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Cite this: DOI: 10.1039/c6lc01237j

Received 3rd October 2016,Accepted 5th December 2016

DOI: 10.1039/c6lc01237j

www.rsc.org/loc

Slanted, asymmetric microfluidic lattices as size-selective sieves for continuous particle/cell sorting†

Masumi Yamada,*a Wataru Seko,a Takuma Yanai,a Kasumi Ninomiyab and Minoru Sekia

Hydrodynamic microfluidic platforms have been proven to be useful and versatile for precisely sorting par-

ticles/cells based on their physicochemical properties. In this study, we demonstrate that a simple lattice-

shaped microfluidic pattern can work as a virtual sieve for size-dependent continuous particle sorting. The

lattice is composed of two types of microchannels (“main channels” and “separation channels”). These

channels cross each other in a perpendicular fashion, and are slanted against the macroscopic flow direc-

tion. The difference in the densities of these channels generates an asymmetric flow distribution at each

intersection. Smaller particles flow along the streamline, whereas larger particles are filtered and gradually

separated from the stream, resulting in continuous particle sorting. We successfully sorted microparticles

based on size with high accuracy, and clearly showed that geometric parameters, including the channel

density and the slant angle, critically affect the sorting behaviors of particles. Leukocyte sorting and mono-

cyte purification directly from diluted blood samples have been demonstrated as biomedical applications.

The presented system for particle/cell sorting would become a simple but versatile unit operation in micro-

fluidic apparatus for chemical/biological experiments and manipulations.

Introduction

As represented by the selection of circulating tumor cells(CTCs) from peripheral blood samples,1 the purification ofproperly differentiated cells from stem cell-derivedpopulations,2 and the detection of fetal nucleated erythrocytesfor prenatal diagnosis,3 cell sorting has recently become moresignificant in biological studies and clinical applications.Many types of conventional techniques are available forsorting cells, ranging from highly sophisticated fluorescenceactivated cell sorting (FACS) to relatively simple operationsincluding centrifugation, filtration, and immunomagneticseparation. However, there is a trade-off between the sortingprecision and the operability in terms of cost and throughputin these conventional techniques. Therefore, new methodolo-gies that allow for highly precise cell sorting using simple op-erations/devices are required.

In the past decade, microfluidic technologies have beenrecognized as practical tools for precisely manipulating bio-logical particles such as animal/plant cells, bacteria, andextracellular vesicles.4,5 The size of typical microchannelstructures is similar to the size of a single cell, so the han-

dling of cells with micrometer-scale precision is possible, butcannot be realized using conventional bulk-scale techniquesor systems. Many types of unique cell sorting mechanismshave been devised, utilizing the stable laminar flows inmicrochannels. Cell sorting schemes using microfluidictechnologies are roughly classified into two types: batch andcontinuous processes. Representative examples of the formerinclude cell trapping/capture techniques using micro-chambers,6 microdroplets,7 antibody-coated surfaces,8 micro-vortices,9 and physical filtration.10 These techniques are moresuitable for the analysis of rare cell populations rather thanpreparative sorting. On the contrary, continuous processesare preferable when a cell sample of a relatively large quan-tity is handled and when cells are sorted into multiple frac-tions. The microchannel system for continuous sorting alsofacilitates integration with upstream/downstream cell manip-ulations, including sample pre-treatment, post-sorting pro-cessing, cell characterization and analysis, and cultivation.Furthermore, the tandem connection of multiple cell sortingprinciples based on different factors is possible.11,12 Re-searchers have developed cell sorting techniques that com-bine laminar flows with outer force-driven cell migration, in-cluding dielectrophoresis,13,14 acoustophoresis,15,16 gravity-driven separation,17,18 magnetophoresis,19,20 and centrifugalsorting.21 Many types of purely hydrodynamic techniqueshave also been reported, including deterministic lateral dis-placement (DLD),22–24 pinched-flow fractionation (PFF),25,26

hydrodynamic filtration (HDF),27–29 hydrophoresis,30,31 cell

Lab ChipThis journal is © The Royal Society of Chemistry 2016

aDepartment of Applied Chemistry and Biotechnology, Graduate School of

Engineering, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan.

E-mail: m-yamada@faculty.chiba-u.jp; Fax: +81 43 290 3398; Tel: +81 43 290 3398b Asahi Kasei Corp, 2-1 Samejima, Fuji-shi, Shizuoka 416-8501, Japan

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6lc01237j

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margination,32,33 Dean flow-based sorting,34 and inertialmicrofluidics.35,36

It is generally accepted that size is one of the most basiccellular characteristics closely associated with cellular pheno-type, function, maturity, and differentiation. For example,CTCs are generally larger than other blood cells, and this sizedifference has been utilized for enrichment and selection ofCTCs. In addition, several types of somatic cells with stem- orprogenitor-cell-like properties are smaller than other cells, asrepresented by small hepatocytes37 and limbal corneal epi-thelial side population cells.38 Furthermore, size-based classi-fication of blood cell population (e.g., selection of leukocytesand their phenotypic sub-sorting) is essential for clinical di-agnosis, treatment, and biomedical studies. Previously devel-oped hydrodynamic microfluidic cell sorting methods achievepassive and highly precise size-based sorting simply by intro-ducing a cell suspension into specific microchannel struc-tures. To date, the sorting of blood cells,23 hepatic cells,29

CTCs,12,39 mesenchymal stromal cells,40 bacteria,41 and em-bryoid bodies42 has been demonstrated. In addition, the ap-plicability to sorting based on other parameters, includingcell shape,43,44 density,18,21 and deformability,45,46 has beensuccessfully proven. Other applications of these methods in-clude sub-micron particle sorting,47 particle concentration,48

bead-based biochemical analysis,49 cell cycle synchroniza-tion,31,50 and multistep carrier fluid-exchange for cell treat-ment.51,52 These examples clearly demonstrated the useful-ness and high versatility of hydrodynamic microfluidic cellsorting methods as new platforms for precisely manipulatingbiological particles. However, there are problems with generalmicrofluidic cell sorters, including channel clogging and rela-tively low throughput, which should be solved to realize moreversatile systems with facile operations. Microfluidic cellsorting systems based on new concepts are of great impor-tance to the development of new tools for the biological andbiomedical research fields.

In this study, we propose a new microfluidic mechanismfor continuous particle/cell sorting using a slanted, asymmet-rically arranged microfluidic lattice structure, which works asa size-selective sieve (Fig. 1). As in most of the previoushydrodynamic microfluidic particle/cell sorting techniques,continuous sorting is achieved simply by introducing a parti-cle suspension and buffer flows into the microchannel net-work. In the lattice structure, the difference in the lateral po-sitions of the small/large particles is gradually enhanced,achieving size-based particle sorting, as in the DLD tech-nique. The critical size for particle sorting is controllable byaltering the lattice geometry such as the densities of the per-pendicularly crossing channels of which the lattice is com-posed. This scheme is advantageous because the arrayed con-figuration of the microchannels possibly enables relativelyhigh-throughput sorting. In addition, the lattice pattern isrobust against the problem of microchannel clogging becausebypass flows would be generated even when several pointsare clogged. In the experiment, we first examined the effectof the lattice geometries on the sorting performances of

model particles. Then, the separation of blood cells (erythro-cytes and leukocytes) and the direct enrichment of monocytesfrom a diluted blood sample were examined. Furthermore, amicrodevice with multiple separation units was designed andfabricated in order to increase the throughput of cell sorting.

Sorting mechanism

Fig. 1 shows the mechanism of size-based particle sortingusing the lattice-shaped microchannel network. This micro-channel is composed of several inlet/outlet channels and arectangular lattice region. The lattice region is composed oftwo types of channels that cross each other in a perpendicu-lar pattern. The main channels are slanted to one direction(lower right in the figure), whereas the separation channels

Fig. 1 (a) Schematic of the microfluidic lattice device for continuouscell/particle sorting. (b) An enlarged image showing the particlebehaviors at a crossing point. (c) Parameters characterizing the latticestructure. Density factor n is defined as indicated.

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are slanted in the opposite direction. The most importantgeometric feature to note is the significantly large differencebetween the densities of these channels. The density of theseparation channels is 30–100 times greater than that of themain channels. In addition, there are straight side channelson both sides of the lattice region. The channel depth wasuniform across the entire microchannel network.

A particle suspension is continuously pumped into thismicrochannel from the central inlet, whereas buffer solutionswithout particles are introduced from the inlets on bothsides. We assumed a uniform flow rate profile throughout thelattice region (i.e., uniform flow velocities in the y directionat arbitrary points on the x axis) in a macroscopic view. Basedon this assumption, it was expected that only a small amountof the fluid flow is split into the separation channel at eachcrossing point (Qsep/Qmain ≪ 1 in Fig. 1(b)) because the den-sity of the separation channels is significantly higher thanthat of the main channels. This indicates that the flow profileis in a zig-zag pattern in a microscopic view, and a very thinflow region near the sidewall (with the boundary width of wb)is split at each crossing point (Fig. 1(b)). Particles with sizessmaller than the critical value flow along the streamline, andtherefore, they enter the separation channels when they flownear the sidewall. Hence, small particles enter the separationchannel at a certain frequency and show a zigzag motion. Incontrast, particles with sizes larger than the critical value donot enter the separation channels and just flow along themain channels, because the center positions of such particlesdo not exist in the thin flow region entering the separationchannel. The behavior of these particles is similar to that inthe HDF scheme, in the sense that the virtual boundary widthof the flow region wb determines the boundary size for the par-ticle sorting.27,28 The distance between the lateral positions ofthe smaller and the larger particles is gradually enhanced asin the case of the DLD technique,22 resulting in continuoussize-based particle sorting. When large particles reach the endof the main channel, they flow along the side channel. Sepa-rated particles are individually recovered through different out-let channels primarily depending on their sizes.

There are several factors affecting the critical size for parti-cle sorting. The value of wb is considered to be nearly equalto the maximum radius of the particles that can enter theseparation channel. This value of wb is determined by themain channel width wmain and the ratio of the flow rate ofparticles entering the separation channel at the crossingpoint, Qsep/Qmain, when a parabolic flow-rate profile in the x–y plane is assumed and the flow rate distribution in the z(depth) direction is neglected. The following geometrical rela-tionship exists between these parameters:

(1)

Assuming uniform flow velocity within the entire latticeregion, and neglecting the boundary conditions (i.e., the

splitting/recombining flows on the side channels), the ratioof the flow rate division into the separation channel at eachcrosslinking point, Qsep/Qmain, is determined by the ratio of thedensities of the main and separation channels and the slantangle θ of the main channels, and is expressed as follows:

(2)

where n is the density factor, which is explicitly shown inFig. 1(c) and defined by the following equation:

(3)

where dmain and dsep are the distances between the center po-sitions of the neighboring main channels and separationchannels, respectively. From eqn (1) and (2), it was expectedthat the boundary width, wb, is simply dependent on the den-sity factor n and the main channel width wmain. The followingrelationship exists between these parameters:

(4)

The critical size for the particle separation, which is dou-ble the value of wb, is therefore predictable only from themicrochannel geometries, on the condition that the particleinertia is not taken into consideration. For example, for a lat-tice channel with wmain, dmain, dsep, and θ of 25 μm, 930 μm,35 μm, and 15°, respectively, wb becomes 1.5 μm, suggestingthat spherical particles with radii equal to or smaller than 1.5μm (diameter of 3.0 μm) can flow into the separation chan-nels. Although this is a rough estimation, we designed micro-channels based on this concept and attempted to control thecritical size for particle sorting.

Experimental sectionMicrochannel fabrication and design

PDMS-glass microfluidic devices were fabricated by usingstandard soft lithography and replica molding processes, asdescribed in previous studies.27,53 Briefly, SU-8 structureswere patterned on a silicon wafer via soft lithography to pre-pare the mold. Then, PDMS prepolymer was cast onto thismold and cured. After the crosslinking reaction, the PDMSplate with microgroove structures was peeled off from themold, and inlet/outlet holes were punched into the structure.The PDMS plate was then bonded to a flat glass slide aftertreatment with O2 plasma. Silicone tubes (outer Φ = 1 or 2mm) were attached to the inlet/outlet holes, and then gluedinto place. We designed and prepared several types of micro-devices with different lattice geometries. The designs of thestandard devices (microdevices A–E) are shown in Fig. 2. Forall of these devices, the widths of the main and separation

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channels were 25 μm and 15 μm, respectively, and the chan-nel depth was a consistent 25 μm. The slant angle θ waschanged from 15° to 45°. The density factor n was changedfrom 35 to 100. There were pillar structures in the three inletchannels with an inter-pillar distance of 20 μm. The pillarsfunction as pre-filters to prevent the introduction of largeparticle aggregates into the lattice region. There were sixoutlet channels for the individual recovery of separated parti-cles/cells. In addition to the single lattice structures, we fabri-cated a microdevice equipped with eight parallel lattice chan-nels for the purpose of increasing the throughput of cellsorting.

Sorting of model particles

Fluorescent polystyrene microbeads with different diameters(3.0 μm, 4.8 μm, and 9.9 μm) were obtained from ThermoFisher Scientific (MA, USA). These particles were suspendedin an aqueous solution of 0.5% tween 80 and 18% sucrose;sucrose was used to adjust the fluid density and to preventparticle precipitation. The particle concentrations were 6.7 ×105, 4.2 × 105, and 2.8 × 105 particles mL−1 for 3.0 μm, 4.8μm, and 9.9 μm particles, respectively. Before conductingparticle-sorting experiments, the PDMS microdevice wasdegassed in a desiccator; this operation facilitated the re-moval of trapped air in the channel via adsorption. The parti-cle suspension and the buffer solution without particles werecontinuously introduced into the microchannel using syringepumps (KDS200, KD Scientific, MA, USA). The behavior offlowing particles was observed using a fluorescence micro-scope (IX71, Olympus Corp., Tokyo, Japan) equipped with aCCD camera (DP80, Olympus). Sorted particles were recov-ered, and the number of the recovered particles was countedusing a hemocytometer. In addition, the output flow rate

through each outlet was evaluated by continuously introduc-ing distilled water from all three of the inlets and measuringthe output volumes by weighing.

High-speed microPIV

The flow-rate profile in the lattice region was visualized usinga high-speed confocal micro-particle image velocimetry(microPIV) system (Confocal Scanning Micro PIV System,Seika Corp., Tokyo, Japan). Green-yellow colored fluorescent0.5 μm microparticles (Life Technologies, CA, USA) weresuspended in an aqueous solution of 0.5% tween 80 at a con-centration of 1.8 × 1010 particles mL−1. This suspension wasintroduced into the microchannel from the three inlets, at atotal flow rate of 1.2 μL min−1. Confocal fluorescence imageswere captured at 2000 frames s−1, and the flow velocities anddirections were calculated using image-analyzing software(Koncerto, Seika Corp.).

Blood cell sorting

Peripheral blood samples were obtained from healthy volun-teers in accordance with an institutional review board (IRB)-approved protocol. The volunteers submitted written in-formed consent for blood analyses, which were performed inaccordance with protocols approved by the IRB. For leukocyteseparation, the blood samples were diluted at 1/5–1/20 with abuffer solution (phosphate buffered saline containing 0.2%bovine serum albumin). Hoechst 33342 (Life Technologies)was added to the diluted blood sample at a final concentra-tion of 1 μg mL−1. This sample was introduced from inlet 2,whereas the buffer solution was introduced from inlets 1 and3. After recovering the sorted blood cells from each outlet,the numbers of leukocytes and erythrocytes were counted.

Fig. 2 (a) Designs of five types of lattice microchannels with different geometries. (b) Enlarged image of the lattice structure. (c) Photograph ofmicrodevice A. (d) Micrographs showing the inlet region, lattice region, and outlet region of microdevice A.

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Smears were also prepared on glass slides, and then cellswere stained with Wright staining.

For sub-sorting of leukocytes and purification of mono-cytes, condensed blood samples were initially prepared toreproduce the leukocyte-rich blood sample obtained byleukapheresis. A peripheral blood sample was mixed with ananti-coagulation reagent (ACD-A solution, Terumo Corp.,Tokyo, Japan) at a volume ratio of 8 : 1. This sample was thenmixed with an equal volume of DPBS (Life Technologies) andthen poured onto Ficoll-Paque PREMIUM (GE Healthcare,Little Chalfont, UK). After centrifugation for 15 min at 500 ×g at room temperature, the buffy coat layer with enrichedmononuclear leukocytes was recovered. Cells were concen-trated by centrifugation and added to a peripheral bloodsample, to obtain a condensed blood sample. This samplewas diluted with PBS/BSA at 1/5, and used for microfluidic ex-periments. For analyzing the subpopulation of sorted leuko-cytes, an automated haematology analyzer (XS-800i, SysmexCorp., Hyogo, Japan) was used. The monocyte population wasprecisely determined as CD14+ cell population by flow cytom-etry; cells were stained with PE-conjugated mouse anti-human CD14 antibodies (A07764, Beckman Coulter, CA, USA)and analyzed using a flow cytometer (Cytomics FC500,Beckman Coulter). In addition, monocyte enrichment by den-sity gradient centrifugation was performed for the condensedblood sample, as a control experiment. The condensed bloodsample was mixed with an equal volume of DPBS, and gentlypoured onto Ficoll-Paque PREMIUM. After centrifugation for30 min at 500 × g at room temperature, the buffy coat layerwith enriched mononuclear cells was obtained. After washingthe cells twice with DPBS via gentle centrifugation, the cellpopulation was analyzed.

Results and discussionSorting of standard particles

Before conducting particle-sorting experiments, we evaluatedthe volumetric flow rates to each outlet using microdevice A.Distilled water was continuously pumped into the latticedevice through the three inlet channels with a total flow rateof 120 μL min−1. As a result, the output flow rates from thesix outlets were almost equal, but the flow rate from outlet 1was slightly higher (∼22%) than the others (14–18%), asshown in Fig. S1.† The ratio of the input flow rates from thethree inlets did not affect the output flow rates (data notshown). These results indicated that the flow rate profile wasalmost uniform along the x axis, which is a prerequisite forprecisely sorting particles/cells based on the presentedconcept.

Next, we observed the behaviors of standard microparti-cles and examined if particle sorting actually takes place.Fig. 3(a and b) shows the introduced particles flowing nearthe center of the lattice region of microdevice A, when Q1, Q2,and Q3 were 30, 40, and 50 μL min−1, respectively. The red-colored 3.0 μm particles flowed into the separation channelsat a certain frequency. On the other hand, large 9.9 μm parti-

cles did not enter the separation channels even though theyflowed near one sidewall of the main channel, and finallyreached the right side channel (side channel near outlet 6).This was because only a limited, thin flow region near thesidewall of the main channel was split into the separationchannel at each crossing point. Medium-sized 4.8 μm parti-cles sometimes flowed into the separation channels. As a re-sult, these three types of particles were recovered from differ-ent outlets, as shown in Fig. 3(c and d) and Fig. 4(a).Although there was a difference between the theoretical esti-mation and the actual particle behavior, especially in thesense that the 4.8 μm particles flowed into the separationchannels contrary to predictions, we confirmed that thepresented lattice channels function as hydrodynamic filtersfor continuous size-based particle sorting. It is noted that thetotal flow rate did not significantly affect the sorting behav-iors of the particles, when the total flow rate was changedfrom 30 to 120 μL min−1 (Fig. S2†). This result indicates thatthe particle inertia did not have a significant impact on parti-cle/cell sorting under these flow rate conditions.

Next, we investigated the effect of the initial position ofparticle introduction by changing the ratio of the input flowrates, because this parameter may affect the sorting results.High separation resolution is expected when the relative flowrate Q2 is decreased, but we kept Q2 at 40 μL min−1 and thetotal flow rate at 120 μL min−1, in order to ensure a relatively

Fig. 3 Sorting of fluorescent standard microparticles (3.0 μm red, 4.8μm green, and 9.9 μm green particles). The input flow rates Q1, Q2,and Q3 were 30, 40, and 50 μL min−1. Microdevice A was used. (a andb) Particles flowing near the center of the lattice region, (c–f) particlesflowing near outlets 3–6 of the lattice region.

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high sorting throughput. As a result, the introduced particleswere separated for all of these conditions. The output posi-tions of the three types of particles were shifted to the right(direction to outlet 6) when the relative value of Q1 was in-creased and that of Q3 was decreased, because the initial po-sitions of the particles shifted to the right (Fig. 4(b and c)).This result suggested that the input position of particle intro-duction is the critical factor dominating the output positionsof particles. It was expected that the sorting precision, i.e.,the ability to distinguish particles with different sizes, wouldbe improved if the relative flow rate of the particle suspen-sion (Q2) was decreased. In the following experiments, the ra-tio of the flow rates was fixed at 3 : 4 : 5 unless otherwisenoted.

Flow-profile visualization using confocal microPIV

We assumed that the thin flow region near the sidewall ofthe main channel (with a width of wb) is divided into the sep-aration channel, which is determined by the ratio of thedivided flow at a crossing point. We then investigated if theratio of the divided flow rate actually corresponds to the theo-retical estimation. The flow profile near the center of thelattice region of microdevice A was visualized using a high-speed confocal PIV system and 0.5 μm fluorescent particlesas tracers. The total flow rate was 1.2 μL min−1, which wassuitable for clearly visualizing the flow profile. This value wassignificantly lower than that of the particle sorting experi-ments, but we considered that the flow profiles would not besignificantly changed because of the stable laminar flow andthe low Re value in the main channel (Re in the main chan-nel was ∼10 when the total flow rate was 120 μL min−1).

The obtained flow rate profile in an x–y plane at z = 12.5μm (center in the depth direction) is shown in Fig. 5. Themaximum flow speed in the main channel was ∼3.5 mm s−1,whereas that in the separation channel was ∼0.076 mm s−1.Considering that the cross-sectional area of the main channel(25 × 25 μm) was larger than that of the separation channel(15 × 25 μm), the ratio of the volumetric flow rate split into aseparation channel was ∼1/80 of that flow through a crossingpoint. This value is slightly different from but reasonablyconsistent with our theoretical estimation, in which the volu-metric flow rate split into the separation channel at a cross-ing point is 1/100. This slight difference would be the main

reason why the output flow rate from outlet 1 was higherthan those from other outlets (Fig. S1†). Based on theobtained value of Qsep/Qmain = 1/80, the value of wb was esti-mated to be ∼1.65 μm from eqn (1), indicating that particleslarger than ∼3.3 μm would not flow into the separation chan-nels. It is notable that there is a variation in the value ofQsep/Qmain, depending on the position in the lattice region,especially near the side channels.

Effect of microchannel geometry on the particle sortingefficiency

Various geometrical parameters of the lattice channel may af-fect the sorting efficiency of particles, including the widths ofthe main/separation channels, the density factor n, the slantangle θ, and the crossing angle of the main and separationchannels. Here, we examined two factors, n and θ, byemploying several types of microdevices, as shown in Fig. 2.The flow rates were kept constant (Q1, Q2, and Q3 were 30,40, and 50 μL min−1) for all of these microdevices. Fig. 6shows the sorting results for standard particles using lattice

Fig. 4 Sorting results of standard particles using microdevice A, when the input flow rates Q1, Q2, and Q3 (a) 30, 40, and 50 μL min−1, (b) 40, 40,and 40 μL min−1, and (c) 50, 40, and 30 μL min−1, respectively. Each data represents the mean ± SD from 3 experimental results.

Fig. 5 Visualization of the flow profile in microdevice A using high-speed confocal micro-PIV. Fluorescent particles with a diameter of 0.5μm were used as the tracer. This image shows the flow-rate distribu-tion in an x–y plane at z = 12.5 μm (center in the depth direction). Themicrochannel wall was visualized by white lines.

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channels with different values of n (microdevices B and C,with n of 55 and 35, respectively). In contrast to the sortingresults using microdevice A with an n value of 100 (Fig. 4(a)),3.0 μm and 4.8 μm particles were not separated. On theother hand, 9.9 μm particles were separated from thesesmaller particles, but the outlet position was slightly shiftedto the left (direction to outlet 1) compared to microdevice A.It was theoretically expected that the ratio of the flow ratesplit into the separation channel at the crossing point wouldincrease when n decreased, which results in the increase ofthe critical size for sorting and a shift of the particle outputposition to the left. The obtained result was consistent withthis theoretical tendency and indicates that the critical sizefor particle sorting is tunable by controlling the value of n.

Next, we used microdevices D and E to examine the effectof θ on the separation efficiency. In these devices, θ was 30°and 45°, respectively, and the distances between the neigh-boring main channels, dmain, were changed in order to keepthe density factor n and the boundary width wb constant. Theresults of the particle sorting are shown in Fig. 7. When θ

was 30°, 9.9 μm particles were separated from other smallerparticles and mainly distributed to outlet 6, similar to thecase with microdevice A (Fig. 4(a)). In contrast, 3.0 μm and4.8 μm particles were not separated. This result indicatedthat the critical size for particle sorting was increased com-pared to microdevice A with a θ of 15°. Interestingly, the in-troduced particles were not separated when θ was 45°, andthey were mainly distributed to outlet 2, regardless of the par-ticle sizes. These results clearly suggest that the slant angle isalso a critical factor that dominates particle behavior.

Blood cell sorting

The efficient sorting of cells is one of the most importantapplications of microfluidic systems. In particular, thesorting and phenotyping of specific blood cell populationsare in high demand for clinical diagnosis, treatment, andbiomedical research. We first applied the lattice device to thepurification of leukocytes. A peripheral blood sample was di-luted at 1/20 and introduced into microdevice A from inlet 2.The inlet flow rates Q1, Q2, and Q3 were 30, 40, and 50 μL

min−1, respectively. Fig. 8(a) shows the flowing blood cellsnear the outlets of the lattice region. Erythrocytes weremainly distributed to outlets 2, 3, and 4, whereas leukocyteswere recovered from outlets 5 and 6. During the cell sortingoperation for ∼30 min, cell separation behaviors werestable and we did not observe any microchannel clogging.

Fig. 6 Results of particle sorting using microchannels with differentvalues of the density factor n; (a) n = 35 (microdevice C) and (b) n = 55(microdevice B). Q1, Q2, and Q3 were 30, 40, and 50 μL min−1,respectively. Each dataset represents the mean ± SD from 3experimental results.

Fig. 7 Results of particle sorting using microchannels with differentvalues of the slant angle θ; (a) θ = 30° (microdevice D) and (b) θ = 45°(microdevice E). Q1, Q2, and Q3 were 30, 40, and 50 μL min−1,respectively. Each dataset represents the mean ± SD from 3experimental results.

Fig. 8 Blood cell separation using microdevice A. (a) Cells flowingnear outlets 2 and 5 of the lattice region. (b) Micrographs showingcells before separation and separated and recovered from outlets 2, 4,and 5 of microdevice A. Cells were stained with Wright's stain. (c)Distribution of erythrocytes and leukocytes to each outlet. Eachdataset represents the mean ± SD from 3 experimental results.

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After collecting cells from the outlets, the numbers of cellsdistributed to each outlet were calculated. As shown inFig. 8(b and c), the separation results for erythrocytes weresimilar to those for the 3.0 μm particles shown in Fig. 4(a). Itwas previously reported that erythrocytes behave like spheri-cal solid particles with a size of 2–3 μm in a hydrodynamicseparation scheme using microfluidic devices,26 which corre-sponds to the current separation results. On the other hand,leukocytes were mainly distributed to outlet 5. Consideringthat the 9.9 μm particles were mainly recovered from outlet 6under the same operational conditions (Fig. 4(a)), and thefact that there should be leukocytes with sizes larger than 10μm, the leukocytes probably deformed and flowed into theseparation channels. The ratios (purity) of leukocytes in therecovered samples from outlets 5 and 6 were ∼80% and∼95%, respectively, indicating that direct and nearly perfectfractionation of leukocytes/erythrocytes was possible usingthe presented sorting system.

Purification of monocytes from condensed blood samples

In this section, we further demonstrated the sub-sorting ofleukocytes. We attempted to purify monocytes, which are thelargest cells among leukocyte populations and constitute ap-proximately 5% of leukocytes. Monocytes are clinically impor-tant as they play an important role in the immune systemand angiogenesis, and are used for transplantation therapyfor ischemia.54 In addition, monocytes can differentiate intodendritic cells55 and pancreatic β-cell-like cells,56 which arecritical for cancer immunotherapy and for treating type I dia-betes mellitus, respectively. In conventional methods, mono-cytes are purified by density gradient centrifugation usingPercoll,57 Ficoll-Hypaque,58 or OptiPrep,59 which achieveslarge scale purification. For cancer immunotherapy, mono-cytes are isolated from patients using two-step procedures: (i)leukocyte-rich, condensed blood samples are obtained byleukapheresis and (ii) monocytes are subsequently purifiedby density gradient centrifugation.60,61 However, centrifugalseparation techniques usually do not enable high purificationefficiency and recovery rates. In addition, it is known that themedium for density gradient centrifugation degrades themonocyte functions and alters the surface characteristics.62,63

A simple technique for purifying monocytes, which can re-place the conventional density gradient centrifugation,should facilitate the clinical application of monocytes. Untilnow, microfluidic devices have been used to analyze bloodcell phenotypes including monocytes,64–66 and the immuno-logical separation and/or depletion of monocytes have alsobeen demonstrated.67,68 However, there have been only a fewreports demonstrating hydrodynamic methods for monocyteseparation/enrichment,23,69 and hence, it can be said that ap-proaches for label-free sorting/enrichment of monocytesusing microfluidic devices have not been fully developed.

To achieve monocyte purification, we designed and fabri-cated another lattice device shown in Fig. 9(a), in which nwas 41 and θ was 30°. Compared to microdevice A, the out-

lets for leukocyte recovery were expected to shift to the left(direction to outlet 1), in order to sort leukocytes into severalfractions. In addition, the position of inlet 2 was shifted tothe left to change the initial position of the cells, in order toeffectively use the entire lattice region for cell sorting. The re-sult of standard particle sorting, when Q1, Q2, and Q3 were10, 10 and 40 μL min−1, respectively, is shown in Fig. 9(b),and the measurement of the output flow rates is shown inFig. S3.† Compared to the particle sorting result using micro-device D (Fig. 7(a)), the output positions of the three types ofparticles shifted to the outlets with smaller numbers, primar-ily because of the decreased n and the shift of the introduc-tion positions of the particles.

Next, we investigated if the presented microfluidic latticecan replace the centrifugal monocyte purification process,which is combined with leukapheresis and employed for can-cer immunotherapy. We prepared condensed, leukocyte-richblood samples that reproduced the leukapheresis products,and performed microfluidic cell purification for these sam-ples. The cell composition of the condensed blood sample, incomparison with normal peripheral blood, is shown in Fig.S4.† The distribution of blood cell population using an

Fig. 9 Direct purification of monocytes. (a) Microfluidic device used tosort monocytes. In this device, θ, n, dmain, dsep, and wb were 30°, 41,850 μm, 35 μm, and 2.3 μm, respectively. (b) Sorting results for modelparticles using this microdevice. (c) Sorting results for the blood cellsanalyzed by an automated haematology analyzer and flow cytometer(monocytes). Samples from two outlets (1 and 2, 3 and 4, and 5 and 6)were combined. As a control, density gradient centrifugation was alsoperformed. In (b) and (c), each dataset represents the mean ± SD from3 experimental results.

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automatic analyser and flow cytometry is shown in Fig. 9(c),where samples from outlets 1 and 2, 3 and 4, and 5 and 6were analyzed together. Erythrocytes were separated and re-covered mainly from outlets 1 and 2. Leukocytes were recov-ered from outlets 1–6, but monocytes were mainly distributedto outlets 5 and 6 (74 ± 20%, n = 3), and the purity of mono-cytes in this sample fraction was 78 ± 14%. As a controlexperiment, we performed density gradient centrifugationusing Ficoll-Paque for the same condensed blood samples,which resulted in a poorer monocyte recovery of 52 ± 17%with a purity of 20 ± 3%. These results clearly demonstratedthe high ability of the presented microfluidic system formonocyte separation in comparison to the conventionalmethods. Aside from the recovery/purity, the presented pro-cess is advantageous because it enables efficient enrichmentof intact monocytes without the use of a density gradientmedium or immunological labeling that possibly affectsmonocyte functionalities. The prepared monocytes areexpected to be available for various downstream biomedicalapplications, especially transplantation therapies and furtherdifferentiation studies.

High-throughput sorting by parallel arrangement

The processing speed for cell samples of the single lattice de-vice is of the order of several tens of microliters per minute,which sometimes might not be satisfying for conducting gen-eral medical/biological applications. Hence, we designed amicrodevice with a parallel lattice arrangement to increasethe throughput of particle/cell sorting. We employed the lat-tice structure shown in Fig. 9(a) as the unit structure, anddesigned and fabricated 8-unit parallel devices as shown inFig. 10. The microchannel network was formed on two levels(Fig. 10(a)). The parallel lattice structures were located on thebottom layer, whereas the branched inlet/outlet channels for

effective fluid delivery and recovery were on the top layer. Ineach lattice unit, the outlet ports 1–4 and outlet ports 5 and 6shown in Fig. 9(a) were united to sort particles into 2 frac-tions (outlets 1 and 2). The inlet/outlet channels were evenlybranched in a tournament-tree format and were connected tothe 8 lattice units. The resistances of the outlet channels tooutlets 1 and 2 were precisely adjusted (1 : 4.4), so that theratio of the output flow rates became almost equal to that forthe single unit device (82% to outlets 1–4, 18% to outlets5 and 6).

The results of particle sorting are shown in Fig. 10(c).Almost all the 9.9 μm particles were recovered from outlet 2,whereas the smaller particles were from outlet 1, as in the caseof the single unit microdevice (Fig. 9(b)). We changed the totalinput flow rate from 300 to 1200 μL min−1, but the sorting effi-ciency was not affected, showing the robustness against theinput flow rate. Under the highest flow rate conditions, theflow rate of the particle suspension was 400 μL min−1. Theseresults showed that the numbering-up strategy is effective foreasily increasing the sorting throughput. Recently, microfluidicdevices enabling ultra-high-throughput particle/cell sortinghave been developed using submillimeter-sized micro-channels,39 but in general, the sorting precision is not suffi-ciently high when the throughput is dramatically increased.The presented microdevice with parallel unit structures wouldbe advantageous because it does not degrade the sorting preci-sion even under high flow-rate conditions. If multiple PDMSplates with the bottom layer are laminated and one plate withthe top layer is placed on the top, the throughput of particlesorting will be further increased.

Conclusions

We have successfully demonstrated a new method for parti-cle/cell sorting using simple lattice-shaped microchannel

Fig. 10 (a) Illustration showing the parallel microchannel incorporating 8 lattice structures. The lattice channel shown in Fig. 9(a) was arranged inparallel. The device was composed of a two-level microchannel network. (b) Photograph of the fabricated device. (c) Sorting results of model par-ticles with different flow rates. The particle suspension was introduced from inlet 2 with a flow rate of Q2.

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networks, which work as size-selective sieves. We identifiedimportant geometrical parameters that affected the particlesorting efficiencies, including the density factor and the slantangle of the main channel. As a practical application of thismicrofluidic system, we demonstrated direct leukocyte/mono-cyte purification from blood samples, and the obtained re-sults showed the versatility and feasibility of the lattice de-vice, especially for cancer immunotherapy. One of the mostimportant characteristics of the presented system is its ro-bustness against the problem of microchannel clogging,which was confirmed by the experiments of blood cellsorting. The presented concept is universal and versatile, andwould be applicable not only to cell/particle separations butalso to general bioparticle manipulations including particle/cell concentration, treatment, and characterization, as in thePFF, HDF, and DLD techniques. We have not optimized someof the geometrical/operational parameters, including thecrossing angle of the main/separation channels, the widthand length of the entire lattice area, fluid viscosity, and parti-cles' physicochemical characteristics, and hence, further ex-perimental and theoretical studies would be supportive tomaximizing the efficiency of the presented approach.

Acknowledgements

This study was supported in part by Grants-in-aid for Scien-tific Research (23106007 and 26286032) from Ministry of Edu-cation, Culture, Sports, Science, and Technology, Japan. Wethank Ms. Yuko Matsuda for technical assistance.

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