Article pubs.acs.org/ac
Magnetophoresis-Integrated Hydrodynamic Filtration System for Size- and Surface Marker-Based Two-Dimensional Cell Sorting Masahiro Mizuno, Masumi Yamada,* Ryusuke Mitamura, Kohei Ike, Kaori Toyama, and Minoru Seki Department of Applied Chemistry and Biotechnology, Graduate School of Engineering, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan S Supporting Information *
ABSTRACT: A simple microfluidic system has been presented to perform continuous two-parameter cell sorting based on size and surface markers. Immunomagnetic bead-conjugated cells are initially sorted based on size by utilizing the hydrodynamic filtration (HDF) scheme, introduced into individual separation lanes, and simultaneously focused onto one sidewall by the hydrodynamic effect. Cells are then subjected to magnetophoretic separation in the lateral direction, and finally they are individually recovered through multiple outlet branches. We successfully demonstrated the continuous sorting of JM (human lymphocyte cell line) cells using anti-CD4 immunomagnetic beads and confirmed that accurate size- and surface marker-based sorting was achieved. In addition, the sorting of cell mixtures was performed at purification ratios higher than 90%. The proposed system enables two-dimensional cell sorting without necessitating complicated setups and operations, and thus, it can be a useful tool for general biological experiments including cell-based disease diagnosis, stem cell engineering, and cellular physiological studies. conventional techniques such as centrifugation and filtration. Specific equipment such as a centrifugal elutriator17,18 has to be employed to sort cells into multiple fractions. Microfluidic systems for continuous size-based cell sorting have also been developed, including deterministic lateral displacement,19 pinched-flow fractionation (PFF),20,21 hydrodynamic filtration (HDF),22,23 and hydrophoresis.15,24 Another widely used cellular property is the expression of surface markers. For example, hematopoietic stem cells and circulating tumor cells are separated from cell mixtures based on the positive expressions of CD3425 and CD326 (EpCAM),26 respectively. Magnetic-activated cell sorting (MACS), which utilizes antibody-conjugated magnetic particles, is one of the most frequently employed conventional methods for surface marker-based cell separations.27 When selecting minor cell populations from highly complex cell samples, multiple cellular properties should be simultaneously employed. The fluorescence-activated cell sorting
A
process for selecting specific cells from heterogeneous suspensions is indispensable in medical and biological sciences. In the rapidly advancing field of stem cell-based tissue engineering, target cells with the desired differentiation characteristics are enriched while depleting undifferentiated cells. Cell sorting technologies have significantly contributed to the generation of functional hematopoietic cell,1 myogenic cell,2 hepatocyte,3,4 cardiomyocyte,5,6 neuron,7 and photoreceptor cell8 populations from embryonic stem (ES) cells or induced pluripotent stem (iPS) cells. In addition, techniques for selecting specific cell populations are essential for subsequent clinical diagnosis, including the detection/characterization of circulating tumor cells (CTCs)9,10 and prenatal diagnosis using fetal nucleated erythrocytes separated from maternal blood.11 Among the various cellular physicochemical properties used for cell selection, cell size is a basic but significantly important characteristic dominating the cell type, cycle,12 maturity,13 and differentiation capability.14 Size-based separation allows us to perform synchronization of cell cycles and the enrichment of progenitor/immature cells.15,16 In general, however, it is not easy to perform accurate size-based cell separations by using © 2013 American Chemical Society
Received: November 22, 2012 Accepted: July 22, 2013 Published: July 22, 2013 7666
dx.doi.org/10.1021/ac303336f | Anal. Chem. 2013, 85, 7666−7673
Analytical Chemistry
Article
Figure 1. Principle of two-dimensional (2D) cell sorting system integrating hydrodynamic filtration (HDF) and magnetophoresis. Cell suspension and sheath flow are continuously introduced from Inlets 1 and 2, respectively. Cells are aligned on the lower sidewall of the main channel after passing through the multiple branch points connected to Drain 1. The cells are then sorted into each separation lane based on the size on the basis of the HDF concept. In the downstream, a magnetic field is applied perpendicularly to the flow direction in order to move the cells with a larger number of attached immunomagnetic beads toward the magnet. The separated cells are individually recovered from each outlet.
marker. Our system combines the previously developed HDF scheme for size-dependent cell sorting with magnetophoresis50−52 for surface marker-based cell selection. In the experiment, we initially characterized the fabricated microchannel network in terms of the flow distributions and the sorting efficiency of standard particles. To prove our concept, we sorted JM cells using CD4 as the target surface marker that was labeled with immunomagnetic beads, and the expression of this marker was quantified. We also demonstrated the ability to select specific cells from a complex cell mixture. The presented two-dimensional (2D) cell sorter can be used for various biochemical, tissue engineering, and diagnosis applications.
(FACS) system is a powerful method to achieve multiparameter cell sorting. In FACS systems, the cell size and the expression of surface markers are detected from the light scattering and antibody-derived fluorescence profiles, respectively. This is followed by cell encapsulation into individual droplets and subsequent sorting by applying an electric field.28 FACS systems achieve high detection/sorting rates of tens of thousands of cells per second with extremely high accuracies, and it has become an essential tool for biomedical research. However, despite its high utility and powerful capabilities, commercial benchtop FACS systems are bulky and costly. Hence, they have not satisfied the increasing cell-sorting requirements in typical biomedical experiments conducted in individual laboratories. A simple but highly precise multiparameter cell sorting system is therefore required. Microfluidic technologies have recently become a versatile method for manipulating small biological objects such as cells, organelles, and biomacromolecules by utilizing the inherent laminar flow profiles.29 Various types of state-of-the-art microfluidic systems have been proposed for continuous cell sorting, with the help of freely designable cell-size microchannel configurations.30 In addition to the cell size, various cellular properties have been utilized for microfluidic cell sorting, including surface marker,31,32 deformability,33−36 adhesion property,37−40 dielectric property,41,42 density,43−45 and shape.46,47 Unlike FACS systems, wherein the target cells are sensed and then actively sorted, the passive mechanisms of most of these microfluidic cell sorters lead to considerably simpler experimental setups and operations. However, microfluidic cell selection systems based on multiple independent parameters have not been fully developed, with several exceptions,48,49 primarily because of the difficulties in connecting multiple continuous separation principles in tandem. In this study, we propose a simple but efficient microfluidic cell sorting system based on two factors, size and surface
■
EXPERIMENTAL SECTION Principle. The basic principle of the 2D cell sorting system is shown in Figure 1. Prior to cell sorting, target cells are labeled with immunomagnetic beads. A cell suspension and a sheath flow are continuously introduced into the microchannel network. In the HDF scheme, the virtual width of the flow region entering a side channel, w1, dominates the sizes of the particles that can enter the side channel; cells with a radius larger than w1 hardly flow into the side channel.22,53 The value of w1 is controlled by the ratio of the volumetric flow rates distributed to the main/side channels, a (= Q1/(Q1 + Q2)), and the main channel width, w0. The ratio of flow rates, a, is determined by the hydrodynamic resistances R of each microchannel segment. After passing through the multiple branch points to Drain 1, cells are aligned onto one (lower) sidewall in the main channel, and then they are sorted into multiple separation lanes (N = m) based on size. Following the size-based sorting, surface marker-based cell separation is conducted by employing magnetophoresis. It is anticipated that cells would be focused onto one (right) sidewall just after entering the individual separation lanes, which enables the subsequent magnetophoretic separation without necessitating additional cell focusing processes. By 7667
dx.doi.org/10.1021/ac303336f | Anal. Chem. 2013, 85, 7666−7673
Analytical Chemistry
Article
applying the magnetic field perpendicularly to the flow direction, cells conjugated with more immunomagnetic beads move toward the magnet more than those with fewer beads. Finally, the cells are sorted into individual outlet branch channels (N = n) connected with the end of the separation lanes and individually recovered from m × n outlets. In this manner, sequential 2D cell sorting according to the size and expression of surface marker is realized. Microdevice Fabrication, Design, and Evaluation. Materials used in this study are described in the Supporting Information. Polydimethylsiloxane (PDMS)-glass hybrid microdevices were fabricated using rapid prototyping and replica molding techniques as described elsewhere.54 The microchannel design is shown in Figure 2a; it comprised two inlets, two drains, and 6 × 4 outlets for the recovery of the separated
cells. A total of 50 side channels were connected to Drain 1, each of which was composed of narrow (20 μm) and broad (30 μm) segments, with properly adjusted flow resistances. The width of the main channel, w0, was 35 μm, and the widths of the six separation lanes (Lanes 1−6) were changed from 34 to 58 μm. The channel depth was uniform at 30 μm. The theoretical value of w1 was 4.0 μm for all the 50 side channels connected to Drain 1, and hence, cells smaller than 8 μm would be removed through these side channels. The theoretical values of w1 for Lanes 1−6 were 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 μm, respectively, which implied that cells with sizes of 8−10, 10−12, 12−14, 14−16, 16−18, and 18−20 μm would be sorted into these six separation lanes, respectively. The neodymium magnet was placed 1 mm from the lower sidewall of Lane 1. The end point of each separation lane was branched into 4 outlet branch channels (branches 1−4) connected to an individual outlet reservoir (Φ = 1 mm). In order to efficiently deplete the nonmigrated cells at the magnetophoresis stage, Branch 1 was designed to be broader (50 μm) than the other three branches (25 μm). The theoretical retention times of the fluid in the magnetic field-applied regions in the separation lanes were 23.5−27.9 s when the total introduced flow rate was 10.0 μL min−1. We conducted several tests to examine whether the fabricated microdevice works as designed. First, the ratios of the flow rates distributed to 2 drains and 24 outlets were measured. The volumetric flow rates distributed to Drains 1 and 2 were estimated by introducing distilled water from Inlets 1 and 2 with an equal flow rate of 5.0 μL min−1 and measuring the output volumes. On the other hand, the flow rates through 6 separation lanes and 24 outlet branch channels were estimated by counting the flowing 1-μm fluorescent particles introduced from Inlets 1 and 2. In addition, size-based separation behaviors of model particles (green fluorescent 9.9-μm particles and nonfluorescent 15-μm and 20-μm particles) were observed. Furthermore, the retention times of the cells through the 40 mm-long magnetic field-applied region in the separation lanes were directly measured by introducing cell suspension and PBS from Inlets 1 and 2, respectively. The retention times of 30 individual cells were averaged. Cell Sorting Experiments. JM cells (human T-lymphocyte cell line) and HeLa cells (human cervical cancer cell line) were obtained from RIKEN BRC and cultured at 37 °C in a humidified atmosphere with 5% CO2. After harvesting from cell culture dish or flask, JM cells and HeLa cells were stained with blue (Hoechst 33342) and red (VybrantDiI) dyes, respectively, and subsequently labeled with immunomagnetic microbeads (Φ = 50 nm). These cells were suspended in PBS containing 1% bovine serum albumin and 5% Through Path, a nontoxic surfactant solution for preventing cell adhesion on the microchannel surface, at a concentration of ∼2 × 106 cells mL−1. Cell suspension and PBS were introduced from Inlets 1 and 2, respectively, with an equal flow rate of 5.0 μL min−1. For the sorting experiments on JM cells and the mixture of JM and HeLa cells, anti-CD4 immunomagnetic beads were used. Immunomagnetic beads were added to the cell suspension and incubated at 4 °C for 15 min. To quantify the amount of CD4 expression on the JM cell surface, the cells were initially conjugated with R-phycoerythrin (PE)-conjugated anti-CD4 antibody and then conjugated with anti-PE immunomagnetic microbeads. In addition, CD4 expressionson JM and HeLa cells were examined by using a flow cytometer (MACSQuant VYB,
Figure 2. (a) Schematic diagram showing the microchannel design, (b) enlarged image showing the structures of the inlet channels, side channels, and separation lanes, (c, d) micrographs showing the (c) separation lanes and (d) the branch point and outlet branch channels, and (e) photograph of the entire microdevice. Image b is not to scale. The unit in image b is micrometers. 7668
dx.doi.org/10.1021/ac303336f | Anal. Chem. 2013, 85, 7666−7673
Analytical Chemistry
Article
μm, respectively, showing ∼10% discrepancies from the theoretical values (3.6% and 4.0 μm, respectively). The recalculated ratios a for Lanes 1−6 were 7.4, 9.0, 11.0, 15.9, 20.9, and 25.1%, respectively, and the corresponding virtual widths w1 were 5.8, 6.5, 7.2, 8.8, 10.3, and 11.5 μm, respectively. This difference would slightly shift the separation sizes of the particles from the theoretical values, but this does not essentially affect the cell separation performances. This assertion was consistent with the experimental results of model particle sorting, as shown in Figure S1a,b in the Supporting Information. Particles with sizes of 9.9, 15, and 20 μm were mainly sorted into Lanes 1, 3, and 6, respectively, based on the principle of HDF. After completing the magnetophoresis, the cells in each separation lane are sorted into four fractions. The outlet branch channels were asymmetrically designed, as in the case of the asymmetric PFF,21 to effectively eliminate nonmigrated cells through Branch 1. It was expected that ∼55% of the flow in each separation lane will be distributed to Outlet 1. As shown in Figure 3b, we confirmed that the measured flow rates corresponded well with the theoretical values with errors less than 5%. This result indicates that the ratio of the flow rates to each outlet could also be precisely adjusted. Furthermore, it is necessary to ensure sufficient retention times for the magnetophoretic cell migration in the separation lanes. We estimated the magnetophoretic migration speed of magnetically labeled JM cells inside a straight microchannel in advance and obtained the average cell migration speed of 1.32 μm s−1. Since the widths of Lanes 1−6 were 34−58 μm, the retention times should be ∼20 s, and thus, relatively long separation lanes with a length of ∼45 mm were employed. The results for the retention times obtained from direct measurement of 30 individual JM cells for each separation lane, together with the theoretical retention times of the fluid, are shown in Figure 3c. Although the measured values were slightly shorter than the theoretical values, sufficient retention times were achieved for the 6 separation lanes (20−22 s). The centers of the cells were at a distance from the microchannel surface, thereby possibly making the flowing speed of cells to be slightly higher than the average fluid velocity. Cell Focusing. For conducting the second stage of the cell sorting by magnetophoresis, the lateral positions of the cells at the entrance of each separation lane should be uniform and preferably focused onto one sidewall. In the presented microdevice, the cell positions will be expected to be automatically focused onto the downstream sidewall at the entrance of each separation lane, as a result of the size-based sorting. We examined if cell focusing is actually achieved, by using JM cells with an average size ± SD of 13.2 ± 3.9 μm. To clearly visualize the cells, cell nuclei were stained with Hoechst 33342, and the lateral positions were enumerated by image processing. Figure 4a shows a superimposed image of the nuclei of the flowing JM cells being sorted at the entrance of the separation lanes. The distributions of the centers of the cell nuclei, along the x-axis located at 100 μm from the branch points, are shown in Figure 4b. For all the 6 separation lanes, nuclei of more than 90% of the JM cells intersected the x-axis between 70% and 100% from the left sidewall. The distributions of the cell positions were primarily caused by the variations in cell size; for example, cells with diameters between 8 and 10 μm were expected to be introduced into Lane 1. Cell shapes might also affect the cell positions since we had previously observed the rotatory movements of non-
MiltenyiBiotec GmbH, BergischGladbach, Germany). For the sorting experiments of white blood cells (WBCs) from a blood sample, we added JM cells to a human peripheral blood sample, which was obtained from a healthy volunteer and was diluted at 1/20 with PBS; WBCs were labeled with PE-conjugated antiCD4 antibody and then labeled with anti-PE immunomagnetic microbeads, and their nuclei were stained with Hoechst 33342. Cell suspension and sheath flow were continuously introduced into the microdevice at identical flow rates of 5.0 μL min−1. The sorting behaviors of the cells were monitored under a microscope (IX71, Olympus) and a CCD camera (DP72, Olympus), and the cells flowing into each outlet reservoir were counted.
■
RESULTS AND DISCUSSION Evaluation of Microdevice. The most important prerequisite for performing size-based cell sorting in the HDF scheme is the correspondence of the theoretical and actual flow-rate ratios distributed at each branch point. The hydrodynamic resistance of a microchannel segmentis inversely proportional to the fourth power of the microchannel diameter. As a result, even a slight discrepancy between the designed and actual microchannel dimensions may induce a large error in the cell sorting performance. Hence, we first investigated whether the fabricated microchannel network works properly in terms of the fluid delivery. The measured volumetric flow rates distributed to each drain and separation lane are shown in Figure 3a. Despite the relatively complicated microchannel
Figure 3. Validation of the 2D cell sorter: (a) distributions of the volumetric flow rates (average ± SD) to 2 drains (N = 6) and 6 separation lanes (N = 3), (b) distribution of the volumetric flow rates to 4 outlet branch channels (N = 3), and (c) retention times of JM cells (average ± SD, N = 30) in the magnetophoresis regions with a length of 40 mm in each separation lane.
network comprising 50 long side channels (24 mm) and 6 separation lanes (∼45 mm), the measured values were in good agreement with the theoretical values; for example, the errors between these values for Lanes 1−6 were less than 0.2%. From these results, we recalculated the w1 values, which were almost equal to the radii of the maximum cells flowing into the side channels and separation lanes. The obtained a and w1 values, averaged for the 50 branch points to Drain 1, were 4.1% and 4.3 7669
dx.doi.org/10.1021/ac303336f | Anal. Chem. 2013, 85, 7666−7673
Analytical Chemistry
Article
Subsequently, magnetophoretic cell separation was performed. In order to achieve efficient surface marker-based cell separation using a simple experimental setup, we applied the magnetic field by placing a neodymium magnet. As the target surface marker to sort JM cells (a human T-lymphocytic leukemic cell line), we employed CD4; the CD4 antibody recognizes approximately 65% of peripheral blood T cells.55 Figure 5a,b shows JM cells flowing near the end point of Lane
Figure 4. Cell focusing at the entrance of the separation lanes: (a) superimposed image of the nucleus positions of JM cells. Microchannel walls were outlined with white dotted lines. (b) Distributions of the centers of the cell nuclei along the x-axis, located 100 μm from the branch point as shown in part a.
spherical particles/cells near the branch point in the HDF microchannel.46 Our obtained results imply that almost all the cells that were introduced into each separation lane flow through Branch 1, if they do not migrate in the lateral direction. Therefore, exact magnetophoretic cell separation is possible without necessitating additional complicated cell focusing mechanisms, owing to the effects of this simultaneous cell focusing. 2D Cell Sorting. Next, as the first stage of the 2D cell sorting, we examined the size-based sorting of JM cells. In this study, the flow rates from Inlets 1 and 2 were kept identical, 5.0 μL min−1, for all the experiments described below in order to ensure perfect cell alignment after passing through the 50 branch points. The results of the size-based sorting are shown in Figure S2 in the Supporting Information. There was a clear difference in the sizes of the cells recovered from each outlet, although the cell sizes were overlapping between the neighboring fractions, possibly because of the nonspherical cell morphologies. Each fraction contained uniform-sized cell populations with coefficient of variation (CV) values for diameters of ∼15%. We confirmed that the presented cell sorter was capable of sorting the cells according to size, as the first stage of the 2D sorting. Size is one of the most basic factors that characterize cells, and, for example, young and undifferentiated cells are generally small in size. FACS systems are almost the only conventional technique capable of size-based sorting in a continuous manner; however, their principle is based on the relative quantification of the forward scattering of the laser light, and thus, absolute size-based sorting is not usually conducted. The presented system is advantageous in the sense that the simple introduction of the cell suspension enables highly effective absolute size-based cell sorting into multiple fractions.
Figure 5. 2D sorting of JM cells based on size and expression of surface marker (CD4). (a, b) Micrographs of JM cells at the branch point of Lane 1 with and without applying the magnetic field. Nuclei of JM cells were stained in blue with Hoechst 33342. (c, d) Ratios of JM cells recovered from each outlet with and without applying the magnetic field. The sum of the ratios for each separation lane was standardized to be 100%.
1. The ratios of the cells recovered from each outlet, with and without applying the magnetic field, are shown in Figure 5c,d), where the sum of the values for each separation lane was normalized to be 100%. When the magnetic field was not applied, most of the cells were recovered through Branches 1 and 2 for all 6 separation lanes. The sum of the ratios of the cells recovered from Outlets 1 and 2 was 89%, whereas the ratios from Outlets 3 and 4 were 9% and 2%, respectively. The small cell populations recovered from Outlets 3 and 4 can be attributed to the hydrodynamic lift force56 and the cell 7670
dx.doi.org/10.1021/ac303336f | Anal. Chem. 2013, 85, 7666−7673
Analytical Chemistry
Article
collisions. On the other hand, with the application of the magnetic field, the flow positions was shifted toward the magnet’s (lower/left) direction by magnetophoresis, and a certain amount of cells was recovered from Outlets 3 and 4. The ratios of the cells recovered from Outlets 3 and 4 increased to 21% and 22%, respectively, although there were still considerable amounts of cells recovered from Outlets 1 and 2 (∼57%). We then investigated the reasons for the presence of nonmigrated JM cells in the magnetophoresis stage. The most probable reason is the difference in the amount of surface marker expressions on JM cells; in flow cytometric analysis, for example, it is typical for the fluorescence signal intensity to be distributed with a range of one or two orders even for a cell group known to express specific surface markers.57 To examine this issue, we performed two-step magnetic labeling of JM cells and conducted relative quantification of the expression amounts of CD4. Cells were initially labeled with an anti-CD4 antibody conjugated with R-phycoerythrin (PE) with red fluorescence, and subsequently, cells were labeled with immunomagnetic beads conjugated with an anti-PE antibody. After separating the JM cells, the red fluorescence intensities derived from PEconjugated anti-CD4 were analyzed via image processing using Image J. The results of the CD4 expressions for the cells separated from Outlets 1−4 of Lane 3 are shown in Figure 6. Despite using different types of immunomagnetic beads, the ratios of the cells sorted into each outlet did not significantly change as compared to the direct labeling. We found a clear difference in the CD4 expression between the cell populations sorted into each outlet. In particular, the cells recovered from Outlet 4 showed a significantly high fluorescence profile, whereas fluorescence was hardly detectable from the cells from Outlet 1. The result of the flow cytometric analysis of JM cells before sorting (Figure S3 in the Supporting Information) also indicated that there were ∼50% JM cells that do not express CD4. Although we did not analyze the absolute amounts of CD4, our results clearly demonstrate that the difference in the surface marker expression was effectively utilized for cell sorting. Unlike standard FACS systems, in which fluorescence signals are often binarized for each surface marker, the proposed microfluidic system achieves cell sorting into multiple fractions with stepwise expressions of surface markers and this is combined with accurate size-based sorting. Sorting of Cell Mixture. Finally, we demonstrated 2D sorting of a mixture of two cell types as a model separation of actual cell samples with considerable heterogeneity. HeLa cells, which are the widely used human cervical cancer cell line, and JM cells were used for the model separation. The size of HeLa cells was measured to be 14.1 ± 2.9 μm and, thus, separating these cells solely based on the difference in cell sizes would be impossible. These cells were independently labeled with different dyes, and this was followed by the immunomagnetic labeling of CD4 on cell surfaces by adding anti-CD4 immunomagnetic beads. HeLa cells were expected to be not magnetically labeled; flow cytometric analysis also showed that the ratio of CD4 positive HeLa cells was low, although a small amount of nonspecific binding was observed (Figure S3 in the Supporting Information). Figure 7 shows the results of the 2D sorting of the cell mixture. As shown in Figure 7b,c, which represents the separated cell samples from Outlets 2 and 4 of Lane 3, selective enrichment of JM cells was successfully performed. The ratio of
Figure 6. Relative quantification of the amount of surface marker expressions on JM cells. (a−d) Fluorescence micrographs of JM cells sorted into each outlet of Lane 3. Red fluorescence was derived from PE-conjugated anti-CD4 on the cell surface. Cells are outlined by white broken lines. Scale bar, 50 μm. (e) Fluorescence intensity profile of cells retained in each outlet of Lane 3, analyzed via image processing.
the JM cells in the sample recovered from Outlet 4 was increased to ∼92% from that in the original sample, 41%. On the other hand, HeLa cells were mainly recovered from Outlets 1 and 2 for all the separation lanes. The ratios of HeLa cells in the sample recovered from Outlets 1 and 2 of Lane 3 increased to 67% and 68%, respectively. Nonmigrated JM cells were recovered from these outlets, which resulted in insufficient enrichment of HeLa cells. Figure 7e shows the recovery ratios of the two cell types from each outlet. We confirmed that the enrichment of JM cells occurred for all 6 separation lanes when the magnetic field was applied. In addition, it was possible to performed the 2D sorting of WBCs from a diluted human peripheral blood sample. The results shown in Figure S4 in the Supporting Information clearly demonstrated that our system can be applicable to the sorting of actual biological samples containing cells with wide variations in size and surface-marker expression.
■
CONCLUSIONS In this study, a simple but efficient multiparameter cell sorting system has been proposed by integrating HDF and magnetophoresis. Simultaneous cell focusing resulted from the HDF 7671
dx.doi.org/10.1021/ac303336f | Anal. Chem. 2013, 85, 7666−7673
Analytical Chemistry
Article
CD38.58 Since the proposed system is continuous and passive, it can be integrated with an immunochromatography method or cell rolling on the microchannel surface38,40 by using other types of antibodies. In addition, there are various microfluidic technologies for continuous cell separation using different factors such as density, deformability, and charge, and hence, simultaneous connections with such cell sorting systems would be useful. In this study, we have just demonstrated the sorting of two types of cells; however, future studies may make it possible to purify cell populations with specific characteristics, which are differentiated from ES or iPS cells.
■
ASSOCIATED CONTENT
■
AUTHOR INFORMATION
S Supporting Information *
Materials and supporting figures (Figures S1−S4) showing the results of size-based sorting of model particles, size-based sorting of JM cells, flow cytometric analysis of JM and HeLa cells before sorting, and 2D sorting of white blood cells (WBCs) from diluted human blood. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Author
*Fax/phone: +81-43-290-3398. E-mail: m-yamada@faculty. chiba-u.jp. Notes
Figure 7. Separation results of a mixture of JM and HeLa cells. (a−c) Fluorescence micrographs of the cells before separation and recovered from Outlets 2 and 4 of Lane 3. JM cells and HeLa cells were stained with blue and red dyes, respectively. Scale bar, 50 μm. (d) The ratios of the JM cells and HeLa cells recovered from each outlet in Lane 3. (e) The ratios of the JM and HeLa cells recovered from 6 × 4 outlets. The averaged ratios of JM cells recovered from Outlets 1, 2, 3, and 4 were 31%, 24%, 23%, and 23%, respectively, whereas those of HeLa cells were 52%, 35%, 10%, and 2%, respectively.
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS This study was supported in part by Grants-in-aid for Scientific Research (Grant 23106007) and for Improvement of Research Environment for Young Researchers from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. We thank Prof. D. Umeno at Chiba University for technical assistance in performing flow cytometric analysis.
■
separation was effectively utilized for the subsequent magnetophoresis without employing additional cell focusing operations. Our proposed system was capable of conducting accurate cell sorting based on both size and surface marker. By using antiCD4 immunomagnetic bead, we were able to conduct 2D cell sorting of cell mixtures into 6 × 4 fractions. In this manner, we have demonstrated the feasibility of the presented system as a new microfluidic cell sorting approach. Unlike current techniques of cell sorting based on multiple factors, like FACS systems, the presented 2D cell sorting system is able to continuously, passively, and accurately separate cells with simple mechanisms, and thus, it can be effectively applied to typical biological experiments. In future work, several improvements must be made so that the proposed system can be applied to actual biomedical engineering and clinical uses. First of all, the throughput of the system, ∼100 cells s−1, should be increased. This value can be increased by aligning multiple microfluidic systems in parallel or fabricating high-aspect-ratio microchannel structures. A system to apply strong magnetic fields, such as an electromagnet placed near the microchannel, would also be effective, since it can speed up the magnetophoretic migration and significantly increase the input flow rates and the sorting throughput. The second improvement refers to the increase in the number of factors for cell selection. For example, separation of stem cells by using FACS systems sometimes utilizes multiple target surface markers, as represented by the purification of hematopoietic stem cells using CD34 and
REFERENCES
(1) Matsumoto, K.; Isagawa, T.; Nishimura, T.; Ogaeri, T.; Eto, K.; Miyazaki, S.; Miyazaki, J.; Aburatani, H.; Nakauchi, H.; Ema, H. PLoS One 2009, 4, e4820. (2) Chang, H.; Yoshimoto, M.; Umeda, K.; Iwasa, T.; Mizuno, Y.; Fukada, S.; Yamamoto, H.; Motohashi, N.; Miyagoe-Suzuki, Y.; Takeda, S.; Heike, T.; Nakahata, T. FASEB J. 2009, 23, 1907−1919. (3) Song, Z.; Cai, J.; Liu, Y.; Zhao, D.; Yong, J.; Duo, S.; Song, X.; Guo, Y.; Zhao, Y.; Qin, H.; Yin, X.; Wu, C.; Che, J.; Lu, S.; Ding, M.; Deng, H. Cell Res. 2009, 19, 1233−1242. (4) Si-Tayeb, K.; Noto, F. K.; Nagaoka, M.; Li, J.; Battle, M. A.; Duris, C.; North, P. E.; Dalton, S.; Duncan, S. A. Hepatology 2010, 51, 297−305. (5) Yang, L.; Soonpaa, M. H.; Adler, E. D.; Roepke, T. K.; Kattman, S. J.; Kennedy, M.; Henckaerts, E.; Bonham, K.; Abbott, G. W.; Linden, R. M.; Field, L. J.; Keller, G. M. Nature 2008, 453, 524−528. (6) Matsuura, K.; Masuda, S.; Haraguchi, Y.; Yasuda, N.; Shimizu, T.; Hagiwara, N.; Zandstra, P. W.; Okano, T. Biomaterials 2011, 32, 7355−7362. (7) Singh Roy, N.; Nakano, T.; Xuing, L.; Kang, J.; Nedergaard, M.; Goldman, S. A. Exp. Neurol. 2005, 196, 224−234. (8) Lamba, D. A.; McUsic, A.; Hirata, R. K.; Wang, P. R.; Russell, D.; Reh, T. A. PLoS One 2010, 5, e8763. (9) Paterlini-Brechot, P.; Benali, N. L. Cancer Lett. 2007, 253, 180− 204. (10) Sheng, W.; Chen, T.; Kamath, R.; Xiong, X.; Tan, W.; Fan, Z. H. Anal. Chem. 2012, 84, 4199−4206. (11) Bianchi, D. W.; Flint, A. F.; Pizzimenti, M. F.; Knoll, J. H.; Latt, S. A. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 3279−3283.
7672
dx.doi.org/10.1021/ac303336f | Anal. Chem. 2013, 85, 7666−7673
Analytical Chemistry
Article
(46) Sugaya, S.; Yamada, M.; Seki, M. Biomicrofluidics 2011, 5 (45), 24103. (47) Beech, J. P.; Holm, S. H.; Adolfsson, K.; Tegenfeldt, J. O. Lab Chip 2012, 12, 1048−1051. (48) Adams, J. D.; Kim, U.; Soh, H. T. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 18165−18170. (49) Kim, U.; Soh, H. T. Lab Chip 2009, 9, 2313−2318. (50) Pamme, N.; Wilhelm, C. Lab Chip 2006, 6, 974−980. (51) Xia, N.; Hunt, T. P.; Mayers, B. T.; Alsberg, E.; Whitesides, G. M.; Westervelt, R. M.; Ingber, D. E. Biomed. Microdev. 2006, 8, 299− 308. (52) Shen, F.; Hwang, H.; Hahn, Y. K.; Park, J.-K. Anal. Chem. 2012, 84, 3075−3081. (53) Yamada, M.; Seki, M. Anal. Chem. 2006, 78, 1357−1362. (54) McDonald, J. C.; Duffy, D. C.; Anderson, J. R.; Chiu, D. T.; Wu, H.; Schueller, O. J. A.; Whitesides, G. M. Electrophoresis 2000, 21 (40), 27−55. (55) Parnes, J. R. Adv. Immunol. 1989, 44, 265−311. (56) Nieuwstadt, H. A.; Seda, R.; Li, D. S.; Fowlkes, J. B.; Bull, J. L. Biomed. Microdev. 2011, 13, 97−105. (57) Barnett, D.; Storie, I.; Wilson, G. A.; Granger, V.; Reilly, J. T. Clin. Lab. Haematol. 1998, 20, 155−164. (58) Reya, T.; Morrison, S. J.; Clarke, M. F.; Weissman, I. L. Nature 2001, 414, 105−111.
(12) Tzur, A.; Kafri, R.; LeBleu, V. S.; Lahav, G.; Kirschner, M. W. Science 2009, 325, 167−171. (13) Barrandon, Y.; Green, H. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 5390−5394. (14) Hung, S. C.; Chen, N. J.; Hsieh, S. L.; Li, H.; Ma, H. L.; Lo, W. H. Stem Cells 2002, 20, 249−258. (15) Choi, S.; Song, S.; Choi, C.; Park, J. K. Anal. Chem. 2009, 81, 1964−1968. (16) Overturf, K.; Al-Dhalimy, M.; Finegold, M.; Grompe, M. Am. J. Pathol. 1999, 155, 2135−2143. (17) Bauer, J. J. Chromatogr., B 1999, 722, 55−69. (18) Morijiri, T.; Yamada, M.; Hikida, T.; Seki, M. Microfluid. Nanofluid. 2013, 14, 1049−1057. (19) Davis, J. A.; Inglis, D. W.; Morton, K. J.; Lawrence, D. A.; Huang, L. R.; Chou, S. Y.; Sturm, J. C.; Austin, R. H. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 14779−14784. (20) Yamada, M.; Nakashima, M.; Seki, M. Anal. Chem. 2004, 76, 5465−5471. (21) Takagi, J.; Yamada, M.; Yasuda, M.; Seki, M. Lab Chip 2005, 5 (120), 778−784. (22) Yamada, M.; Seki, M. Lab Chip 2005, 5, 1233−1239. (23) Yamada, M.; Kano, K.; Tsuda, Y.; Kobayashi, J.; Yamato, M.; Seki, M.; Okano, T. Biomed. Microdev. 2007, 9, 637−645. (24) Choi, S.; Song, S.; Choi, C.; Park, J. K. Lab Chip 2007, 7, 1532− 1538. (25) 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.; Thomas, E. D. Blood 1991, 77, 1717−1722. (26) 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. (27) Miltenyi, S.; Muller, W.; Weichel, W.; Radbruch, A. Cytometry 1990, 11, 231−238. (28) Herzenberg, L. A.; Parks, D.; Sahaf, B.; Perez, O.; Roederer, M. Clin. Chem. 2002, 48, 1819−1827. (29) Pamme, N. Lab Chip 2007, 7, 1644−1659. (30) West, J.; Becker, M.; Tombrink, S.; Manz, A. Anal. Chem. 2008, 80, 4403−4419. (31) Inglis, D. W.; Riehn, R.; Austin, R. H.; Sturm, J. C. Appl. Phys. Lett. 2004, 85, 5093−5095. (32) Estes, M. D.; Do, J.; Ahn, C. H. Biomed. Microdev. 2009, 11, 509−515. (33) Mohamed, H.; Murray, M.; Turner, J. N.; Caggana, M. J. Chromatogr., A 2009, 1216, 8289−8295. (34) Hou, H. W.; Bhagat, A. A.; Chong, A. G.; Mao, P.; Tan, K. S.; Han, J.; Lim, C. T. Lab Chip 2010, 10, 2605−2613. (35) Hur, S. C.; Henderson-MacLennan, N. K.; McCabe, E. R.; Di Carlo, D. Lab Chip 2011, 11, 912−920. (36) Choudhury, D.; Ramsay, W. T.; Kiss, R.; Willoughby, N. A.; Paterson, L.; Kar, A. K. Lab Chip 2012, 12, 948−953. (37) Kwon, K. W.; Choi, S. S.; Lee, S. H.; Kim, B.; Lee, S. N.; Park, M. C.; Kim, P.; Hwang, S. Y.; Suh, K. Y. Lab Chip 2007, 7, 1461− 1468. (38) Karnik, R.; Hong, S.; Zhang, H.; Mei, Y.; Anderson, D. G.; Karp, J. M.; Langer, R. Nano Lett. 2008, 8, 1153−1158. (39) Li, P.; Gao, Y.; Pappas, D. Anal. Chem. 2011, 83, 7863−7869. (40) Choi, S.; Karp, J. M.; Karnik, R. Lab Chip 2012, 12, 1427−1430. (41) Flanagan, L. A.; Lu, J.; Wang, L.; Marchenko, S. A.; Jeon, N. L.; Lee, A. P.; Monuki, E. S. Stem Cells 2008, 26, 656−665. (42) Ling, S. H.; Lam, Y. C.; Chian, K. S. Anal. Chem. 2012, 84, 6463−6470. (43) Huh, D.; Bahng, J. H.; Ling, Y.; Wei, H. H.; Kripfgans, O. D.; Fowlkes, J. B.; Grotberg, J. B.; Takayama, S. Anal. Chem. 2007, 79, 1369−1376. (44) Petersson, F.; Aberg, L.; Sward-Nilsson, A. M.; Laurell, T. Anal. Chem. 2007, 79, 5117−5123. (45) Morijiri, T.; Sunahiro, S.; Senaha, M.; Yamada, M.; Seki, M. Microfluid. Nanofluid. 2011, 11, 105−110. 7673
dx.doi.org/10.1021/ac303336f | Anal. Chem. 2013, 85, 7666−7673
