Microbioassay System for Antiallergic Drug Screening Using

Apr 5, 2005 - This article describes an antiallergic drug-screening system by the detection of histamine released from mast cells (suspension cells) o...
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Anal. Chem. 2005, 77, 3309-3314

Microbioassay System for Antiallergic Drug Screening Using Suspension Cells Retaining in a Poly(dimethylsiloxane) Microfluidic Device Takahito Tokuyama, Shin-ichiro Fujii, Kiichi Sato, Mitsuru Abo, and Akira Okubo*

Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi, Bunkyo, Tokyo 113-8657, Japan

This article describes an antiallergic drug-screening system by the detection of histamine released from mast cells (suspension cells) on a multilayer microchip. In this study, the elastmeric material, poly(dimethylsiloxane) (PDMS), was employed to fabricate microchannels and microchambers. The microchip consists of two sections: a histamine-releasing one, which has a cell chamber, and a histamine-derivatizing one. Both were laminated to one microchip. Rat peritoneal mast cells were retained in the cell chamber (1.2 µL) with a filtering system using a cellulose nitrate membrane. This filtering system could easily retain suspension cells without cell damage. Mast cells were viable for a sufficient time to conduct the assay on the cell chamber. The cells were stimulated with a chemical release compound 48/80 (C48/80), and then histamine flowed into the lower layer, where it was derivatized to the fluorescent molecules with o-phthalaldehyde and its fluorescence was detected on the microchip. This flow system could detect the time course of the histamine release, and this microchip system required only 20 min for the assay. By this integrated system, 51 pmol of histamine released from 500 cells was detected, and the number of cells required for the assay was reduced to 1% compared with conventional bulk systems. By comparing the released histamine levels with and without drugs, their effect could be evaluated. The inhibition ratio of C48/80 induced-histamine release using an antiallergic drug, disodium cromoglicate (DSCG), was related to the concentration of DSCG. This flow system was applicable for antiallergy drug screening by rapid measurement of the inhibition of histamine release from a very small amount of mast cells. Cellular analysis using living cells, which enables researchers to perform a complex analysis of life, is one of the most important methods in the biological fields. For example, a bioassay, an experiment that uses living things to test the effect of the chemicals, is an indispensable technique for drug screening, a safety evaluation of chemicals, and other basic life science research. The conventional bioassay, however, involves troublesome handling procedures such as pipetting and requires many valuable cells and reagents. In recent years, micro total analysis * To whom correspondence should be [email protected]. Fax: +81-3-5841-8027. 10.1021/ac048288o CCC: $30.25 Published on Web 04/05/2005

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© 2005 American Chemical Society

systems (µTAS) have been of great interest to biological researchers for cellular analysis. A prominent characteristic of µTAS is the capability of constructing highly functional systems on a microchip. Therefore, many processes that were complicated in conventional cellular analysis could be integrated on a microchip. This integration resulted in short-time analysis and easy handling for operation. Moreover, integrated systems had advantages such as a reduction in the consumption of cells, reagents, and samples, real-time analysis, and constancy of experimental conditions.1 As stated above, cellular analysis on a integrated microchip provides numerous benefits. Thus, cellular analysis on the microchip has been rapidly spreading, for example, to applications of cell sorting,2,3 and the introduction of genes into cells.4,5 However, there have been few papers about the integration of all processes of a bioassay using living cells on a microchip.6,7 Mammalian cells used in bioassays can be roughly classified into adherent cells and suspension cells. In most reports of cellular analysis on microchips, adherent cells have been employed, since the cells could adhere onto the channel by their own adhesive strength. Suspension cells such as hemocytes, a kind of cancer cell, and primary cells have been employed in microchips by immobilizing the cells by artificial adherence8 or allowing the cells to flow through channels.9,10 In the artificial adherence system, however, the nature of the cells may be changed, and a long-time assay cannot be realized in the flow system. An efficient and suitable retaining technique for suspension cells on the microchip has not yet been reported. Therefore, in this study, a nonadhesion method to retain cells on a microchip is proposed, and a newly developed cellular analysis system combined with a fluorescent (1) Park, T. H.; Shuler, M. L. Biotechnol. Prog. 2003, 19, 243-253. (2) Fu, A. Y.; Chou H. P.; Spence, C.; Arnold, F. H.; Quake, S. R. Anal. Chem. 2002, 74, 2451-2457. (3) Muller, T.; Gradl, G.; Howitz, S.; Shirley, S.; Schnelle, T.; Fuhr, G. Biosens. Bioelectron. 1999, 14, 247-256. (4) Lin, Y, C.; Li, M.; Fan, Y. S.; Wu, L. Y. Sens. Actuators, A 2003, 108, 1219. (5) Pioufle, B. L.; Surbled, P.; Nagai, H.; Murakami, Y.; Chun, K. S.; Tamiya, E.; Fujita, H. Mater. Sci. Eng. C-Biomimet. Supramol. Syst. 2000, 12, 7781. (6) Matsubara, Y.; Murakami, Y.; Kobayashi, M.; Morita, Y.; Tamiya, E. Biosens. Bioelectron. 2004, 19, 741-747. (7) Tanaka, Y.; Sato, K.; Yamato, M.; Okano, T.; Kitamori, T. Bunseki Kagaku 2004, 20, 411-413. (8) Kato, K.; Umezawa, K.; Funeriu, D. P.; Miyake, M.; Miyake, J.; Nagamune, T. BioTechniques 2003, 35, 1014-1021. (9) Schilling, E. A.; Kamholz, A. E.; Yager, P. Anal. Chem. 2002, 74, 17981804. (10) McClain, M. A.; Culbertson, C. T.; Jacobson, S. C.; Allbritton, N. L.; Sims, C. E.; Ramsey, J. M. Anal. Chem. 2002, 74, 1798-1804.

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labeling reaction and detection of released materials from cells is described. This system was applied to a bioassay system using living suspension cells, in which the cell-retaining region and the reaction and detection area were integrated. In this study, application to a screening system for antiallergic drugs was attempted, and primary mast cells, which are suspension cells, were retained in the microchip by the filtration method using a membrane filter. Mast cells are responsible for a type-I allergic reaction, represented by atopic dermatitis and pollen hypersensitivity. Stimuli applied to mast cells by allergens cause a release of mediators, such as intracellular histamine, according to the strength of the stimuli. The histamine, in turn, causes numerous allergic reactions. Thus, by examining histamine behavior in response to stimuli, the elucidation of a histamine release mechanism in mast cells, as well as of the pathogenesis of allergic diseases, may be accomplished. Moreover, the system was suitable for rapid screening of antiallergic drugs with very small consumption of cells and reagents by comparing released histamine level with and without any drugs. When using primary cells from an animal as in this study, the reduction of the number of cells using the assay is to realize a large number of times of the assay. In summary, this study reports the construction of a system on a microchip wherein mast cells collected from rat were retained and stimulated by a supposed allergen and the amount of histamine released was quantified. EXPERIMENTAL SECTION Reagents and Materials. Histamine diphosphate monohydrate was purchased from Wako Pure Chemicals Ind. (Osaka, Japan). Compound 48/80 (C48/80), o-phthalaldehyde (OPA), and disodium cromoglicate (DSCG) were obtained from Sigma Chemical Co. (Tokyo, Japan). The poly(dimethylsiloxane) (PDMS) elastomer (Sylgard 184) was obtained from Dow Corning Asia (Tokyo, Japan). The saline solution (pH 7.4) contained 145 mM NaCl, 2.7 mM KCl, 1 mM CaCl2, and 5 mM HEPES. The fluorescence-derivatizing solution contained 0.5% OPA, 1% MeOH, and 0.6 M NaOH. As a terminating solution, 2.6 M citric acid was used. Rat peritoneal mast cells were collected from the abdominal cavity of male Wistar rats (Nippon Bio-Supp. Center) weighing 250-300 g.11,12 Apparatus. On-chip fluorescence detection was conducted using a fluorescence microscope, an inverted research microscope, IX71 (Olympus), and accessories. The excitation lamp was a 100-W mercury lamp U-LH100HGAPO. A filter cube, U-MWU2, with an excitation filter (BP 425-445 nm), a dichroic mirror (DM 400 nm), a barrier filter (BA 420 nm), and a 10× objective lens (UPlan Apo ×10) were used for the detection of fluorescence-derivatized histamine. A thermal incubation box, IBMU (Olympus), was mounted on the microscope to incubate the whole system. A photonic multichannel spectral analyzer, PMA-11 (Hamamatsu Photonics), was connected to the microscope, and the fluorescence at 440 nm was measured (time of exposure 0.5 s). In an off-chip detection system, the fluorescence of derivatized histamine was measured using a 500-µL microcuvette and a (11) Akagi, M.; Matsui, N.; Mochizuki, S.; Tasaka, K. Pharmacology 2001, 63, 203-209. (12) Akagi, M.; Fukuishi, N.; Kan, T.; Sagesaka, Y. M.; Akagi, R. Biol. Pharm. Bull. 1997, 20, 565-567.

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Figure 1. Cellular analysis system consisting of a histamine release section (a) which has a cell chamber and a histamine fluorescence derivatizing section (b). (c) and (d) are connecting tubes with syringes. When the releaser solution was reached at the chamber, mast cells released histamine. Released histamine flowed to the lower layer, where it was derivatized to the fluorescent molecules with OPA. Fluorescence derivatized histamine was detected by fluorescence microscope at the downstream of the channels.

fluorometer (FP-6500, Jasco) at an excitation wavelength of 356 nm and emission wavelength of 440 nm. Off-Chip Fluorescence Derivatizing. Histamine was subjected to a highly selective fluorescence-derivatizing reaction with OPA. Off-chip fluorescence derivatization was conducted according to the method described by Shore.13 First, 100 µL of 0.4 M NaOH was added to 200 µL of saline solution containing histamine. After a few minutes of incubation at 37 °C, 100 µL of 1% OPA/10% MeOH solution was added to this solution, followed by incubation for 2 min at 37 °C, and then 2 M citric acid was added to terminate the reaction. Detection was carried out with a fluorometer (excitation wavelength 356 nm, emission wavelength 440 nm). Bioassay Microchip and Assay Scheme. A schematic image of the microchip (consisting of four layers) is shown in Figure 1. Syringes were set on a syringe pump (KD Scientific) and connected with the microchip via fused-silica capillary tubes (100µm i.d.; 200-µm o.d.). For loading of cells, the size of the connecting tube (c) was 2 mm i.d. × 3 mm o.d. × 10 mm. PDMS, by which multilayer structure could be constructed, was selected as a microchip base material to place a filter between the layers. Microchannels (300-µm width and 70-µm depth) were fabricated in a PDMS microchip using a glass template fabricated by a simple microfabrication method.14 The uppermost layer with connecting tubes was simply laid on top of the lower layers with channels. This cellular analysis system consisted of two sections. One was a histamine release reaction section, which had a microchamber for retaining cells, and the other was a histamine fluorescencederivatizing reaction section. Schematic image of the histamine release section (a) and the histamine fluorescence derivatizing section (b) are shown in Figure 1a and b, respectively. Cells introduced through the connecting tubes (c) were retained in the cell chamber. The cell retention chamber was constructed by inserting a filter (cellulose nitrate membrane, pore size 5 µm, Toyo Roshi Kaisha, Ltd.) between the second and the third layers, to hold the cells in the space above the filter. Introduced cells were retained in this space by filtering. The size of the chamber was 2 mm × 2 mm × 0.3 mm (chamber volume (13) Shore, P. A.; Burkhalter, A.; Cohen, V. H. J. Pharmacol. Exp. Ther. 1959, 127, 182-186. (14) Fujii, S.; Tokuyama, T.; Abo, M.; Okubo, A. Analyst 2004, 129, 305-308.

Figure 2. Schematic image of the retention method of cells.

∼1.2 µL), with a filter-holding area (3 mm × 3 mm) (Figure 2). Next, the stimulating substance (C48/80) flowed through the cell chamber to release histamine from mast cells in amounts according to the degree of stimuli, and the histamine was led to the lower histamine fluorescence derivatizing reaction section. A channel was fabricated for each of the sample solution, the reactive solution, and the terminating solution (citric acid). First, the sample was mixed with the reactive solution at the first junction, and the reaction time was adjusted by changing the length of the winding channel. The reaction was then terminated by adding citric acid at the second junction. After the addition of citric acid, the histamine fluorescence derivative flowed toward the fluorescence detection point downstream of the microchip. Fluorescence detection was carried out in the channel with a fluorescence microscope. Retention of Cells. Mast cells were purified to 95% using Percoll density gradient centrifugation.15 The microchip was incubated at 37 °C and then filled with saline solution. A few microliters of cell suspension was introduced into the connecting tubes (Figure 1c) using a micropipet. The tube was connected to a syringe pump via a capillary, and a buffer injection was initiated to send the cells to the chamber. To examine whether the cells were retained in this retention system without leakage, toluidine blue flowed through the line after cell introduction, and the chamber was then observed using a microscope. Furthermore, the eluate of a 10-min flow of the saline solution at 10 µL/min was collected and dyed with toluidine blue to check the cell leakage in the eluate. Viability of Cells on the Microchip. The viability of cells was determined by trypan blue cell staining. After cell introduction to the microchamber, the cells were incubated under the flow of the saline solution at 5 µL/min. The cells were recovered by deconstructing the microchip and stained with trypan blue every 20 min for 2 h. As a control, ∼1 × 106 cells were suspended in 1 mL of the saline solution, incubated at 37 °C under the test tube, and left standing. The viability of cells was calculated as described above. Fluorescence Derivatizing Reaction. The reaction of OPA and histamine is known to be pH-dependent.16 The pH and the mixing ratio of the reactive solution were, therefore, determined to allow for the highest fluorescence intensity (flow rate of the histamine solution:reactant solution:terminating solution ) 10:2: 1) (data not shown). To determine the flow rate necessary to accomplish a sufficient reaction, the channel length was fixed at 26 cm, and the flow rate (15) Nemeth, A.; Rohlich, P. Eur. J. Cell Biol. 1980, 20, 272-275. (16) Allenmark, S.; Bergstrom, S.; Enerback, L. Anal. Biochem. 1985, 144, 98103.

of the histamine solution (1 µM) was varied within the range of 1-20 µL/min. The eluted histamine fluorescence derivatives were collected. Fluorescence intensity was measured using a fluorometer after 50 µL of the elutant had been diluted to 200 µL with distilled water. Furthermore, 10 µM histamine derivative (off-chip preparation) was passed through the channel of the detection point at 1-20 µL/min, and the course of fluorescence intensity was monitored using the fluorescent microscope to decide the optimal flow rate for histamine detection. Adsorption of Histamine and Histamine Fluorescence Derivatives. To determine the percentage of adsorption of histamine and histamine fluorescence derivatives to PDMS microchips, 200 µL of 1 µM histamine solutions (in saline) was fed from a connecting tube (c); the eluted histamine solution was a fluorescence-derivatized off-chip; the rate of recovery was determined from the fluorescence intensity obtained. The histamine fluorescence-derivatizing solution (off-chip preparation) was similarly introduced into the microchip, the eluate was collected, and the rate of recovery was determined. On-Chip Detection of Released Histamine. Cells were retained on a microchip, and histamine that was released from the cells upon stimulation was detected by on-chip fluorometry. In this study, a histamine release model was employed against allergens using nonsensitized mast cells and C48/8017,18 as the stimulating agent.11,12 All solutions were sufficiently degassed before introduction into the microchip. First, all channels were filled with the saline solution. The microchip was placed on the stage of the fluorescence microscope so that it could be brought to focus on the detection point. Reagents and a syringe pump were placed in a thermal incubation box, and the temperature of the whole system was kept at 37 °C. The cell suspension was loaded into the connecting tubes (c), and cells were retained in the chamber. The feeds of the saline solution, the derivatizing solution, and the terminating solution were initiated at the same time. After 10 min of preincubation, the saline solution was substituted with a releaser solution (0.5 µg/mL C48/80 in saline). After the examination, the microchip was able to reuse by washout of the used cells. Next, a histamine release inhibition test was performed. To identify an antiallergic drug, it was necessary to evaluate the degree of inhibition of histamine release. DSCG, which is well known to have a release-inhibiting effect on C48/80, was used as a positive control for this study.19 The experimental procedures were the same as above, where C48/80 was used as the stimulating agent, except for the addition of an inhibitor, DSCG. Cells were preincubated with the DSCG saline solution (20 µg/ mL-5 mg/mL) and then stimulated with C48/80 (0.5 µg/mL) in the DSCG saline solution. The release-inhibiting rate of DSCG was determined by calculating each fluorescence intensity from charts obtained with and without an inhibitor under the cellstimulating condition and assigning them to the following formula:

inhibition (%) ) (A - B) × 100/A (17) Paton, W. D. M. Br. J. Pharmacol. 1951, 6, 499-508. (18) Ennis, M.; Pearce, F. L.; Weston, P. M. Br. J. Pharmacol. 1980, 70, 329334.

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Figure 3. Micrograph of mast cells retained inside the chamber. The black spots are mast cells stained by toluidine blue, and they did not leak from the chamber.

Figure 4. Comparison of viable rate of mast cells retained on the microchamber and microplate (bulk). Open circle, retained in microtiter plate; solid square, retained in the cell chamber.

where A is histamine release level in control cells and B is histamine release level in DSCG-treated cells. RESULTS AND DISCUSSION Retaining Cells on the Microchip. The present analysis system consisted of a histamine release section, which had a cell chamber and a histamine fluorescence derivatizing section (Figure 1). First, mast cells were retained on the microchip by filtration with a cellulose nitrate membrane (Figure 2). In Figure 3, mast cells (∼9-20 µm) dyed with toluidine blue could be seen as black spots; these cells had not leaked outside the chamber. By checking the elutant, it was confirmed that mast cells were not eluted out in the solution filtered through the membrane. These data showed that rat peritoneal mast cells, which were suspension cells, were physically retained within the chamber by the proposed method. It was then tested whether the cells were viable or not by staining with trypan blue. Figure 4 shows the viable rate of mast cells cultured off- and on-chip, i.e., cells left in a 24-well microtiter plate at 37 °C and cells retained in the microchip, respectively. In both the off-chip and the on-chip culture, the number of viable cells decreased in ∼1.5 h because of the difficulties of long-time cultivation of the primary mast cell. Since the total assay time required after the collection of cells was ∼20 min, it could be confirmed that mast cells were viable for a sufficient time to quantify released histamine. (19) Thomson, D. S.; Evans, D. P. Clin. Exp. Immunol. 1973, 13, 537-544.

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Figure 5. Influence of histamine flow rate on the efficiency of fluorescence derivatizing on-chip reaction (n ) 3).

Figure 6. Influence of flow rate of on fluorescence detection intensity. Flowed histamine was derivatized off-chip in advance (n ) 3).

The cell retention system by filtering was suitable for handling cells on the microchip. Since this retention method did not require the adhesive ability of the cell itself, conventional cultivation of cells, which requires from a few hours to a few days, was unnecessary. Therefore, the assay using the retention system could be started immediately after the introduction of only the required number of cells into the microchip. The degree of integration was very high because the cells could be placed directly on the microchannels for reactions and detection. On-Chip Fluorescence Derivatization Reaction. Histamine released from mast cells was derivatized at the third layer (Figure 1b). At the first junction, histamine was mixed with OPA, and the derivatizing reaction proceeded. After sufficient reaction time, citrate flowed together and the pH of the solution was adjusted to stop the reaction (pH 3.3). Figure 5 shows the result of the on-chip fluorescence derivatization reaction with different flow rates of the histamine standard solution (solute in saline solution). When the flow rate was greater than 3 µL/min, the fluorescence intensity decreased. It could be deduced that a sufficient reaction time could not be obtained at a high flow rate. When the flow rate was 3 µL/min, the linear velocity was ∼13 cm/min, demanding 2 min for a sufficient reaction. This value almost matched the reaction time of bulk experiments (data not shown). Since a stable fluorescence intensity could be obtained at 3 µL/min, a flow rate of 3 µL/min or less was employed. In addition, the flow rate affected the fluorescent detection intensity. Figure 6 shows the relationship between the signal intensity and the flow rate of the histamine derivative solution prepared in bulk scale. The signal intensity decreased at a flow

Figure 7. Real-time detection of C48/80-induced histamine release from mast cells. 1 × 103 cells were injected. A 0.5 mM solution of C48/80 (a) and the saline solution without the agent as a blank (b) were introduced at the arrow point.

rate of less than 5 µL/min, and the effect was notable at less than 3 µL/min. From these results, it was thought that a sufficient response would be obtained with feed at a flow rate of 3 µL/min when both the reaction and detection were performed in the microchip. It was therefore decided to employ this flow rate. The fluorescence detection of histamine was carried out under the conditions described above. An linear calibration curve was obtained in the range of 0-40 µM (R2 ) 0.999) with a detection limit of 142 nM (S/N ) 3, n ) 3) and quantification limit of 471 nM (S/N ) 10, n ) 3). The adsorption of histamine and its derivative on the microchip made of PDMS was investigated. The recovery rate of histamine was measured form the eluate when 1 µM histamine and the derivative flowed through the PDMS microchip. The recovery rates of both histamine (96 ( 7%) and the derivative (94 ( 9%) were almost 100% (n ) 3). Histamine Release Reaction by C48/80. Figure 7 shows the histamine release by C48/80 from 1000 cells using the present microchip system. Figure 7a shows the result using C48/80 (0.5 µg/mL) as stimuli, and (b) shows the result without the stimuli (blank). As opposed to the result without stimuli, the release of histamine began ∼5 min after the solution was substituted for a buffer containing a stimulating agent. Since it took ∼2 min for the buffer containing the stimulating agent to reach the cell chamber and a little short of 3 min from the chamber to the detection point, it could be observed that histamine was released soon after the stimulating agent had reached the cells and that the release was terminated within a few minutes. As shown in these results, the system could detect the time course of the histamine release from mast cells unlike a conventional batch system. The operations required for the assay were loading mast cells and preincubating, followed by substituting the saline solution with a stimulating agent, and initiating the feed of the reactant solution and the terminating solution in a one-step operation. This could be done with ease, without the need for a complex operation of pipetting and centrifugation as in the conventional method. Moreover, this microchip system required only 20 min for the assay after 10 min of preincubation, while the analysis time for the conventional method, which was performed with a microtiter plate as a reference experiment, was more than 40 min. Thus, the necessary time was reduced to about half by the integration of the assay.

Figure 8. Relationships between the introduced number of the cells and released histamine (n ) 3).

Figure 9. Inhibition by DSCG (antiallergic drug) (n ) 3).

Released histamine was calculated using the peak area obtained based on the calibration curve above. By varying the amount of cells employed, Figure 8 confirmed that the amount of histamine released corresponded to the number of cells. Histamine was detected in proportion with the number of cells with more than ∼500 cells. The effect of cellular stimuli by allergens could be observed quantitatively with only ∼500 cells when using the present releaser. The amount of histamine released from 500 cells was calculated at 51 pmol. Assuming that 20 pg of histamine was contained per cell, ∼55% of the histamine was released by 0.5 µg/mL C48/80. This result meant that a release rate that was approximately equivalent to that of the bulk system using 1 × 105-106 cells was obtained,12,21 confirming that an appropriate release was carried out in the present microchip system. On the other hand, signals by released histamine could be obtained with only ∼100 cells, although the quantification would be difficult. In a case in which the purpose is to judge whether the substance to be added has the ability of releasing histamine (on/off assay), ∼100 cells would be required. Histamine Release Inhibiting Test. For screening of an antiallergic drug, a histamine release inhibition test is a useful and major bioassay method in which the inhibition rate of histamine release from cells stimulated by an allergen is determined. Figure 9 shows the results of the histamine release inhibiting test with the present microchip system. Release reactions conducted with a DSCG solution showed an inhibited release rate compared to those without DSCG, and a sufficient inhibiting effect on histamine release depending on the concentration of DSCG was observed on the microchip. It is therefore possible to (20) Weng, Q. F.; Xia, F. Q.; Jin, W. R. J. Chromatogr., B 2002, 779, 347-352 (21) Dai, Y.; Hou, L. F.; Chan, Y. P.; Cheng, L.; But, P. P. H. Biol. Pharm. Bull. 2004, 27, 429-432.

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employ the present microchip system to determine whether unknown samples in place of DSCG have an inhibiting effect on histamine release as well as to measure the degree of inhibition of histamine release. Accordingly, the system was proven applicable to drug screening. Although the model system using C48/ 80 as stimuli to mast cells was used in this study, the system will also be useful for the assay of practical allergens to act on sensitized mast cells. Moreover, the system could be applied to the rapid analysis of biological mediators, which are secreted only in very small amounts and immediately diffused, because the areas of cell culture, reactions, and detection were highly integrated on one chip. In addition, the potential of instantly subjecting primary cells

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without cell attachment to the assay indicated the possibility of application to custom medical practices, such as immediate bioassays of a patient’s primary cells, which can only be collected in small amounts. ACKNOWLEDGMENT This research was supported by Grants-in-Aid for Scientific Research 14206014 from Japan Society for the Promotion of Science, Japan. Received for review November 19, 2004. Accepted March 9, 2005. AC048288O