Microfabricated Asanalytical Systems for Integrated Cancer

REVIEW

Microfabricated analytical systems for integrated cancercytomics

Donald Wlodkowic & Jonathan M. Cooper

Received: 2 February 2010 /Revised: 29 March 2010 /Accepted: 3 April 2010 /Published online: 25 April 2010# Springer-Verlag 2010

Abstract Tracking and understanding cell-to-cell variabil-ity is fundamental for systems biology, cytomics andcomputational modelling that aids e.g. anti-cancer drugdiscovery. Limitations of conventional cell-based techni-ques, such as flow cytometry and single cell imaging,however, make the high-throughput dynamic analysis oncellular and subcellular processes tedious and exceedinglyexpensive. The development of microfluidic lab-on-a-chiptechnologies is one of the most innovative and cost-effective approaches towards integrated cytomics. Lab-on-a-chip devices promise greatly reduced costs, increasedsensitivity and ultrahigh throughput by implementingparallel sample processing. The application of laminar fluidflow under low Reynolds numbers provides an attractiveanalytical avenue for the rapid delivery and exchange ofreagents with exceptional accuracy. Under these conditions,the fluid flow has no inertia, enabling the precise dosing ofdrugs, both spatially and temporally. In addition, byconfining the dimensions of the microfluidic structure, itis possible to facilitate the precise sequential delivery ofdrugs and/or functional probes into the cellular systems. Asonly low cell numbers and operational reagent volumes arerequired, high-throughput integrated cytomics on a singlecell level finally appears within the reach of clinicaldiagnostics and drug screening routines. Lab-on-a-chipmicrofluidic technologies therefore provide new opportuni-ties for the development of content-rich personalizedclinical diagnostics and cost-effective drug discovery. It is

largely anticipated that advances in microfluidic technolo-gies should aid in tailoring of investigational therapies andsupport the current computational efforts in systemsbiology.

Keywords Cytomics . Cytometry .Microfluidics .

Lab-on-a-chip . Real-time cell assays . Cell sorting

Introduction

Cell populations represent an intrinsically heterogenic andstochastic system with a high level of spatio-temporalcomplexity [13]. Temporal cell-to-cell variability arisesfrom subtle fluctuations in the concentrations of regulatoryproteins, protein oscillations, position in the cell cycle andthe activation of multiple compensatory and failsafemechanisms (e.g. apoptosis, autophagy) (Fig. 1) [36].Within such heterogenic clusters, multiple variables oftenact at the same time while interconnected molecularpathways provide adaptive and compensatory outcomes(Fig. 1). Importantly, fluctuations at a single cell level oftenlead to profound changes in the structure of particular cellpopulation [710]. These phenomena are particularlyimportant in cancer research where the regulation of cancercell death and survival involves rapid switches betweenboth stochastic and binary signalling events. The level ofcomplexity, with numerous variables acting at the sametime, requires multiparametric and dynamic investigation oflarge numbers of single cells. This feat is still largelyinaccessible by using conventional bioanalytical and diag-nostic approaches [710]. Figure 1 describes a hypotheticalsituation where three cancer cells derived from theseemingly homogeneous population respond differentiallyto a drug stimulus in time. Whilst expression of an arbitrary

D. Wlodkowic (*) : J. M. Cooper (*)The Bioelectronics Research Centre, University of Glasgow,Oakfield Avenue,Glasgow G12 8LT, UKe-mail: [email protected]: [email protected]

Anal Bioanal Chem (2010) 398:193209DOI 10.1007/s00216-010-3722-8

molecule that supports cell proliferation and blocks celldeath rapidly decreases in one cell (denoted cell 1) anothercell responds to the drug with a substantial delay (denotedcell 2). Yet another cell (denoted cell 3) is characterized byonly brief oscillation in the protein level that is followed bya quick recovery (Fig. 1). As a result of stochasticmolecular responses within the population of cells one cellgains a survival advantage while two others are eliminatedby drug-induced reduction of protein level and/or itsactivity (Fig. 1) [5, 79]. The surviving cell can subse-quently divide and expand in the presence of the druggiving rise to a resistant clone (Fig. 1). This hypotheticalexample demonstrates that evolving subpopulation struc-ture can be defined by single cell stochastic reactions.These mechanisms provide e.g. means for repopulation andemergence of resistant clones that are the basis of refractoryand relapsed cancers [1, 7, 1012]. Figure 1 illustrates thus

that understanding the cell-to-cell variability is fundamentalfor systems biology and particularly important in processesof high spatio-temporal complexity such as response ofcancer cells to therapy (Fig. 1) [69]. The latter is a criticalevent that defines tumour growth rate and response to anti-cancer therapy and has recently provided a new frameworkfor the rationally designed and molecular anti-cancertherapeutics. Yet only by obtaining the real-time insightsinto the drugcell interactions can one create information-rich data sets, significantly improving the in vitro validationof molecular drug targets [13, 14]. The possibility ofcontinuously tracking individual cells from the time ofencountering a stress signal, through the decision-makingand execution phases, also provides previously inaccessibleinformation of how complex biological systems progressfrom e.g. life-maintaining to death-allowing steady states[79, 15, 16]. The most promising are, in this respect, the

Fig. 1 Reactions of cancer cellsto therapeutic compounds arestochastic in nature and can leadto a variable therapeutic out-come. a A hypothetical clonalcancer population, respondingdifferentially to a drug stimulus,in time is presented. Stochasticemergence of two different phe-notypes (red, green) in time isseen on the upper panel. Notethat whilst expression of anarbitrary molecule that supportscell proliferation and blocks celldeath rapidly decreases in onecell (denoted cell 1, blue) an-other cell responds to the drugwith a substantial delay (denotedcell 2, red). Yet another cell(denoted cell 3, green) is char-acterized by only brief oscilla-tion in the protein level that isfollowed by a quick recovery ofthe protein level and/or activityprofile (lower chart). Coloursrepresent three distinct subpo-pulations of cells. b Repopula-tion initiated by stochastic cellresponses to an anti-cancer drug.Clonal cell subpopulation gain asurvival advantage whilst twoothers are eliminated by drug-induced reduction of proteinlevel and/or its activity (as dis-cussed in a)

194 D. Wlodkowic, J.M. Cooper

conceptual multidimensional assays where the combinationof gene delivery technology (genomics), functional anddynamic live-cell analysis (cytomics) and intracellularantibody staining of selected proteins, also known asimmunocytochemistry (proteomics) can provide innovative,multivariate assays for high-content data mining andenhanced elucidation of cell signalling pathways [13, 14,1618].

Microfabricated systems for single cytomics

Recent studies in systems biology have shed new light onthe underlying molecular mechanisms of cell-to-cell vari-ability in cancer cell decision making [412]. Mostimportantly it appears that not only genetic and epigeneticdifferences between cancer cells, but also extremely subtlechanges in protein concentrations and the intrinsic stochas-ticity of biochemical reactions within the cell signallingpathways contribute to the observed cell-to-cell variability[412]. As a result even in a theoretically genetically andepigenetically identical population of cells (only sister cellsare deemed to be genetically and epigenetically identical)the responses to anti-cancer drugs will always be stochasticand cellular decisions will be probabilistic in nature [412].Dynamic switches between the stochastic and all-or-nothing events are difficult to record by using conventional,end-point approaches [15, 16, 19]. The major drawback ofsuch binary analysis (e.g. western blotting, ELISA, QT-PCR, fluorimetry, spectrophotometry) is that they are basedon analysis of a total cell population that averages theresults from every given cell [16, 2023]. Importantly theyonly capture a snapshot of the cellular reaction which isinherently a stochastic system [20, 21]. Flow cytometry(FCM) is one of the conventional technologies that can beused to overcome a frequent problem of traditional bulktechniques [2023]. The major advantages of FCM includethe possibility of multiparameter measurements (i.e. thecorrelation of different cellular events at a time), single cellanalysis (the avoidance of bulk analysis) and rapid analysistime (measuring thousand of cells per second) [2126]. Acommon drawback of conventional FCM methods is,however, that cells are suspended in a laminar stream offluid that is rapidly discarded [27, 28]. As a result norepetitive analysis of time-resolved events is possible.Furthermore, as only integrated fluorescence is collectedby photomultiplier tubes (PMTs) and no imaging is used,this design suffers from the loss of both temporalinformation and spatial (subcellular data) that enables thecharacterization of many morphological features [15, 16,27, 28].

Measurement of inter- and intra-cellular complexity thatcan uncover e.g. cell-to-cell variability in cancer cell

decision making requires an in-depth 4D investigation ofcell populations at a single cell level [15, 16, 1921]. Thedevelopment of reliable methodologies to track the behav-iour of single cells within subtle cell subpopulations is alsoof paramount importance in clinical diagnostics andpersonalized therapy. Inherent limitations of traditionalflow cytometry have recently stimulated the fast develop-ment of slide-based cytometers and in-flow imagingcytometers (multispectral imaging cytometry) that combineadvantages of both flow cytometry and fluorescence imageanalysis (FIA) [2729]. These innovative technologiesemploy cytometric principles rather than conventionalimage analysis to collect high-content data on single cells[2729]. When combined with slide-based and/or micro-plate scanning and increasing integration with Nipkowspinning disk confocal modules, these systems providereasonable throughput and high-resolution screening capa-bilities at a subcellular level. Yet a common drawback of allconventional high-throughput analysers is bulkiness, equip-ment and maintenance costs [25, 26]. Moreover, the highpower consumption and requirement for highly trainedservice personnel precludes their widespread use [3032].Furthermore, a considerable number of cells and reagentsare usually required for each conventional cell analysis(typically above 104/mL) and processing of the samplesprior to analysis is time consuming, involving severalcentrifugation steps [21]. As the cost and time savings playan ever increasing role in drug discovery and medicaldiagnostics, enabling strategies that can reduce expendi-tures while increasing throughput and content of informa-tion from a given sample attract a mounting interest withinthe biopharmaceutical community [3336].

Transfer of traditional methods to a microfabricatedformat provides a means to increase both the resolution ofanalysis and sampling throughput while reducing the costsof a single assay [3638]. During the last decade, a range ofmicroarray technologies have been developed [3941].Technological foundations initially developed for DNAmicroarrays have recently provided the starting point forprotein, carbohydrate and tissue microarrays that are slowlyemerging as useful tools in both clinical diagnostics anddrug discovery pipelines [4145]. They offer miniaturiza-tion, low reagent consumption, automation as well asqualitative and quantitative approaches to analyse geneand protein expression on a population level [44]. They do,however, suffer from a lack of capabilities to monitor singleliving cells in real time and as such represent a binarysystem that averages the results from every given cell whilecapturing a snapshot of the intermittent cellular reaction[15, 16, 19, 20]. As a result current experimental evidenceof transient and intermittent physiological processes islikely biased by the intrinsic time delay between reagentaddition, cell reaction and the ensuing analysis. The advent

Microfabricated analytical systems for integrated cancer cytomics 195

Fig. 2 Microfabricated (lab-on-a-chip) bioanalytical systems for cell-based assays allow for extensive integration of on-chip componentsand high-level of miniaturization. a Integrated microfluidic cell cultureand lysis on a chip. Chip with six separate devices filled with a dye forchannel visualization (left panel). A magnified image of one chambershowing the trapping region structure which consists of an array offour cell traps separated by spacers (right panel). Scale bars are1.3 mm. (Reproduced with permission from The Royal Society ofChemistry (RSC) from ref. [119].) b A high aspect ratio microfluidicdevice for high-throughput mammalian cell culture. (Reproduced withpermission from The Royal Society of Chemistry (RSC) from ref.[75].) c Integrated and automated microfluidic cell culture system.

Insets show a close-up of two culture chambers, with the multiplexerflush channel in between them (left), the input multiplexer, with on-chip peristaltic pump, a waste output for flushing the mixer, and thecell input line (right). (Reprinted with permission from ref. [120].Copyright 2007 American Chemical Society.) d A multi-step micro-fluidic device for studying cancer metastasis. A layout of the devicewith 6 reservoirs, inlet ports for cell seeding and Matrigel loadingports (left panel). Cell migration, transmigration and cell invasion areathat comprises 10-mm-wide by 150-mm-long microfabricated gaps(right panel). (Reproduced with permission from The Royal Societyof Chemistry (RSC) from ref. [121])


of microfluidic lab-on-a-chip technologies and their inte-gration to design micro total analysis systems (TAS) isone of the most innovative approaches towards integratedcytomics and improved drug screening routines (Fig. 2)[3638]. Microfabricated lab-on-a-chip devices promise

greatly reduced equipment costs, increased sensitivity andthroughput by implementing massive experimental paralle-lization while performing analysis on a single cell level(Fig. 2) [37, 46, 47]. Most importantly, as only low cellnumbers and operational reagent volumes are required,

Fig. 3 Microfabricated cell arrays for extended cell culture and high-throughput analysis at a single cell level. a Microfabricated cell arrayfor correlative high-content analysis of individual non-adherent cells.Scanning electron microscope (SEM) image of Jurkat T cells insidethe microfabricated glass cell array. Scale bar 20 mm (left panel).Device allows for lucid optical examination of cells by utilizing e.g.wide-field transmitted light (middle panel) and confocal microscopy(two Rhodamine 123-stained MOLT-4 cells are shown) (right panel).(Reproduced with permission from The Royal Society of Chemistry(RSC) from ref. [122].) b Proprietary microfabricated high-density cellarray. Microwells 650650 m were fabricated by anodically bondingthe silicon grid wafer to a 500-m borofloat glass substrate (leftpanel). Cell proliferation analysis on a high-density cell array (right

panel). Long-term clone formation was started with a single K-562cell FACS sorted to one well and cultured for up to 2 weeks.(Reproduced with permission from Picovitro AB, Stockholm, Swe-den.) c Cell microarray platform fabricated by a PEG (poly(ethyleneglycol) diacrylate) hydrogel patterning on glass. SEM image ofMOLT-3 leukaemic cells confined in 15 mm15 mm PEG wells.(Reproduced with permission from The Royal Society of Chemistry(RSC) from ref. [123].) d CellTRAY, a novel micro-etched live-cellscreening technology. Independently addressable regions of glass orplastic microwells allow for a multiplexed and time-resolvedexperimentation at a single cell level. Data courtesy of Dr CathyOwen reproduced with permission from Nanopoint Inc. (Honolulu,Hawaii , USA)


dynamic cytomics on rare cell populations finally appearswithin investigational reach (Fig. 2) [37, 47, 48]. LOCsystems also provide innovative ways to simultaneouslyanalyse large population of single cells where, to uncoverthe stochastic basis of cellular decision making, each cellhas to be isolated from others to minimize the influence ofextrinsic factors such as cell-to-cell contacts and paracrinesignalling. Many microfluidic systems can also track singlecell responses multiparametically, whereby the position ofevery cell is encoded and spatially maintained overextended periods of time. In this respect, microfluidicplatforms can fundamentally enhance the mathematicaloncology and systems biology efforts and provide newvistas for a new generation of rationally designed anti-

cancer drugs. They will be, therefore, a valuable tool for theemerging field of systems oncology and provide new vistasto validate mathematical models on patient-derived cells.To reflect on this microfluidics has already been heralded asan emerging technology with a multitude of applications inhigh-throughput drug screening routines, content-rich per-sonalized clinical diagnostics and diagnostics in resource-poor areas (Fig. 2) [3638, 4953].

Living cell microarrays

In the post-genomic era the functional assessment of newlyidentified genes and validation of potential therapeutic

Fig. 4 Dielectrophoresis (DEP)-based cell arrays. a DEP dynam-ic array cytometer that allows forautomated loading, observationand arbitrary sorting of cells aftertheir optical examination. SingleDEP traps consist of four elec-troplated gold electrodes ar-ranged trapezoidally. (Reprintedwith permission from ref. [70].Copyright 2002 AmericanChemical Society.) b Prototypeof a negative dielectrophoresis(nDEP) trapping array for tran-sient immobilization of singlecell. The ring-shaped nDEP trapswere fabricated from two titani-um/platinum layers with a ben-zocyclobutene (BCB) dielectric(left panel). c Bead and/or cellimmobilization using in a nDEPis possible even in a continuousflow of media (right panel).(Reproduced with permissionfrom The Royal Society ofChemistry (RSC) from ref. [68].)


targets are a primary challenge [54, 55]. The recentintroduction of molecularly targeted anti-cancer therapeu-tics has revealed a significant need for new classes ofclinical bioassays [5659]. The shift from cytotoxicchemotherapy to biomolecular therapeutics requires thatdrug efficacy be assessed at the biologically active dose thatmodulates the target, rather than through a conventionaldosetoxicity relationship [5659]. In this context, func-tional cytomics is slowly becoming an omnipotent part ofthe post-genomic drug discovery pipelines [13, 14].

Multidimensional and high-throughput analysis of live cellscan serve as an excellent dynamic analytical tool, forinstance to (i) understand micro-evolution of cancer cells,(ii) discover function of new genes and (iii) screen large-scale chemical and genomic libraries [1316, 19, 20].Cellular processes such as cancer cell responses to drugshave large cell-to-cell variations, and are often initiated and/or executed in a multi-organelle/multi-pathway fashion [16,19, 60, 61]. Moreover, the majority of events, e.g. (i)mitochondrial or ER/Golgi dynamics during execution of

Fig. 5 Integrated microfluidicdevices for a long-term cellculture. a A microfluidic devicefor a high-throughput mamma-lian cell culture. Stable gradientgeneration can be created acrossthe columns of independentmicrochamber arrays (left pan-el). SEM image of singlemicrochamber (right panel).Multiple perfusion channelssurround the main culturechamber that is 40 m in heightwith a diameter of 1 mm.(Reproduced with permissionfrom The Royal Society ofChemistry (RSC) from ref.[75].) b Microfluidic living cellarray for real-time gene expres-sion studies. Multilayer designconsists of a 1616 array of cellculture chambers that are isolat-ed by sets of reversible PDMSbarriers. PDMS barriers arecontrolled by valve controlmanifolds (left panel). Phasecontrast image of a single cellculture chamber with H35 cellsreaching confluence (right pan-el). (Reproduced with permis-sion from The Royal Society ofChemistry (RSC) from ref.[76].) c Application of a micro-fluidic living cell array (as de-scribed in b) for a real-time geneexpression analysis usingtime-lapse microscopy and ge-netically modified reporter cells.Fluorescence time-lapse imagesof NFB and GRE GFP-reporters in microfluidic cellculture chambers 2, 5, 8, 11, 14and 17 h after stimulation.(Reproduced with permissionfrom The Royal Society ofChemistry (RSC) from ref.[76].)


caspase-dependent apoptosis, (ii) autophagy and (iii)phagocytosis, all have highly ordered interactions betweendifferent intracellular compartments [5, 8, 9, 60]. Monitor-ing of such biological phenomena at a single cell levelrequires real-time and spatial detection systems with drugdelivery resolution on a micrometre scale [15, 16, 19, 20].This is currently not attainable in any macroscale analyticalsystems. As a result many attempts have been made totransfer single cell assays to a microfabricated systemcommonly known as a microarray format [54, 55].Especially work by the group of Sabatini provided newinsights on how to apply static high-density cell arrays toperform discrete and parallel transfection of cells withthousands of RNAi reagents [54, 55]. Adhering andgrowing cells reportedly internalize the nucleic acids andbecome transfected (the remaining cells form a non-transfected monolayer) [55]. Sabatinis group has proventhat this reverse transfection array format is capable ofefficient and spatially resolved cell transfection of humancells [55]. Patterned RNAi cell microarray technology isundoubtedly a major step towards the miniaturization andsimplification of high-throughput cell assays in a micro-array format. Others attempts have been made to use a

high-density living cell array platform where cells aregrown in microwells with sizes of the order 10600 m(Fig. 3) [6267]. These are fabricated in glass or biocom-patible polymers such as poly(dimethylsiloxane) (PDMS)using standard microfabrication techniques used in theelectronics industry (Fig. 3) [6267]. Many differentdesigns have been proposed that support large-scalesingle-cell trapping and real-time cell imaging using micro-well arrays (Fig. 3) [6267]. This is a very promisingtechnique mostly due to its simplicity of fabrication andstraightforward operation [6267]. Trapping single cells insuch static systems can be, however, influenced by manyfactors such as cell size, cell buoyancy, microwell diameter,microwell depth and settle time [6365]. Moreover theprecise delivery and exchange of reagents using static cellmicroarrays still requires macroscale liquid handling equip-ment [66, 67]. Recently another innovative technique togenerate ordered arrays of cells has been proposed by usingelectromagnetic (dielectrophoretic; DEP) pattering (Fig. 4)[6870]. To create spatially defined cell arrays the DEPtechnique generally confines cells by exploiting theirelectric dipoles that are induced in the electric field gradient[47, 48, 6870]. The cell trapping element is usuallycomposed of a metal ring electrode and the adjacent groundplane of the chip that creates a closed electric field cage(Fig. 4) [6870]. DEP allows for a stable immobilization offlowing cells even when they flow in a fluid stream ofrelatively high velocity. Moreover, by controlling individ-ually addressable electrodes, selected single cells or smallclusters of cells can be released from the DEP trappingregion at any given time [6879]. This opens vastopportunities for integrated devices with multiple analyticalcapabilities on one chip [40]. DEP array technologies are,however, complex and expensive to manufacture as theyrequire fabrication of multiple electrode units for everysingle cell trap [40, 6870]. Moreover, they require highconductivity physiological media and generate high inten-sity electric field regions (especially in positive DEP) thatsubject cells to large transmembrane potential changes.New reports suggest, however, that the negative DEP(nDEP) technology can overcome such limitations of DEP(Fig. 4) [68]. nDEP trapping maintains levitating cellsinside the potential energy wells [68].

All platforms described so far are based an open-accessformat. As such they are prone to the substantial evapora-tive water losses that hamper their exploitation in long-termand live-cell screening experiments. Such a format alsoprecludes a straightforward strategy for secure biocontain-ment of infectious specimens such as viral gene vectors orHIV+ and blood samples. Microfluidics, however, isuniquely aimed at manipulating liquids at ultralow volumesin enclosed circuitry on-chip (Fig. 5) [37, 38, 4652]. Atmicroscale, fluids exhibit different physico-chemical prop-

Fig. 6 Microfluidic cell arrays exploiting the hydrodynamic celldocking principle and continuous microperfusion for drug delivery. aDynamic single cell culture array with a branching architecture andindividual chambers containing arrays of micro-mechanical traps (leftpanel). A 3D diagram of the design and mechanism of hydrodynamiccell trapping (middle panel). Traps are fabricated in PDMS andbonded to a glass substrate (right panel). They allow a gentle trappingwith no cell deformation. (Reproduced with permission from TheRoyal Society of Chemistry (RSC) from ref. [77].) b High-densitymicrofluidic array for cell cytotoxicity analysis. Schematic of the 2424 chamber microfluidic cytotoxicity array. Each chamber containseight micro cell sieves for cell trapping (left panel). Microfluidiccytotoxicity array chip assembly with fluidic interconnections (middlepanel). HeLa cell capture in micro cell sieves (right panel). (Reproducedwith permission from The Royal Society of Chemistry (RSC) from ref.[78].) c Microfluidic array cytometer with a triangular chamber thatcontains a low-density cell positioning array (left panel). SEM image ofa trapping array fabricated in a biocompatible elastomer, PDMS (middlepanel). Dynamic analysis of drug-induced cytotoxicity on a microfluidiccell array (right panel). Note the stochastic nature of the anti-cancer drugaction. Gradual increase in staining with annexin V marks theexternalization of phosphatidylserine (PS) residues characteristic of earlyapoptotic stages (red) whereas gradual plasma membrane permeabilityto PI represents progressive destabilization of plasma membranestructure in cells undergoing apoptosis (yellow). d Quantification oftime-resolved analysis at a single cell level. Human promyelocyticleukaemia cells were cultured on a microfluidic array cytometer asdescribed in c. Four representative cells were selected and their fluore-scence following incorporation of PI assessed as a mean fluorescenceintensity (MFI). Note the stochastic response to a pan-kinase inhibitorstaurosporine with a profound variability between cells in population.Black line represents a point of no return where initial destabilization ofplasma membrane to PI is irreversible and cell undergoes rapid celldeath

R


erties, dominated by viscous rather than inertial forces [46,47, 71]. Fluid flow under low Reynold numbers is thereforelaminar and mass transport is dominated by diffusion [46,71]. As a result, several parallel streams of reagents canflow next to each other without convection. By adjustingthe fluid flow rate, the interface between such adjacentstreams can be precisely controlled [72, 73]. In addition, asthe interial forces are minimal, when pumping of the fluidceases so does the movement of the fluid, enabling theprecise delivery of the reagent, in both time and space.These properties uniquely position microfluidic lab-on-a-chip technology to culture living cells for extended periodsof time and deliver cell-permeable fluorescent probes, drugsand growth factors to defined subcellular microdomains(Fig. 5) [7274]. The confining dimensions of the micro-fluidic structures also facilitate precise positioning of singlecells and sequential delivery of drugs and/or functionalprobes while continuously monitoring the cell microenvi-ronment and cell responses (Fig. 5) [37, 47, 48, 7274].

Accordingly, recent works have proven that microfluidicdevices are useful for high-throughput drug screening andparticularly suitable for 4D studies on rare subpopulations,such as cancer and haematopoietic stem cells (Figs. 5 and6) [16, 19, 20, 47, 7578]. Several groups have recentlyproposed conceptual designs that enable culture of cells on-chip (Fig. 5) [16, 19, 20, 47, 7578], some of which allowfor a rapid hydrodynamic positioning of single cells insidethe microfluidic chips (Fig. 6) [16, 19, 20, 77, 78]. At thecentre of this cell microarray technology lays a set ofmicro-mechanical traps, designed to passively immobilizeindividual cells into a predefined pattern (Fig. 6) [16, 19,77, 78]. Living cell microarrays constructed from abiologically compatible polymeric substratum allow quan-titative analysis of the dynamic events at a single cellresolution (Fig. 6) [15, 16, 19, 77, 78]. Unlike flowcytometry, however, measurements are made at multipletime points, and in contrast to conventional time-lapsemicroscopy, image analysis is greatly simplified by arrang-ing the cells in a spatially defined pattern and by theirphysical separation (Fig. 6) [15, 16, 19, 77, 78]. Wepostulate that the combination of microfluidic cell arrayswith integrated on-chip gene delivery technology(genomics), functional and dynamic live-cell analysis(cytomics) and intracellular antibody staining of selectedproteins (proteomics) can provide innovative, multivariateassays for high-content data mining and enhanced elucida-tion of cell signalling pathways (Figs. 5 and 6) [15, 16, 19,63, 79]. Microfluidic cell array technologies are thereforeemerging as a novel high-throughput experimental tech-nique that makes it possible to correlate pre-stress cellphenotypes and post-stress outcomes at a single cellresolution [54, 55]. Like DNA microarrays, it is poised tobring breakthrough discoveries in oncology, immunology

and neuroscience, particularly if used in conjunction withRNAi library screens, gene reporter systems, dynamicfunctional bioassays and single cell proteomics [54, 55,63, 76, 79].

Microflow chip-based cytometry

As discussed above, flow cytometry is a powerfulanalytical and diagnostic tool that leverages the multipa-rameter and high-speed measurements at the single celllevel [2129]. It suffers, however, from a high cost,complex operation and limited portability. Microfluidicsoffers here an innovative route to supersede these dis-advantages through the development of innovative micro-flow cytometers (FCM), micro fluorescently activated cellsorters (FACS) and micro magnetically activated cellsorters (MACS) (Figs. 7 and 8) [8087]. The majoradvantage of microflow cytometry chips is that they samplea greatly reduced number of cells when compared withconventional FCM [8093]. This is of particular valuewhen studying e.g. rare patient-derived cells, and monitor-ing their reactions to therapeutic compounds. Cells onplanar microfluidic chips can be hydrodynamically focusedto generate a single file which can be interrogatedsequentially by independent laser beams (Fig. 7) [80, 81,8793]. Recent developments of nonplanar microchipsopen many innovative opportunities to obtain confinementand regulation of laminar streams of cells in two or even inthree dimensions (Fig. 7) [80, 81, 8793]. The enclosednature of microflow cytometers make them particularlysuitable for the analysis of highly infective samples. Thismay be of particular importance during e.g. production ofmammalian viral vectors; monitoring of HIV infections,viral-induced cell death in pulmonary diseases or monitor-ing of cancer-targeted adenoviral therapy.

Pressure-based microflow cytometers can be devel-oped with flow rate controlled either by step motor-driven syringe pumps, positive air pressure applied toinput reservoirs or vacuum applied onto output reservoirs(Fig. 7) [80, 81, 8793]. Whilst, in conventional flowcytometers the transfer rates through the flow chamber canbe as high as 104106 cells/s, most microfluidic planarchips maintain are much lower transfer rates of 10300 cells/s [8790]. This is advantageous for preservationof live cells. The reduced transfer rates can be, in turn,effectively compensated for by parallel processing andsimultaneous analysis of multiple and parallel streams ofcells on each chip [80, 81, 8793]. Some of the mostrecent innovations in the FCM chip design also facilitatethe collection of undiluted cells following the microcytometric analysis, a feature not attainable in anyconventional systems [8890]. This allows for many


Fig. 7 Innovative microflow cytometers (FCM). a Prototype of theelectrical microcytometer with 3D hydrofocusing. Experimental setup:syringe pumps control sample and sheath flows through the sensingregion, which is connected to the external circuit via micro-manipulators.(Reproduced with permission from The Royal Society of Chemistry(RSC) from ref. [91].) b Prototype of the lab-on-a-chip cytometer withintegrated microfluidic dye laser, optical waveguides, microfluidicnetwork and photodiodes. (Reproduced with permission from TheRoyal Society of Chemistry (RSC) from ref. [108].) c Single-layerplanar microcytometer utilizing innovative drifting based 3D hydrody-namic focusing. (Reproduced with permission from The Royal Societyof Chemistry (RSC) from ref. [92].) d Prototype of the microfluidic flowcell with a two-stage cascaded hydrodynamic focusing, integrated

mirrors (green), grooves for optical fibres (yellow) and fluidicconnections (blue) to externally mounted electrodes. (Reproduced withpermission from The Royal Society of Chemistry (RSC) from ref. [93].)e CellLab Chip (Agilent Technologies, Santa Clara, CA, USA). Cross-sectional view of the microfluidic chip with an optical layout of theAgilent 2100 Bioanalyzer (Agilent) providing an external interface tothe chip-based cytometer. Excitation light sources (LED and solid-statelaser) are depicted together with light paths and corresponding detectors(FL1 and FL2). Note that substantial fluorescence signal separation (FL1em 525 nm vs. FL2 em 685 nm) alleviates need for a spectralcompensation. Disposable microfluidic cartridge and microfluidicnetwork that allows for a 2D hydrodynamic focusing of cells into asingle file (dotted panel on right)


functional studies to be performed post-analysis on thegiven cell sample.

Apart from the traditional fluorescence detection, spec-tral impendence using the Coulter principle has also beenadapted for on-chip cytometers to study the function of cellsize, cytoplasmic resistance and membrane capacitance[9496]. Precise differential white blood cell counts havealready been demonstrated by using the on-chip Coulter

principle [97, 98]. Recent reports suggest, however, thateven more high-throughput data can be obtained by usingin-flow dielectric spectroscopy on-chip [99, 100]. In thisregards innovative high-throughput screening (HTS) tech-nologies that are developed in a miniaturized formatinclude the capacitance and impedance cytometry [100102]. Moreover, a number of unconventional cytometrictechnologies have recently been proposed for a non-


invasive and real-time cell analysis on microfluidic chips.These include real-time studies on a single cell level such astime-of-flight (TOF) optophoresis and scanning thermallens microscopy (TLM) [103107].

Current progress in on-chip cytometry leverages manyrecent advances in microfluidic technology for the singlecell analysis with the ultimate outcome to produce user-friendly, reasonably priced and portable devices capable ofmultiparameter fluorescent interrogation of single cells insuspension (Fig. 7). The ultimate challenges for micro-fabricated cytometers remain in their robust coupling withthe laboratory macroenvironment (Fig. 7) [108].

Microfabricated cell sorters

The accurate detection, quantification and separation ofsingle cell clones is of paramount importance in clinicaldiagnostics, drug discovery pipelines and ultimately inpatient-tailored therapies [2326]. In this respect, flowcytometry still remains the technology of choice, especiallyfor rapid quantification and cell separation using high-speedfluorescently activated cell sorting (FACS) [2326]. Indeed,

modern electrostatic sorters support sorting algorithmsbased on up to 16 optical parameters from a single cell,with acquisition rates that exceed 25,000 events per second[2326]. This has opened new horizons for cell biology,immunology and cancer research [2326]. The widespreaduse of FACS is, nevertheless, severely limited because of itshigh complexity, power consumption and resulting intrinsiccost of the equipment and need for specialized training.This restricts such equipment to only centralized corefacilities [87]. As large reagent expenditures and multiplesample processing steps are also required, it profoundlydecreases efficiency and makes separation of clinicallyrelevant cell subpopulations particularly challenging. More-over, the high-pressure and electrostatic charge applied tocells during FACS can adversely affect recovery of fragilecells, such as apoptotic cells and cancer stem cells [87].

Not surprisingly, there is an increasing interest anddemand for cost-effective and portable cell sorting systemsthat will supplement conventional FACS especially in (i)high-throughput cell separation during drug screeningroutines, (ii) clinical grade cell sorting, (iii) diagnostic inresource-poor areas, (iv) military operations and (v) humanspace exploratory missions [37, 8186, 109118]. In thiscontext microfluidics has an immense potential to meetthese demands, due to the inherent ease of rapid prototyp-ing, potential of flexible and scalable designs, enhancedanalytical performance and economical fabrication (Fig. 8)[37, 8186, 109118]. Recently, development of manyinnovative microfluidic cell sorters has been reported(Fig. 8). These include (i) fluorescently activated cellsorters (FACS), (ii) in-flow micro magnetic cell sorters(MACS) capable of rapidly deflecting paramagneticallylabelled cells in a continuous stream of isotonic buffer, (iii)integrated optofluidics microsystems for Raman-activatedcell sorting (RACS) and (iv) functionalized micropostarray sorter [8186, 109118].

Immunomagnetic cell separation on-chip is particularlyadvantageous mainly due to its simplicity. It typicallyemploys monoclonal antibodies conjugated with super-paramagnetic particles used to separate cell subpopulationof interest [86, 110115, 117]. Advantages of this techniqueover FACS include minimal power consumption, substan-tial portability and simplicity of operation. Great simplifi-cation of these laboratory procedures will promote thefurther development of the MACS technology [86, 110115, 117]. Furthermore, by employing enclosed, disposablechip sorting cartridges, these designs will enable clinicalgrade, sterile sorting without undesired aerosol formationusually associated with conventional FACS [86, 110115,117]. Such design characteristics enable a secure sorting ofhighly infectious specimens without containment cabinetsas opposed to the dedicated rooms necessary for biohazardFACS sorting. Ultimately, we also envisage implementation

R Fig. 8 Innovative microfluidic cell sorters. a Schematics of theintegrated microfabricated cytometer and high-throughput fluores-cently activated cell sorter. (Reproduced with permission from TheRoyal Society of Chemistry (RSC) from ref. [82].) b SEM image ofthe micro cell sorter chip with integrated holding/culturing chamber asdescribed in a: a sheathing buffer inlet, b chimney sample inlet, cdetection zone, d holding/culturing chamber, e sieve to allow diffusionof nutrients and confinement of cells, f channel for draining excessliquid during sorting and for feeding fresh media to the cells duringcultivation. (Reproduced with permission from The Royal Society ofChemistry (RSC) from ref. [82].) c Integrated optofluidic Raman-activated cell sorter (RACS) that combines multichannel microfluidicdevices with laser tweezers Raman spectroscopy (LTRS) for delivery,identification and sorting of individual cells. (Reproduced withpermission from The Royal Society of Chemistry (RSC) from ref.[116].) d Principles of the continuous flow magnetic separation on-chip. An inhomogeneous magnetic field is applied perpendicular tothe direction of flow. Magnetic particles or magnetically labelled cellsare attracted into the field and thus deflected from the direction oflaminar flow. (Reproduced with permission from The Royal Societyof Chemistry (RSC) from ref. [113].) e Microfluidic chip forcontinuous sorting by using free-flow magnetophoresis. (Reproducedwith permission from The Royal Society of Chemistry (RSC) fromref. [115].) f Prototype of the micromagnetic-microfluidic bloodcleansing device (MMBCD). Transverse merging of microfluidicchannels allow for the rotation of inlet fluid streams about thelongitudinal axis of the separation channel. The MMBCD devicegenerates magnetic field gradients across vertically stacked channelsto enable continuous and high-throughput separation of fungi fromflowing whole blood. The device was successfully used to cleanse80% of living fungal pathogens from human whole blood flowing at arate of 20 mL/h. (Reproduced with permission from The RoyalSociety of Chemistry (RSC) from ref. [117].)


of low-cost optofluidics modules that would delivercomplementary on-chip flow cytometric analysis. This canbe particularly valuable e.g. for CD4+ lymphocytes count-ing and isolation in HIV/AIDS disease monitoring in sub-Saharan Africa. Successful applications of micro magneticcell sorting have already been shown in a gentle separationof human lymphocytes, fibroblasts and apoptosing cancercells [86, 110115, 117]. Considering the simplicity of theon-chip cell sorting protocols, these platforms have a widepotential to be used for automated diagnostic and laboratoryroutines [37].

Although micro magnetic cell sorting on-chip is muchslower than any currently available FACS sorters, innova-tive lab-on-a-chip designs can provide substantial improve-ments. By leveraging e.g. potential for modular designincreased throughput can be achieved by a parallel sampleprocessing paradigm. For this purpose one can takeadvantage of a well-known precedent in microprocessordesign and implement a multi-core sorting module config-uration. We are convinced that micro magnetic cell sortingtechnology will be especially valuable in challengingenvironments, such as biomedical research in developingcountries, field exploratory missions and possibly evenspace medicine and exobiology.

Future outlook

DNA and protein microarray technologies are rapidlyevolving fields that paved the way to modern clinicaldiagnostics, predictive toxicology and molecular pharma-cology [4045]. Therapeutic targets revealed by genetic andproteomic screens have to be, however, thoroughly validat-ed by functional live-cell assays that resolve the spatial andtemporal interrelationships in molecular signalling net-works at a large scale [13, 14]. Yet it is still particularlychallenging to quantify rapidly changing, i.e. dynamic,phenomena, which are by definition impractical to study byusing conventional binary approaches [16, 19, 20]. Dis-secting such signalling complexity at the single cell levelcan be only obtained by microsystems, whereby theposition of every cell is registered and maintained overextended periods of time [16, 19, 37, 48]. In this context,microfluidic lab-on-a-chip technologies provide uncompli-cated and effective solutions for low-cost and high-throughput screening routines at a single cell level [37,4649].

As discussed above, the microfluidic cell arrays providenew opportunities for multivariate single cell analysis atreasonably high data acquisition speeds [15, 16, 19, 20].They are, thus, particularly attractive for the clinical anddiagnostic laboratories as they allow rapid analysis of onlysmall amounts of patient-derived cells [15, 16, 19, 20].

Most importantly, they provide sensitivity that often cannotbe easily achieved with any conventional analytical plat-forms. As such they can be applied in a number of areasincluding accelerated anti-cancer drug discovery andtherapy, particularly in high-throughput and high-contentdrug screening routines [3638, 54]. The key challengesstill, however, lie ahead and include on-chip integration andsimplification of many functional components such asexcitation and collection optics, fluidics, electronics andtheir robust incorporation with the clinical and screeninglaboratories infrastructure [46]. Recent advances in the fieldhave recently provided new solutions such as computer-controlled microvalve arrays, drug mixers and dispensersworking at sub-nanolitre volumes [46]. Miniaturization ofother analytical components, such as organic light emittingdiodes (OLEDs) and detectors, is also poised to revolution-ize future design of clinical chip-based diagnostic devices[46, 93]. We believe that progress in microfluidic solutionswill provide new milestones for the advancement ofbenchtop and user friendly flow cytometers and integratedcell sorters [5052, 80, 82, 88]. Lab-on-a-chip has alreadybeen realized as an emerging technology with a multitudeof applications in high-throughput drug screening routines,content-rich personalized clinical diagnostics and improvedanalytical capabilities for resource-poor areas. Furtherprogress in this field will lead to stand-alone portabledevices that warrant accelerated drug discovery, andultimately personalized therapeutic regimens.

Acknowledgments Supported by Biotechnology and BiologicalSciences Research Council (BBSRC); Engineering and PhysicalSciences Research Council (EPSRC) and the Scottish FundingCouncil, under RASOR Program (Radical Solutions for Researchingthe Proteome).

Authors thank Dr Sara Lindstrm from Picovitro AB (Stockholm,Sweden) and Dr Cathy Owen from Nanopoint Inc. (Honolulu, Hawaii,USA) for providing exemplary data on proprietary microfabricatedanalytical systems. The authors declare no conflicting financialinterest.

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Microfabricated analytical systems for integrated cancer cytomicsAbstractIntroductionMicrofabricated systems for single cytomicsLiving cell microarraysMicroflow chip-based cytometryMicrofabricated cell sortersFuture outlookReferences

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Microfabricated Asanalytical Systems for Integrated Cancer

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