreased with increasing sample size. Figure 2D shows the FPAP/S values for mEBs formed using various initial cell numbers per drop (200 to 10 000). Regardless of the initial cell number, FPAP/S decreased

initial position, to precisely overlay the SECM and optical images. In Figure 5, overlay was achieved by decreasing the opacity of the SECM image layer and superimposing this with the optical micrograph using graphical software (Paint.NET).

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RESULTS AND DISCUSSION ALP Activity of Cellular Suspension Prepared from mEBs by Absorbance Detection. Figure 1A shows the total ALP activity in individual mEBs that were formed using various initial cell numbers per drop. Absorbance measurement was carried out for cell suspensions that were reprepared from mEBs on days 2, 5, and 8. For all mEBs formed with initial cell numbers of 200−10 000, the total ALP activity per mEB increased in proportion to the cultivation period due to the proliferation of individual mEBs. Figure 1B shows the ALP activity per volume for mEBs. For all mEBs formed with initial cell numbers, the ALP activity per cell decreased in proportion to the cultivation period due to the progress of the differentiation state of the ESCs at individual cell levels. Importantly, on day 2, the ALP activity per cell was found to be independent of the initial cell number, which ranged from 200 to 10 000, and was sustained at identical levels. On day 5, the ALP activity per cell was sustained at a lower level relative to that on day 2 and was found to be independent of the initial cell number. On day 8, the ALP activity per cell was reduced further and fluctuated, depending on the initial cell number. Figure 1C shows the total ALP activity in individual mEBs under various initial cell numbers per drop as a function of the mEB radius. This plot shows that the total ALP activity was linear to the third power of the mEB radius, even though mEBs that formed with different initial concentrations were plotted. A theoretical curve was obtained using the least-squares method at the power range, which indicated that the ALP activity was linear to the power of 2.92, 2.86, and 3.04 of the mEB radius for mEBs on days 2, 5, and 8, respectively. The variance in ALP activity increased over time, resulting in a decrease in coefficient of determination values (R2) (0.9968 on day 2, 0.6945 on day 5, and 0.5692 on day 8). The longer cultivation period likely enhanced variation in the differentiation state of the cell in mEBs. As described above, results obtained by the ALP assay of the cell suspension through absorbance detection indicated that it was impossible to detect heterogeneity in the differentiation state of the ESCs within the mEB. Additionally, mEBs used for the ALP assay could neither be reused for further cultivation nor be used for different experiments. ALP Activity of mEBs Measured by SECM. Before conducting the SECM-based ALP evaluation, we also performed conventional ALP staining to confirm the distribution of undifferentiated cells at the surface of the mEBs that formed under various experimental conditions. Regardless of the initial cell number used to form mEBs, ALP activity gradually decreased in proportion to the cultivation period, which strongly suggested the progress of differentiation within and at the surface of mEBs (Figure 2A). However, because the staining reaction was affected by several parameters, including the permeation of the enzymatic substrate and/or dye product, types and concentration of surfactant in the solution, and the incubation time of staining, it was difficult to compare the ALP amount at the surface to that inside the mEB from the amount of stained dyes alone. Cellular proliferation and the decrease in ALP activity were confirmed to have occurred simultaneously. 9650

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Figure 3. (A) Fluorescent and optical images of concentric layer patterning of coculture spheroids on days 1, 2, and 4. ESC (green) and MCF-7 (orange) spheroids were fused on day 1. Scale bars: 200 μm. (B) Optical image of mEBs and ESCs (inner)MCF-7 cells (outer) coculture spheroids on day 4. Four conditions (initial cell numbers of 250, 500, 1000, and 2000) of MCF-7 cell spheroids fused with mEBs (initial cell number is 250 cells only) on day 1. Scale bars: 200 μm. (C) ALP activity (FPAP) of mEBs and coculture spheroids obtained by SECM. (D) Normalized by the total ALP activity of the inner sphere of mEBs (FPAP/absorbance at 405 nm) relative to the value of mEBs only.

Figure 4. (A) ALP activity (FPAP) obtained by SECM vs radius of mEBs on day 8, in which spontaneous beating was either observed (circle) or was not observed (cross) on day 13. (B) Normalized by the surface area of the data contained in (A). (C) Average radius of mEBs on day 8, in which spontaneous beating was observed (n = 11) or was not observed (n = 19) on day 13. (D) Average ALP activity per surface area (FPAP/S) of mEBs on day 8, in which spontaneous beating was observed (n = 11) or was not observed (n = 19) on day 13. P < 0.01, Student’s t-test.

proportionally to the cultivation period. On day 2, FPAP/S values were essentially constant and were independent of the

initial cell number used to prepare mEBs. On day 5, FPAP/S values decreased relative to those on day 2 but were sustained 9651

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Figure 5. (A) Schematic illustration of SECM imaging, coculture spheroid (left, B−D), and outgrowth area (right, E−G). (B) Fluorescent and optical images of Janus spheroids; coculture spheroids fused on day 3 (250 ES−2000 MCF‑7). ESC (green) and MCF-7 cell (orange) spheroids made contact on day 3. (C and F) Respiration activity imaging (left) at −0.5 V vs Ag/AgCl and merged with (B or E) (right). (D and G) ALP activity imaging at +0.3 V vs Ag/AgCl and merged with (B or E) (right). (E) Imaging area of outgrowth cells from coculture spheroid. Scale bars: 200 μm.

spheroids on day 4. The mEB-MCF7 coculture spheroids were prepared using MCF-7 cells of different sizes (with initial cell numbers of 250−2000 MCF-7 cells for M250−M2000). Figure 3C shows FPAP measured by SECM for mEBs and mEB-MCF7 coculture spheroids. The detected FPAP value was markedly diminished with increasing numbers of MCF-7 cells; the MCF7 cell layer hindered the mass transport of PAPP and thus decreased the SECM signal. With respect to mEB-MCF-7 coculture spheroids, oxidation currents detected by SECM were almost completely blocked by MCF-7 cells regardless of the thickness of the MCF-7 cells layer. However, the results also strongly suggest that the permeability of the PAP through the cellular aggregate was not zero because the PAP oxidation current could still be detected in the mEB-MCF-7 coculture, but it could not be detected in the spheroid formed with MCF7 cells alone. Next, the FPAP measured by SECM was normalized by the total ALP activity of the inner sphere of the mEB (Figure 3D). For this purpose, mEB-MCF-7 coculture spheroids were resuspended for an ALP assay on the basis of absorbance

at the same level, independent of the initial cell numbers. On day 8, FPAP/S values were further reduced from those on day 5, and fluctuations in FPAP/S values were enhanced. To summarize the results of this section, ALP activities measured using SECM should be compared by FPAP/S values in order to disentangle the cellular proliferation from the ALP activity at the cellular level. This analytical procedure may allow for effective comparisons of ALP activity in mEBs of different sizes. One must carefully keep in mind that although the SECM response was sensitive for ALP activity at the surface of the mEBs, it did not reflect the total ALP activity in mEBs. ALP Activity Evaluation of Coculture Spheroids by Combining SECM and Absorbance Detection. Figure 3A shows the sequential spheroid fusion process of the mEBMCF7 concentric layer patterning of coculture spheroids on days 1, 2, and 4. We found that when fused on day 1, MCF-7 cells and mEBs always reached into the outer and inside layers, respectively, regardless of the initial cell number of MCF-7 cells and mEBs (Supporting Information, Figure S3). Figure 3B shows optical micrographs of mEB and mEB-MCF7 coculture 9652

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dramatically shifted to the ESC region (Figure 5D). Consequently, we achieved sequential imaging of respiratory and ALP activities in the same sample, which identified the region where ESCs existed selectively in the model sample of the coculture spheroid. Moreover, the coculture spheroid sample was transferred onto a gelatin-coated dish and was further cultured for 2 days. We performed SECM imaging of the outgrowth area of the coculture spheroid (Figure 5E). Morphologically, it was difficult to discriminate ESCs from MCF-7 cells. The SECM image of respiration activity allowed visualization of the region with a lower reduction current, approximately −1.25 nA, which matched precisely to the region where cells were crowded, although there was no distinguishable difference in current values between ESCs and MCF-7 cells. However, the SECM image of ALP activity clarified the region where ESCs existed as the area of the higher PAP oxidation current (>0.7 nA) (Figure 5F,G). Thus, SECM has the potential to discriminate undifferentiated ESCs in the coculture system by using noninvasive and nonlabeled techniques. For more detailed observations and discussion26,27 concerning the negative feedback effect in constant height mode, cross sections are shown in Supporting Information, Figure S5. In summary, using coculture spheroids of ESCs and MCF-7 cells as model samples with heterogeneous ALP activities, we confirmed that the electrochemical response of the ALP of the cellular aggregate mostly originated from the surface reaction. Coculture systems have been used in various studies of differentiation induction,28 cancer cell invasion,29 and biophysical models,30 and these systems also have conceivable applications for mass transfer analysis. Furthermore, we performed SECM imaging of respiration and ALP activity to Janus spheroids and achieved discrimination between undifferentiated and differentiated ESCs in the coculture system by using noninvasive and nonlabeled methods. Therefore, these techniques should prove helpful for initial screenings of undifferentiated cells.

detection after the SECM measurement. The ALP activities in mEB-MCF-7 coculture spheroids were found to be constant and were approximately 0.17 times that of the ALP activity in uncovered EB, regardless of the thickness of the outer layer of MCF-7 cells. With respect to these results, we conclude that regardless of the presence or absence of enzymatic activity in the interior of the cellular aggregate, nearly 80% of the detected FPAP value originated from the surface reaction. Relevance of ALP Activity and Cardiomyocyte Differentiation. FPAP was noninvasively measured on day 8, and mEBs were further cultured on gelatin-coated dishes to promote differentiation of cardiomyocytes, which were then observed until day 13. As SECM measurement allows the recultivation of measured samples, we examined the relevance of ALP activity and differentiation into cardiomyocytes after measuring ALP activity. mEBs that were formed using various initial cell concentrations (500, 1000, 2000, and 5000 cells/20 μL drop) were prepared. A colony was judged to be beating or not beating under optical microscopy observation. Figure 4A,B show the plots of FPAP versus the EB radius (rs) and FPAP/S versus rs, respectively. FPAP was measured on day 8. Circles in the plot indicate the mEB samples for which beating behavior was observed by day 13, whereas crosses indicate mEBs that were not beating. Statistical analysis was performed (Student’s t-test) to compare means between the beating and no-beating groups. There was no difference in the radius (P = 0.977), but there was a significant difference in FPAP/S on day 8 (P = 0.0045) between beating and no-beating mEBs, at least in the radius range of 150−300 μm (Figure 4C,D). For comparison, we also calculated ΔCPAP, FPAP, and FPAP/V values of the beating and no-beating groups, as shown in Supporting Information, Figure S4. This figure shows that normalization by the sample surface allowed for the most discrimination of the beating samples relative to other parameters because the SECM measurement detected the ALP near the mEB surface. From these results, we determined the relevance of ALP activity and differentiation into cardiomyocytes, which suggests that noninvasive measurement of ALP activity enables the selection of EBs with high potential to differentiate into cardiomyocytes. SECM Imaging of Heterogeneous ALP Activities in the Coculture System. In the experiment described above, the activity per mEB was quantified from the concentration gradient of the PAP by scanning in the Z-direction. Subsequently, we performed the imaging of ALP activity in the coculture spheroid by scanning in the XY-direction (Figure 5A). One of the advantages of electrochemical detection is that different substances can be visualized noninvasively for the same samples by simply changing the tip electrode potential. We first detected respiration activity (oxygen reduction current) at −0.5 V versus Ag/AgCl, and we then detected ALP activity (PAP oxidization current) at +0.3 V versus Ag/ AgCl for the identical coculture spheroid. The target sample was the Janus spheroid and coculture spheroids fused on day 3 (250 ES−2000 MCF‑7). When fused on day 3 or later, MCF-7 cells did not cover ESCs completely (Supporting Information, Figure S3B). We prepared a Janus spheroid, which had a small area of ESCs (green) and a large area of MCF-7 cells (orange) (Figure 5B). The SECM image of respiration activity showed a minimum oxygen reduction current at the vertex of the coculture spheroid (Figure 5C) that had a concentric oxygen gradient. In contrast, as observed in the SECM image, the ALP activity that appeared at the position of the highest PAP oxidation current

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CONCLUSION Here, we described an SECM-based ALP activity evaluation of mEBs. Using this system, we were able to compare ALP activity in mEBs of different sizes that were normalized by the surface area of the sample and to monitor the decrease in ALP activity during the cultivation period. Additionally, we showed that mEBs exhibiting low ALP activity on day 8 could differentiate well into cardiomyocytes, suggesting that noninvasive measurement of ALP activity enables effective selection of EBs with a high potential to differentiate into cardiomyocytes. In this study, we used ALP activity as only one indicator; however, a multiparameter analysis that integrates other information, for example, morphological observations, respiration activity (quality of cells), and dopamine (selecting more differentiated to neural cells), would enable more accurate sorting of homogeneous cellular groups. Moreover, coculture spheroids of ESCs and MCF-7 cells were used as model samples with heterogeneous ALP activities, which confirmed that the SECM response of the ALP were almost completely blocked by outer MCF-7 cells layer. In other words, due to the hindering of mass transfer, the electrochemical response mostly originated from the surface reaction. Furthermore, we performed SECM imaging of respiration and ALP activities in Janus spheroids, and we achieved discrimination of undifferentiated ESCs in the coculture system by using noninvasive and nonlabeled 9653

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Analytical Chemistry

Article

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methods. These techniques should prove helpful for initial screenings of undifferentiated cells. Thus, the SECM system described herein, which takes advantage of nonlabel and noninvasive techniques, is expected to contribute to various types of stem cell research in the future.

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ASSOCIATED CONTENT

* Supporting Information S

Additional information as noted in the 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] (H.S.). *E-mail: [email protected] (T.M.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was partly supported by the Cabinet Office, Government of Japan, through its “Funding Program for the Next Generation of World-Leading Researchers” (to H.S.) and by a Grant-in-Aid for Scientific Research (A) (No. 25248032) from the Japan Society for the Promotion of Science (JSPS).

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REFERENCES

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dx.doi.org/10.1021/ac401824q | Anal. Chem. 2013, 85, 9647−9654