High-Density Microcavity Array for Cell Detection - American Chemical

Jun 1, 2009 - High-Density Microcavity Array for Cell Detection: Single-Cell Analysis of Hematopoietic Stem Cells in Peripheral Blood Mononuclear Cell...
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Anal. Chem. 2009, 81, 5308–5313

High-Density Microcavity Array for Cell Detection: Single-Cell Analysis of Hematopoietic Stem Cells in Peripheral Blood Mononuclear Cells Masahito Hosokawa, Atsushi Arakaki, Masayuki Takahashi, Tetsushi Mori, Haruko Takeyama, and Tadashi Matsunaga* Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan Detection and isolation of specific cell types from limited biological samples have become a major challenge in clinical diagnosis and cell biology research. Here, we report a high-density microcavity array for target cell detection in which thousands of single cells were neatly arrayed onto 10 000 microcavities with high efficiency at approximately 90% of the loaded cells. Cell-specific immunophenotypes were exclusively identified at the singlecell level by measuring fluorescence intensities of cells labeled with antibodies targeting cell surface markers, and the purity of hematopoietic stem cells (HSCs) within human peripheral blood analyzed by this system was correlated with those obtained by conventional flow cytometry. Furthermore, gene expression of the stem cell marker, CD34, was determined from HSCs by isolating single cells using a micromanipulator. This technology has proven to be an effective tool for target cell detection and subsequent cellular analytical research at the single-cell level. Technology to detect and isolate specific cells of interest from heterogenic populations is consistently required in the field of cell analysis.1,2 Hematopoietic stem cells (HSCs) in particular have been significant targets3,4 due to their profound ability to regenerate and sustain the production of all the hematopoietic lineages over the lifespan of an individual and have been widely studied for application in medical transplantation to patients with hematological malignancies or solid tumors.5,6 Thus far, flow cytometry has served as a key technology that enables high-throughput * Corresponding author. Fax: +81-42-385-7713. Phone: +81-42-388-7020. E-mail: [email protected]. (1) Nagrath, S.; Sequist, L. V.; Maheswaran, S.; Bell, D. W.; Irimia, D.; Ulkus, L.; Smith, M. R.; Kwak, E. L.; Digumarthy, S.; Muzikansky, A.; Ryan, P.; Balis, U. J.; Tompkins, R. G.; Haber, D. A.; Toner, M. Nature 2007, 450, 1235–1239. (2) Fuchs, A. B.; Romani, A.; Freida, D.; Medoro, G.; Abonnenc, M.; Altomare, L.; Chartier, I.; Guergour, D.; Villiers, C.; Marche, P. N.; Tartagni, M.; Guerrieri, R.; Chatelain, F.; Manaresi, N. Lab Chip 2006, 6, 121–126. (3) Warren, L.; Bryder, D.; Weissman, I. L.; Quake, S. R. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 17807–17812. (4) Ramos, C. A.; Bowman, T. A.; Boles, N. C.; Merchant, A. A.; Zheng, Y.; Parra, I.; Fuqua, S. A.; Shaw, C. A.; Goodell, M. A. PLoS Genet. 2006, 2, e159. (5) Berenson, R. J.; Bensinger, W. I.; Hill, R. S.; Andrews, R. G.; Garcia-Lopez, J.; Kalamasz, D. F.; Still, B. J.; Spitzer, G.; Buckner, C. D.; Bernstein, I. D. Blood 1991, 77, 1717–1722.

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enumeration and isolation of specific cells such as HSCs, based on immunophenotype analysis using fluorescent labeled-antibodies or other marker molecules.7,8 However, although known to be highly applicable, size, cost, and the necessity for highly trained personnel have constrained researchers from taking advantage of such a technology. As such, recent attempts for cheaper and miniaturized devices have promoted the development and application of mammalian cell-directed microfluidic analytical devices. Integrated with various advanced features including environmental control, measurements on fast time scales, and image processing,9-12 the combination of such devices with array-based platforms, designed for cell entrapment, have further allowed direct imaging of large cell arrays to be conducted and the opportunity to investigate and study cell biology right down to the single-cell level.13,14 In addition, isolation of trapped target cells using techniques such as optical scattering force,15 micromanipulators16,17 and micropallet18 have also facilitated the retrieval of target cells from these devices. However, current array-based microfluidic devices are challenged by low cell entrapment efficiencies, in which many of these devices require additional or excessive loading of cells which is not favorable for the detection and isolation of target cells and for high-throughput analysis. (6) Langenmayer, I.; Weaver, C.; Buckner, C. D.; Lilleby, K.; Appelbaum, F. R.; Longin, K.; Rowley, S.; Storb, R.; Singer, J.; Bensinger, W. I. Bone Marrow Transplant. 1995, 15, 241–246. (7) Gratama, J. W.; Orfao, A.; Barnett, D.; Brando, B.; Huber, A.; Janossy, G.; Johnsen, H. E.; Keeney, M.; Marti, G. E.; Preijers, F.; Rothe, G.; Serke, S.; Sutherland, D. R.; Van der Schoot, C. E.; Schmitz, G.; Papa, S. Cytometry 1998, 34, 128–142. (8) Sutherland, D. R.; Anderson, L.; Keeney, M.; Nayar, R.; Chin-Yee, I. J. Hematother. 1996, 5, 213–226. (9) Di Carlo, D.; Aghdam, N.; Lee, L. P. Anal. Chem. 2006, 78, 4925–4930. (10) Faley, S.; Seale, K.; Hughey, J.; Schaffer, D. K.; VanCompernolle, S.; McKinney, B.; Baudenbacher, F.; Unutmaz, D.; Wikswo, J. P. Lab Chip 2008, 8, 1700–1712. (11) Ionescu-Zanetti, C.; Shaw, R. M.; Seo, J.; Jan, Y. N.; Jan, L. Y.; Lee, L. P. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 9112–9117. (12) He, M.; Edgar, J. S.; Jeffries, G. D.; Lorenz, R. M.; Shelby, J. P.; Chiu, D. T. Anal. Chem. 2005, 77, 1539–1544. (13) Rettig, J. R.; Folch, A. Anal. Chem. 2005, 77, 5628–5634. (14) Biran, I.; Walt, D. R. Anal. Chem. 2002, 74, 3046–3054. (15) Kovac, J. R.; Voldman, J. Anal. Chem. 2007, 79, 9321–9330. (16) Yamamura, S.; Kishi, H.; Tokimitsu, Y.; Kondo, S.; Honda, R.; Rao, S. R.; Omori, M.; Tamiya, E.; Muraguchi, A. Anal. Chem. 2005, 77, 8050–8056. (17) Love, J. C.; Ronan, J. L.; Grotenbreg, G. M.; van der Veen, A. G.; Ploegh, H. L. Nat. Biotechnol. 2006, 24, 703–707. (18) Salazar, G. T.; Wang, Y.; Young, G.; Bachman, M.; Sims, C. E.; Li, G. P.; Allbritton, N. L. Anal. Chem. 2007, 79, 682–687. 10.1021/ac900535h CCC: $40.75  2009 American Chemical Society Published on Web 06/01/2009

Figure 1. High-density microcavity array for cell entrapment. (a) Side view of the microcavity array. (b) Schematic diagram of the cell entrapment device integrated with the microcavity array. (c) The overall view of apparatuses used for cell entrapment, image acquisition, and single-cell isolation, (d) and an image illustration of the process within the array. Cell suspension is introduced into the chamber (1) and trapped onto the microcavities of the microcavity array by applying negative pressure (2). Then, cells were observed under a microscope and target cells were isolated using a micromanipulator (3).

Recently, we introduced a highly efficient cell entrapment platform whereby model mammalian cells were successfully entrapped onto 100 microcavity structures.19,20 In this work, we describe the development of a high-density microcavity array with 10 000 microcavities, which was designed for high efficient entrapment of single cells and applied it in the detection and isolation of CD34+ HSCs which constitute less than 0.15% of the total population of peripheral blood mononuclear cells (PBMCs) in normal individuals.21 Single CD34+ cells were exclusively identified from human PBMCs by image and gene expression analyses. This technology, therefore, shows high potential as a cheap and simple tool for image-based immunophenotyping and for the identification of specific cell types including stem cells, progenitor cells, and tumor cells. EXPERIMENTAL SECTION Cell Entrapment Device Fabrication. A poly(ethylene terephthalate) (PET) plate (20 mm × 20 mm, thickness ) 38 µm) was used as a substrate to fabricate the microcavity array. Microcavities were drilled through the PET substrate with an excimer laser (Optec MicroMaster System; Optec S.A., Belgium) at a wavelength of 248 nm with a frequency of 150 Hz to produce conical apertures with diameters of 2 µm at the top surface (Figure 1a). The distance between each microcavity was 60 µm, with 10 000 cavities arranged in a 100 × 100 array, and the array was fabricated on a typical microscope glass slide. (19) Taguchi, T.; Arakaki, A.; Takeyama, H.; Haraguchi, S.; Yoshino, M.; Kaneko, M.; Ishimori, Y.; Matsunaga, T. Biotechnol. Bioeng. 2007, 96, 272–280. (20) Matsunaga, T.; Hosokawa, M.; Arakaki, A.; Taguchi, T.; Mori, T.; Tanaka, T.; Takeyama, H. Anal. Chem. 2008, 80, 5139–5145. (21) Herbein, G.; Sovalat, H.; Wunder, E.; Baerenzung, M.; Bachorz, J.; Lewandowski, H.; Schweitzer, C.; Schmitt, C.; Kirn, A.; Henon, P. Stem Cells 1994, 12, 187–197.

A poly(dimethylsiloxane) (PDMS) structure, equipped with a vacuum microchannel (i.d. ) 500 µm), was fitted directly beneath the microcavity array to apply negative pressure for cell entrapment while a chamber for cell introduction was constructed by placing a PDMS rim that hems the microcavity array (Figure 1b). PDMS structures were fabricated by pouring a mixture of Sylpot 184 silicone elastomer (Dow Corning Asia Ltd., Tokyo, Japan) and a curing agent (10:1) onto either the master molds or a blank wafer, followed by curing for at least 20 min at 85 °C. Upon curing, the PDMS substrates were carefully peeled off the molds. The cell entrapment setup was constructed by assembling the microcavity array, the PDMS vacuum microchannel, and the PDMS rim using spacer tapes. The vacuum microchannel was connected to a peristaltic pump, and the cell entrapment setup was placed onto a computer-operated motorized stage of an upright microscope (Figure 1c). Cell Culture and Labeling. Raji cells were cultured in RPMI 1640 medium, containing L-glutamine (Sigma-Aldrich, Irvine, U.K.), 10% (v/v) FBS (Invitrogen Corp., Carlsbad, CA), and 1% (v/v) penicillin/streptomycin (Invitrogen Corp.) for 3-4 days at 37 °C with 5% CO2 supplementation. Immediately prior to each experiment, the cells were labeled with CellTracker Red CMTPX or Calcein-AM (Molecular Probes, Eugene, OR), in which labeling was achieved by incubating the cells with the tracking dye (5 µM) for 30 min. Cells were pelleted by centrifugation (200g for 5 min), the supernatant was decanted, and cells were washed twice with PBS to remove any excess dye. Purification and Labeling of PBMCs. Human peripheral blood, collected in tubes containing heparin, was mixed with PBS. It was layered on Histopaque-1077 (Sigma, St. Louis, MO) and centrifuged (600g for 30 min). The plasma-Histopaque interface Analytical Chemistry, Vol. 81, No. 13, July 1, 2009

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containing the mononuclear cell fraction was collected and washed with PBS. PBMCs were stained with a commercial kit (Stem-Kit; Beckman Coulter Inc., Tokyo, Japan) containing FITC-labeled antibody to CD45 (Clone J33), PE-labeled antibody to CD34 (Clone 581, type III), and isoclonic control PE-labeled antibody to CD34. As the control, Stem-Trol control cells (Beckman Coulter Inc.) were spiked into a peripheral blood sample and stained in a similar manner. These control cells are an assayed quality control product and express CD34 class III epitopes and CD45 antigen at the densities approximating normal immature HSCs. Cell Entrapment Operation. Raji cells or PBMCs were resuspended in PBS at a concentration of approximately 1 × 103 cells/µL. A total of 10 µL of cell suspension was dispensed onto a PBS-filled cell-entrapment device equipped with a microcavity array. Subsequently, negative pressure was applied to the cell suspension using a peristaltic pump, connected to the vacuum line of Figure 1b, at a flow rate of 180 µL/min. By applying of a negative pressure to the microcavity array, cells were driven toward and trapped onto the microcavity array (Figure 1d). Cell entrapment efficiency and detection sensitivity of the microcavity array was evaluated by using Raji cells stained with CellTracker Red and Raji cell suspensions spiked with Stem-Trol cells respectively. Image Acquisition and Analysis. After entrapment, cells images were captured using a fluorescence microscope (BX61; Olympus Corporation, Tokyo, Japan), integrated with a computeroperated motorized stage, NIBA and Cy3 filter sets, and a cooled digital camera (DP-70; Olympus Corporation). The Lumina Vision acquisition software (Mitani Corporation., Tokyo, Japan) was used to acquire the images. For morphology observation of the trapped cells, a 3-D image was obtained. Trapped cells stained with Calcein-AM were observed under a fluorescence microscope integrated with a confocal scanner unit (CSU10; Yokogawa Electric Corp., Tokyo, Japan) and a cooled CCD camera (CoolSNAP K4; Nippon Roper, Tokyo, Japan). 3-D images of the trapped cells were attained by capturing adjacent Z-axis optical sections 0.1 µm apart with an excitation wavelength of 488 nm. These sliced images were then reconstructed using the VoxBlast software (Vaytek Inc., Fairfield, IA). To analyze cellular phenotypes and determine the purity of CD34+ cells among PBMCs, the PBMCs trapped on the microcavity array were analyzed accordingly. Arrayed cells were inspected microscopically, and a whole image of the cell arrayed area was obtained. Image scanning of the 10 000microcavity array by two fluorescent wavelengths was completed within 3 min by using a 10× objective lens and the motorized stage. The fluorescence intensities of FITC and PE were analyzed by integrating the difference between the pixel intensities of individual cells and the average background over the entire microcavity array area. On the basis of this singlecell intensity data, the correlation between FITC and PE intensities was investigated. In order to determine a background signal caused by nonspecific adsorption of antibodies, PBMCs stained with FITC-labeled anti-CD45 antibodies and PE-labeled isoclonic control antibodies were analyzed. StemTrol control cells spiked into a peripheral blood sample were also analyzed to define the HSC region in a similar manner. 5310

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Flow Cytometric Analysis. The purity of CD34+ cells was analyzed with an Epics-Altra flow cytometer (Beckman Coulter Inc.) and compared with our system. In a similar manner to image analysis, the correlation between FITC and PE intensities of single cells was determined. Single-Cell RT-PCR. Single cells were isolated from the microcavities by using a micropipet (20 µm diameter) made from a glass capillary using a micromanipulator system (Eppendorf Co., Ltd., Tokyo, Japan). The manipulator was manually operated, and recovered cells were transferred into a 10 µL microtube. The recovered single cells were then directly applied in reverse transcription and PCR amplification by the Primescript RT-PCR Kit (Takara Bio Inc., Shiga, Japan). Messenger RNA extraction and annealing with Oligo-dT Primer were performed at 65 °C for 5 min and 4 °C for 10 min, while reverse transcription was performed at 42 °C for 60 min and 95 °C for 5 min, and PCR amplification for 50 cycles at 94 °C for 30 s and 60 °C for 30 s. PCR products were confirmed by 2% agarose gel electrophoresis. The primer-probe sets (obtained from Takara Bio) were as follows: 5′-TGGCACCCAGCACAATGAA-3′ (forward) and 5′-CTAAGTCATAGTCCGCCTAGAAGCA-3′ (reverse) for β-actin and 5′-ACGGCCATTCAGCAAGACAAC-3′ (forward) and reverse 5′-GCACGTGGTCAGATGCAGAGA-3′ (reverse) for CD34. RESULTS AND DISCUSSION Cell Entrapment on the Microcavity Array. To determine cell entrapment efficiency, Raji cells stained with CellTracker Red were loaded. Entrapment of single cells was completed within 60 s, and successful entrapment was confirmed using a fluorescence microscope (Video S-1 in the Supporting Information). Whole images of the microcavity array were acquired by scanning it using the computer-operated motorized stage, while sectional images (9 × 9 ) 81 images) were captured using a 10× objective lens and were reconstructed to form a single image (Figure 2a). Figure 2b shows a direct correlation between the number of cells loaded and trapped onto the microcavities (r2 ) 0.99), and cell entrapment efficiency was constant within the range of 76-94% when 1 000-9 000 cells were loaded. In a previous report, we showed that cell entrapment efficiency using a 10 × 10 microcavity array was at 70-80%.20 With the use of the advantage of our microcavity array approach, it was then adapted and redesigned to a larger scale in which we targeted our approach for the detection and isolation of specific or target cell types within a cell population such as PBMCs. Interestingly, as shown in the current work, high cell entrapment efficiency was sustained with minimal consumption of cell samples. Higher entrapment efficiency and lesser time for cell entrapment were further achieved by stabilizing the negative pressure. In addition to the evaluation of cell entrapment efficiency, the detection sensitivity of the microcavity array was also performed. StemTrol control cells were spiked into fixed Raji cell suspensions at frequencies ranging from 0.1 to 10%, and the percentage of Stem-Trol cells was determined. As predicted, the percentage of Stem-Trol cells correlated to that of the spiked frequencies (Table S-1 in the Supporting Information). In order to observe morphological changes of cells caused by physical stress, a 3-D image of a single trapped cell was captured by a fluorescence microscope integrated with a confocal scanner unit. Cells stained with Calcein-AM were used to quantify both

Figure 2. Cell entrapment using the 10 000 microcavity array: (a) Fluorescent microphotograph of trapped cells on the array. Raji cells were trapped by applying negative pressure and enumerated under a fluorescent microscope. Scale bar ) 1 mm (whole image) and 100 µm (inset). (b) Cell entrapment efficiency using the array. (c) Threedimensional image of a trapped cell on the microcavity array. Cells stained with Calcein-AM were trapped in the microcavities and observed under a fluorescent microscope integrated with a confocal scanner. Scale bar ) 10 µm.

membrane deformation and integrity when trapped onto a microcavity. Calcein-AM is a cytoplasmic dye that is transported through the cellular membrane into viable cells and is often used as a cell viability indicator. The 3-D image indicated that a part of the cell membrane protrudes into the microcavity (Figure 2c) similar to that observed with the patch clamp method using a microcapillary or microchannels.11,22,23 A cell viability test using Calcein-AM and Propidium iodide (PI) was also performed upon cell entrapment (data not shown). In contrast to Calcein-AM, PI is impermeable to the cell membrane of living cells, permeable to that of dead cells and is commonly used for identifying dead cells. In this study, Raji cells, with an initial cell population viability of 91.7 ± 0.4% were trapped on the microcavity array. No obvious change in cell viability was observed upon PI staining (91.2 ± 4.0%). This observation clearly suggests that the optimization steps and the use of our microcavity array pose very minimal stress or do not damage the cells upon cell entrapment. Detection of CD34+ Cells by Immunophenotyping of Human Peripheral Blood. With the cell entrapment process optimized, the microcavity array was then applied to detect CD34+ (22) Sakmann, B.; Neher, E. Annu. Rev. Physiol. 1984, 46, 455–472. (23) Pantoja, R.; Nagarah, J. M.; Starace, D. M.; Melosh, N. A.; Blunck, R.; Bezanilla, F.; Heath, J. R. Biosens. Bioelectron. 2004, 20, 509–517.

cells from human PBMCs. The CD34 antigen is a standard marker that is used for the identification of stem cells or progenitor cells. Cells stained with PE-labeled anti-CD34 antibodies (targeting HSCs) and FITC-labeled anti-CD45 antibodies (targeting leukocytes) were entrapped on the array (Figure 3a), and cellular phenotypes were identified by evaluating fluorescence intensities of PE and FITC from individual cells. Cell entrapment and image analysis were repeated three times, and the purity of CD34+ cells within the population of PBMCs was calculated. The result indicated that the percentage of CD34+ cells within the PBMCs population was 0.10% (Figure 3b). As a comparison, the same PBMC sample was analyzed by a flow cytometer in a similar approach. Flow cytometric analysis showed that the population of CD34+ cells within the population of PBMCs was 0.11% (Figure 3c). Referring to the analytical range of the microcavity array to flow cytometry, although the amount of sample analyzed in this work is currently limited to 10 000 cells, this issue can be technically resolved by increasing the number of microcavities to simultaneously analyze a larger number of cells (>100 000 cells). Nevertheless, it is shown here that our cell entrapment technology is not only highly efficient in cell entrapment, but immunophenotypic analyses that are reliable and reproducible can also be easily conducted. Identification of CD34+ Cells by Determining mRNA Expression of Stem Cell Marker Genes. To verify the CD34+ cells identified from peripheral blood samples by immunophenotyping, gene expression analysis of the CD34 gene was further conducted. Messenger RNA (mRNA) expression of the CD34 antigen and β-actin (housekeeping gene) were targeted accordingly. Single CD34+ cells showing distinct fluorescence intensities were isolated from the microcavity array using a micromanipulator and subsequently analyzed by single-cell RTPCR. Single leukocytes showing CD34- CD45+ were also isolated and analyzed in the same procedure. On the basis of the results of the single-cell RT-PCR, the expression of β-actin mRNA was observed from 19 isolated CD34+ cells and 5 isolated CD34- cells, indicating that single-cell isolation from the microcavity array using a micromanipulator was successfully performed. Subsequently, the CD34 mRNA expressions of β-actin mRNA positive cells were analyzed. With reference to the gel electrophoresis result, the expression of CD34 mRNA was observed from 11 of the 19 (58%) CD34+ cells while no expression of the mRNA was observed from all the CD34- cells (Figure 4). It was initially predicted, however, that all the CD34+ cells should show a positive result for the expression of the CD34 mRNA. Possible errors during cell selection were ruled out since the selection of these cells was performed manually and the confirmation of amplified β-actin mRNA further supports this ruling. It is therefore speculative that noncontrollable factors such as mRNA degradation during sample handling or low CD34 mRNA expression, since stem cells themselves exist in various conditions or stages,24 may have contributed to the nonamplification of the CD34 mRNA in some of the isolated CD34+ cells. However, this phenomenon was interesting as our values are supported and are consistent with similar analyses conducted against single CD34+ cells.25 In this (24) Baech, J.; Johnsen, H. E. Stem Cells 2000, 18, 76–86. (25) Molesh, D. A.; Hall, J. M. Genome Res. 1994, 3, 278–284.

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Figure 4. Characterization of single cells trapped on the microcavity array with the CD34 marker gene. Gel electrophoresis-like results from samples of peripheral blood mononuclear cells (PBMCs) trapped on the microcavity array. A CD34+ cell and a CD34- cell were isolated from the microcavity array, and the expression of CD34 mRNA and β-actin mRNA was determined.

Figure 3. Detection of hematopoietic stem cells (HSCs) among peripheral blood mononuclear cells (PBMCs) trapped on the microcavity array. (a) Fluorescent microphotograph of PBMCs trapped on the microcavity array. The arrow indicates the CD34+ HSC trapped on the microcavity. PBMCs were stained with PE-labeled anti-CD34 antibodies and FITC-labeled anti-CD45 antibodies. Scale bar ) 1 mm (whole image) and 100 µm (inset). Relationships between FITC and PE fluorescence intensities were determined by (b) image analysis using the microcavity array for cell entrapment or (c) flow cytometric analysis. The dots represent the fluorescence intensity of single cells. Cells were stained with FITC-labeled anti-CD45 antibody and PElabeled anti-CD34 antibody. Gates corresponding to HSCs expressing high levels of CD34 and low levels of CD45 were defined on the basis of an isoclonic control and a Stem-Trol control cell as represented in the dot plots. These gates were used to calculate the purity of HSCs within the population of PBMCs. 5312

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work, single-cell RT-PCR was directed to further demonstrate the applicability of our technology to serve as a tool in further or secondary analyses. Applicability of the Microcavity Array. The high density microcavity array we introduced offers several advantages over current array-based microfluidic devices. First, it was employed to trap large numbers of individual cells in a highly ordered fashion, at high efficiencies. We believe that this is one of the major achievements in the development of such devices. Because of high efficiencies, it allows us to use the system as a useful and potential tool in the detection and isolation of target cell types or possibly rare cells from large heterogenic populations such as HSCs from PBMCs or circulating tumor cells, existing only as 0-10 cells in 1 mL of whole blood from patients with metastatic cancer.1,26 Second, aligning cells on the microcavity array eases cell counting, observation, imaging, and evaluation of bulk or singular cells. This provides a platform technology which takes advantage of high-throughput imaging technologies that are faster and cheaper in data collection for studying molecular phenomena, including the localization and behavior of biomolecules within the cellular system.27,28 Third, it can be used for the entrapment and immobilization of adherent and suspension cell types. Suspension cells, such as leukocytes, in particular, are especially difficult to analyze since these cells are constantly mobile in the medium.29 The microcavity array provides an environment that allows trapped cells to be cultured or maintained by transporting nutrients and oxygen by microfluidic networks, which is an important aspect in cellular activity assays30,31 and can fulfill the requirements needed for precise cell component measurement and long-term monitoring of cells.32 Finally, unlike systems that have been elucidated using stable cell lines or model cell samples,2,26 we believe that applying real samples is of great importance and provides us with better insights on the applicability and feasibility of our technology. (26) Adams, A. A.; Okagbare, P. I.; Feng, J.; Hupert, M. L.; Patterson, D.; Gottert, J.; McCarley, R. L.; Nikitopoulos, D.; Murphy, M. C.; Soper, S. A. J. Am. Chem. Soc. 2008, 130, 8633–8641. (27) Perlman, Z. E.; Slack, M. D.; Feng, Y.; Mitchison, T. J.; Wu, L. F.; Altschuler, S. J. Science 2004, 306, 1194–1198. (28) Conrad, C.; Erfle, H.; Warnat, P.; Daigle, N.; Lorch, T.; Ellenberg, J.; Pepperkok, R.; Eils, R. Genome Res. 2004, 14, 1130–1136. (29) Zuba-Surma, E. K.; Kucia, M.; Abdel-Latif, A.; Lillard, J., Jr.; Ratajczak, M. Z. Folia Histochem. Cytobiol. 2007, 45, 279–290. (30) Di Carlo, D.; Wu, L. Y.; Lee, L. P. Lab Chip 2006, 6, 1445–1449. (31) Li, X.; Ling, V.; Li, P. C. Anal. Chem. 2008, 80, 4095–4102. (32) Harel, M.; Gilburd, B.; Schiffenbauer, Y. S.; Shoenfeld, Y. Clin. Dev. Immunol. 2005, 12, 187–195.

CONCLUSIONS In this study, the high-density microcavity array was designed and applied as a tool for the detection of specific or target cells from cell populations. This technology, proven to be simple, reliable, and highly efficient in cell entrapment, enables phenotypic analyses at the single-cell level based on the evaluation of cell surface marker expressions via microscopic analysis, whereby the detection of single CD34+ HSCs from PBMCs, their isolation, and mRNA expression analysis were successfully demonstrated. Thus, we are confident that our microcavity array technology will not only serve as an important tool in detection and isolation of target cells but also as a low-cost yet efficient approach applicable in cellular analyses and contribute to key research fields including cell-cell interactions studies as well as drug screening. ACKNOWLEDGMENT This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas “Lifesurveyor” Grant No.17066002 and

Young Scientists (B) (Grant No. 20760535) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. This work was also funded by the support program for improving graduate school education of “Human Resource Development Program for Scientific Powerhouse” from the Ministry of Education, Culture, Sports, Science and Technology of Japan to Masahito Hosokawa.

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

Received for review March 13, 2009. Accepted May 12, 2009. AC900535H

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