Advances in microfluidics for drug discovery

1. Introduction

2. Miniaturized platforms for

high-throughput screening

3. Microfluidic methods for

compound synthesis

4. Microfluidic methods for

protein engineering by

directed evolution and protein

crystallization

5. Microfluidic cell sorting

6. Tissue engineering and drug

testing

7. Expert opinion

Review

Advances in microfluidics for drugdiscoveryDario Lombardi & Petra S Dittrich††Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, Switzerland

Importance of the field:Microfluidics is considered as an enabling technology

for the development of unconventional and innovative methods in the drug

discovery process. The concept of micrometer-sized reaction systems in the

form of continuous flow reactors, microdroplets or microchambers is intrigu-

ing, and the versatility of the technology perfectly fits with the requirements

of drug synthesis, drug screening and drug testing.

Areas covered in this review: In this review article, we introduce key micro-

fluidic approaches to the drug discovery process, highlighting the latest and

promising achievements in this field, mainly from the years 2007 -- 2010.

What the reader will gain: Despite high expectations of microfluidic

approaches to several stages of the drug discovery process, up to now micro-

fluidic technology has not been able to significantly replace conventional

drug discovery platforms. Our aim is to identify bottlenecks that have

impeded the transfer of microfluidics into routine platforms for drug

discovery and show some recent solutions to overcome these hurdles.

Take home message: Although most microfluidic approaches are still applied

only for proof-of-concept studies, thanks to creative microfluidic research in

the past years unprecedented novel capabilities of microdevices could be

demonstrated, and general applicable, robust and reliable microfluidic

platforms seem to be within reach.

Keywords: cell sorting, compound synthesis, high-throughput screening, lab-on-chip

technology, microdroplets, microfluidics, tissue engineering

Expert Opin. Drug Discov. (2010) 5(11):1081-1094

1. Introduction

The field of lab-on-chip technology has inspired many researchers from variousdisciplines in the last decades. At the beginning, analytical applications were thefocus of interest, and miniaturization of existing instrumentation led to improvedefficiency of well-established methods [1-3]. Likewise, early concepts of microflui-dic technology were applied in sensing devices, where the microfluidic compo-nent, for example, a straight channel, served as a simple means to deliver a smallamount of analytes to the sensor [4,5]. These concepts were quickly expanded tofurther applications in the life sciences, for example, in medical diagnostics [6].Further promising developments have raised expectations that microfluidics willrevolutionize methods for pharmaceutical industry, too, particularly in the drugdiscovery process. These developments include: i) the finding that chemical reac-tions can be performed faster, with a higher yield and unprecedented specificity inmicroreactors [7-10]; ii) the demonstration of improved cell handling, cell culturingand cell analysis including advances in tissue engineering [11-14] and iii) the advan-ces in compartmentalization and handling of small liquid volumes such assegmented flow and integrated valves [15-17].

10.1517/17460441.2010.521149 © 2010 Informa UK, Ltd. ISSN 1746-0441 1081All rights reserved: reproduction in whole or in part not permitted

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Numerous studies have demonstrated that microfluidictechnology provides novel methods for cell biology, and inrecent times, many of the presented systems are truly complexdevices that are capable of performing several tasks in parallelor in series [18-22]. These are important advancements, as thedrug discovery process involves the identification of targets,synthesis of drug molecules, selection of powerful candidates,and characterization of the potential drug concerning efficacyand toxicity including the investigation or improvement oftheir absorption, distribution, metabolism and excretion(ADME) properties (Figure 1) [23].Each of these processes has specific demands, for example,

occurrence on different time scales and requirement of spe-cific analytical methods. This variability is certainly one ofthe obstacles to dispatch a fully integrated miniaturized devicewith various functional modules on a single platform andraises the question which kind of general platform could fulfillthe demands. In the following, we introduce common micro-fluidic platforms and highlight recent advances of microflui-dics technology that were dedicated to drug synthesis anddiscovery thereby discussing their advantages and limitations.Moreover, microchip technology provides another intriguingpossibility: the engineering of tissues and organ mimics onmicrofluidic platforms is a fast developing field, and recentachievements demonstrate the promising impact on drug test-ing prior to clinical studies. For more general introductionsinto microfluidics and lab-on-chip technology, refer recentreview articles and textbooks [24-27].

2. Miniaturized platforms for high-throughputscreening

The drug discovery process at its early stage primarily con-sists of experiments that can prove the interaction of a com-pound (future drug candidate) and a specific receptor(target). Nowadays, high-throughput screenings (HTS) arethe principal tool for pharmaceutical companies to investi-gate the properties of new chemical entities [23,28]. Tradition-ally, multiple-well plates (e.g., 96, 384 and 1536 well plates)are used to perform screenings. Further miniaturization,however, is important to reduce sample consumption andreaction time and, hence, costs of the process (Figure 2). Sev-eral possible miniaturized formats have been suggested inorder to achieve drug or library formation and drug testing(Figure 3). Miniaturized reaction chambers, micro- or evennanowells have been used to perform reaction and analysissteps on short time scales, consuming minute reagent vol-umes. Direct downscaling of the current standard formatshas the advantage that they are easily adaptable to existingassays, pipetting systems, and analytical and other peripheryinstrumentation. However, microfluidics technology canprovide further approaches: i) fully continuous reactors,ii) continuous processing on a rotating disc, where fluidsare driven by centrifugal forces, iii) continuous produc-tion of microreactors (referred to as droplet-based micro-fluidics, segmented flow or digitial microfluidics), iv) asemi-continuous approach, in which microreactors aredefined by integrated valves and v) other formats for exam-ple, the movement of two plates in opposite directions. Con-tinuous and semi-continuous strategies are conceptuallyintriguing, as they promise faster operation and throughputand improved automation prospects; integration of severalprocesses, however, is much harder to achieve, particularlybecause many chemical protocols involve washing steps andsolvent exchange, which are typically discontinuous pro-cesses. In the following, we briefly introduce and discussthe most often used formats.

2.1 Microwell arrays and m-contact printingMicrowells -- and even nanowells -- have been fabricated toimmobilize cells and particles (Figure 3A). Likewise, adhe-sion-supporting (or preventing) patterns with submicrome-ter resolution [29,30] have been created by micro contactprinting, a technique that utilizes a polydimethylsiloxane(PDMS) stamp to transfer molecules onto a surface. Bothmethods can be integrated with microfluidic systems andenable HT investigations of large numbers of samples, forexample, immobilized cells, liposomes or particles andmake these devices attractive for a wide range of assays indrug discovery [31-39]. The ability to position many cell typeson a single chip is useful for studying the effects of com-pounds exposed for a very short time, or for investigatingvarious compounds supplied in a series [40]. Using finitevolume reactors and flow systems technologies, cell-to-cell

Article highlights.

. Various stages of the drug discovery process can benefitfrom miniaturization, by saving time and costs forchemical compounds, by increasing efficiency ofchemical reactions and analytical methods and byautomation of multistep processes.

. Innovative microfluidic platforms dedicated to the drugdiscovery processes have been introduced,demonstrating that microfluidic technology providesnovel methods for chemical reactions, cell and tissueengineering and handling of small liquid volumes.

. Each of these platforms, running either in a continuous,semi-continuous or discontinuous operation mode, hasbenefits and limitations so that no generalrecommendation for one or the other could be given.

. The implementation of different functional units on asingle device for fully continuous operation is usually notachievable and not practicable due to the very specificmaterials or working conditions that every unit wouldneed to work optimally.

. Some of the microfluidic techniques have reached anadvanced level to be transferred into standard routineoperations such as platforms for chemical synthesis,separation systems, platforms for protein crystallizationand cell analysis platforms.

This box summarizes key points contained in the article.

Advances in microfluidics for drug discovery

1082 Expert Opin. Drug Discov. (2010) 5(11)

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differences, originating from regulatory circuits and distinctmicroenvironments, can now be explored [41].

2.2 Integration of valves by multilayer microfluidicsMicrofluidic multiplexed systems consisting of tens or hun-dreds of on-chip valves can be fabricated by multilayer softlithography [42-47]. The microchip consists of two layers, sepa-rated by a thin membrane (Figure 3B). One layer contains thefluidic channels and the other control layer contains the chan-nels that determine the position of the pneumatic valves. Thevalves are impressed into the fluidic channels on actuation bynitrogen pressure. They define reaction chambers and are

used to deliver fluids to each chamber independently. Fastand automated actuation of the valves facilitates nearly contin-uous processing. This approach can be easily adapted to a broadrange of synthetic, analytical and cell biological applications bymodification of the design [48-52] and, furthermore, can be fullyintegrated with microarray technologies [53,54]. Althoughextremely versatile, the channel dimensions (and aspect ratios)are limited in order to ensure complete closure of the valves.Furthermore, this microchip format requires the use of a flexi-ble polymer (i.e., PDMS) for chip fabrication, which is incom-patible with many organic solvents and, moreover, manycompounds adhere or diffuse into PDMS.

• System biology• Metabolomics• Proteomic

• Platforms for cell culture, single cell studies

• Platforms for cell and tissue studies

• Structure-based drug discovery and protein crystallization• Ligand fishing• Molecular evolution

• Metabolism and cytotoxicity• Cellular activity• Tissue studies

• Generation and handling of small liquid volumes• Microcitometry and cell sorting

• Planner and three- dimensional micromixers and microreactors

• Combinatorial chemistry• Serialized and parallelized synthesis

Librarygeneration

Targetidentification

Leadidentification

Leadoptimization

(sections 2 and 5)Mic

roflu

idic

met

hods

(sections 2 and 5)(section 3) (section 6)

Figure 1. The figure depicts the key stages of the drug discovery process, including the respective microfluidic methods that

are valuable for individual steps in the process.

Macroscopic reactionsystem filled with twocompounds

Miniaturization

Serial (downstream)processing

Parallelization

m × n × d n × d d 3 d

Figure 2. A sketch illustrating the benefits of miniaturization. A macroscopic reaction system is divided into smaller volumes,

requiring now fewer compounds for carrying out the same reaction. The time for diffusion of a compound across the entire

volume is reduced. As a consequence of miniaturization, the surface:volume ratio increases and, hence, heat transport into

and out of the microreactor is enhanced likewise. Downscaling of reaction systems can improve the efficiency of reactions and

analyses in terms of speed, costs and yield or sensitivity. In microfluidic systems, the integration of several functional modules

along a microchannel system provides the requirement for downstream processing and automation. A high level of

parallelization dramatically increases the entire throughput.Image adopted from [24]. Copyright Macmillan Publisher 2006.

Lombardi & Dittrich

Expert Opin. Drug Discov. (2010) 5(11) 1083

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2.3 Droplet-based microfluidicsDroplet-based microfluidics (often referred to as segmentedflow, digital microfluidics or biphasic microfluidics) facilitatesthe formation of microreactors with volumes ranging frompicoliters to nanoliters utilizing two immiscible fluids. Byinjecting a solution (typically, aqueous solution) into a carrierstream (typically, hydrophobic compound such as oil of fluori-nated hydrocarbons), microdroplets of homogeneous size arecontinuously generated (Figure 3E) [55,56]. Inside the micro-droplets, reagents are quickly mixed and incubated withoutevaporation and minimum exposure of compounds to theatmosphere [17,57]. Much effort in recent years has led to thedevelopment of numerous manipulation tools for microdrop-lets, including splitting [58], fusion by electrical means [59] andpassive fusion techniques [60], sorting [61], droplet storage [62,63],positioning [64], implementation with electrophoretic separa-tion [65], injection of cells and particles [66-69] and interfacingwith mass spectrometry [70,71]. Applications of droplet-basedmicrofluidics include synthesis, kinetic measurements (typicaltime scales from seconds to minutes), single cell measure-ments and protein crystallization and production [72-81]. Partic-ularly interesting for genetic analysis is the development ofdroplet-based microreactors to perform PCR [82,83].Although the droplet-based approach is fascinating and

intriguing, so far it has only been used for applications thatrequire aqueous buffers. Moreover, one of the difficulties of

droplet-based microfluidics is the large, dynamic interfacebetween the droplets and the carrier solution, which, in con-sequence, frequently results in partitioning of sample intothe carrier and in cross-contamination. Further developmentsof more suitable surfactants may help to reduce these losses.Another aspect that has to be addressed is the implementationof simple processes such as solvent evaporation or samplewashing and purification.

2.4 Continuous reactors and microfluidic gradient

devicesContinuous flow reactors are usually simple, planar micro-chips that consist of a few inlet channels for reagent supplythat merge into one single channel, where the reaction takesplace (Figure 3F). Fast reactions occur immediately at theinterface of two co-flowing streams. As a consequence ofthe laminar flow regime present in microfluidic devices, thereagents mix due to diffusion. This phenomenon can beexploited to generate gradients across a microfluidic chan-nel [84]. The creation of concentration gradients is useful forstudying cellular response to chemical stimuli [85-87]. Fordrug discovery, they can be used for testing multiple drugdoses simultaneously or for lead optimization [88]. Cells canbe stimulated in order to study the effects of drug concentra-tion on chemotaxis [89,90] and gradients can be applied to cellbehavioral studies on stem cell differentiation [91,92].

Side view Side view

Fluid

N2 N2A. B.

D. E.

C.

F.

Figure 3. Microfabricated platforms for drug screening and testing. Depicted are discontinuously (A-C) and continuously

operating systems (D-F). A. Small microwells, capturing single cells, are shown in the inset. B. Integrated pneumatic valves

compartmentalize reaction chambers and regulate liquid supply. The valves are closed by impressing a flexible membrane

into a fluid channel underneath (inset, here partly open). C. Microcompartments and connection channels are fabricated on

two (glass) plates. Movement of opposing plates in opposite direction connects formerly disconnected compartments. D.

Microfluidics on a disc format. Here, the flow is induced by centrifugal forces on rotation of the disc. E. Microdroplet-

based microfluidics. An aqueous solution is injected into a continuous carrier stream (an immiscible fluid, e.g., oil) resulting in

continuous formation of droplets. F. Continuous flow reactor. Co-flowing of two (or more solutions) can be used to induce

reactions at the interfaces or to establish gradients (e.g., concentration gradients, temperature gradients). The interface is

broadening downstream due to diffusion of compounds across the channel (inset).



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3. Microfluidic methods for compoundsynthesis

Miniaturized systems for carrying out chemical reactions incontinuous flow can have advantages over bulk synthesis [7].Two chemical compounds will react in the same way, regard-less whether the reaction takes place in a large, bulky reactoror in a micrometer-size reactor if the conditions during thereaction are identical. However, in a micrometer-sized reac-tor, these conditions can be much better controlled and repro-duced, particularly if temperature distribution and mixingtimes are clearly defined. Under these considerations, it isnot surprising that many chemical reactions are much fasteron the microscale and result in higher yields with improvedproduct specificity [93,94]. The combination of multiple reac-tion steps in flow or the reagent immobilization and rapidserial processing in flow for the reaction steps addressestraditional bottlenecks in workup and purification [95-98].

Moreover, microfluidic devices represent a valuable tool forDNAor peptide synthesis [99]. This ability plays a significant roleon encoded DNA libraries [100]. Another possible applicationis the combinatorial synthesis of compound libraries [101].

Recently, Wang et al. [102] impressively demonstrated howlarge-scale synthesis and screening of > 1000 products on asingle chip could be accomplished. They developed an inte-grated microfluidic device able to perform 1024 parallel reac-tions using a bovine carbonic anhydrous II 1024 in situ clickchemistry system (Figure 4). Rapid hit identification approachusing SPE purification and electrospray--ionization mass spec-trometry was adopted in order to improve the sensitivity andthroughput of the downstream analysis. The current platformsignificantly reduces reagent consumption and screening time(17 s for the reaction, 15 s for hit identification vs 40 minin the original LC-MS-based method [102]) and allowsfuture automation.

In another study, Theberge et al. [103] developed a devicethat enables Suzuki--Miyaura coupling reactions in aqueousmicrodroplets with catalytically active fluorous interfaces.Such a device can generate a thousand small droplet reactorstotally analogous to the traditional chemists’ flask. Addition-ally, the method enables the generation of droplet reactorswith catalytically active walls by using a fluorous ligand whichboth solubilized a transition metal catalyst in fluorous solventswhile also acting as a surfactant and hence accumulating at thefluorous--water interface [103]. Although the synthesis inmicrodroplets suffers from limitations of solvents that canbe used (preferentially, aqueous buffer) and of compounds(preferentially, hydrophilic compounds that cannot permeateinto the hydrophobic carrier), it is a powerful method forcompound library synthesis and screening. Ongoing effortson developing novel surfactants that assemble at the interfaceof droplet and carrier solutions are aimed at preventingdiffusion of compounds across this interface. This mayhelp improve the versatility of droplet-based microfluidicsfor chemical synthesis. Furthermore, combination of

droplet-based microfluidics with separation techniques(electrophoresis, chromatography) may allow filling drop-lets individually with pure compounds and gradients ofconcentrations starting from chemical mixtures [103].

4. Microfluidic methods for proteinengineering by directed evolution andprotein crystallization

A particularly interesting application of microfluidic devices isdirected evolution [104-106], which is a means of producingproteins with tailor-made activities, and has led to new classesof drugs [104], as well as improved enzymes [105] and strains ofmicroorganisms for industrial applications [106]. Just recently,Agresti et al. [107] have reported the development of an ultra-HTS and sorting platform using droplet-based microfluidicdevices for in vitro evolution of the enzyme horseradish perox-idase (HRP). Picoliter-volume aqueous droplets dispersed inan inert oil as reaction vessels containing yeast cells displayeda variant of the enzyme on their surface; then, these dropletswere sorted at rates of thousands per second, enabling thescreening of a library of 108 in about 10 h, using a total reagentvolume of < 150 µl. The method discovered variants of HRPthat are > 10-fold active than the original enzyme. Comparedto state-of-the-art robotic screening, this is 1000-fold fasterand uses 10 million-fold less volume of reagent, representinga cost savings estimated to be about 10 million-fold.

While directed evolution allows the search of proteins witha desired activity without knowledge of mechanisms of theprotein functioning, the investigation of proteins and theirstructure by X-ray crystallography is essential for a rationaldesign of a specific drug. Again, in this context, droplet-based microfluidics was applied to the formation of crystalsin solution and yielded insights into crystallization processesto obtain parameters such as solubilities, habits, existence ofpolymorphs and estimations of nucleation/growth rates. Inaddition, microfluidic devices enable several features, that is,high-throughput data acquisition using crystallization assaysdown to 1 nl or design specific kinetic routes using the excel-lent control of mass and heat transfers [108-110], and providenew experimental conditions to investigate crystallization(e.g., no turbulence, no or little gravity effect, confinement,large surface:volume ratio). Additionally, the small volumesof microfluidics are of special interest for nucleation as theyenable the observation of only one nucleation event: thismononuclear mechanism is essential for estimating nucleationkinetics and investigate polymorphism [111].

The revolutionary benefits enabled by the microfluidicenvironment have triggered a commercial interest on auto-mated devices for high-throughput crystallization (see systemsfrom Fluidigm and Emerald BioSystems). For example,Emerald BioSystems, Inc. developed a microcapillary proteincrystallization system (MPCS) based on a semi-automatedplug-based crystallization technology, which enablednanoliter-volume screening of crystallization conditions [78].

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Moreover, it enabled direct in situ X-ray diffraction studies ofprotein crystals. The MPCS integrated the formulation ofcrystallization cocktails with the preparation of the crystalliza-tion experiments by using 10 -- 20 nl volume droplets, eachrepresenting a microbatch-style crystallization experimentwith a different chemical composition (Figure 5).A different approach addressing several aspects of crystalli-

zation screening is represented by the work of Ismagilov andco-workers [112,113]. They developed a chip (the so-calledSlipChip) that enabled multiplexed microfluidic reactionswithout pumps or valves (scheme, see Figure 3C). It consistedof two plates contacted through a thin layer of oil; one platecontained microwells preloaded with reagents while the otherplate acted as a lid for the wells with reagents. The plates canalso have a fluidic path consisting of ducts in one plate and

wells in the other with a design that enables the connectionof wells and ducts only when the two plates are aligned in aspecific configuration. Samples and reagents were loadedinto the wells and ducts of both plates; then, one plate wasslipped till the two plates are aligned, so that the patternsof wells and ducts in the plates were connected enablingdiffusion and reactions among the samples on the two plates.Besides screening for crystallization conditions, this chipdesign has been used for carrying out immunoassays andPCR [114,115].

5. Microfluidic cell sorting

Pharmaceutical research is demanding high-performancecell-based assays to efficiently screen and validate potential

PBS

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(iv)PTFE tube for collectingin situ clik chemistry reactions

Cul

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Figure 4. A. Schematic representation of an integrated microfluidic platform for organic synthesis of > 1000 compounds. The

operation of the circuit was computer controlled using color-coded pressure-driven valves: red: positive pressure, off/on;

yellow: peristaltic pumping; green: vacuum. B. Optical image of the actual device. The various channels were loaded with

dyes to visualize the different components: red, yellow and green as in part (A) and blue indicated the fluidic channels.

C. PTFE tubing for off-chip incubation and storage of the reaction mixture slugs. Again, blue and red dyes are used for

visualization. Black scale bars are 3 mm.Image adapted from [102] with permission from the Royal Society of Chemistry. Copyright 2009.

PTFE: Polytetrafluoroethylene.



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drug candidates in the initial stage of drug discovery. The pre-diction of the clinical response to drug compounds often relieson cell culture models that should be as faithful as possible tothe in vivo behavior. For fast screenings of large cell popula-tions, the method of choice is fluorescence activated cell sort-ing (FACS). Miniaturized versions of FACS devices have beendeveloped in the past [116-118]. Although most examples arestill much slower than commercial machines with through-puts of several tens of thousand cells per second, the micro-chip cell sorters have the advantage of implementingreactions (e.g., for labeling) prior to the sorting process andallow implementation of further reaction or analysis schemesafterwards. More recently, the scientific community hasbeen focusing on methods that avoid the use of biochemicallabels; numerous intrinsic biomarkers have been explored toidentify cells including size, electrical polarizability andhydrodynamic properties [119,120]. The microfluidic techni-ques applied to accomplish cell separation and sorting covera broad range of methods, for example, the purely physicalseparation achieved by the use of microscale filters [121].Next, a number of techniques that manipulate microscale

fluid dynamics, including hydrodynamic filtration, field-flow fractionation, flow due to microscale structures andinertial microfluidics are available [122-132]. A particularinterest has been driven by methods that achieve continuousseparation by field-based techniques of magnetophoresis,acoustophoresis and dielectrophoresis [124,133-138]. For exam-ple, Thevoz et al. [139] applied ultrasonic standing waves toachieve cell cycle phase synchronization in mammalian cellsin a high-throughput and reagent-free manner. Their acous-tophoretic cell synchronization (ACS) device was based on avolume-dependent acoustic radiation force within a micro-channel that enabled selective purification of target cells ofdesired phase from an asynchronous mixture based on cellcycle-dependent fluctuations in size. They demonstratedthat ultrasonic separation allowed label-free synchronizationwith high G1 phase synchrony (84%) and throughput(3 � 106 cells/h/microchannel). The results are notable ifcompared to other microfluidic approaches that usuallyachieve the same efficiency with a throughput of approxi-mately an order of magnitude lower than this ACS device.In comparison with conventional techniques, the through-put speed is comparable, while, however, the purity of indi-vidual cell cycle populations is not as good. For example,centrifugal elutriation can achieve up to 90 -- 95%G1 phase synchrony [140].

The aforementioned examples focus on analysis of suspen-sion cells or individual adherent cells. However, in thesemethods, many cell types lack their natural environment, inwhich cells are embedded in extracellular matrices and com-municate among each other. From this point of view, suchmethods are particularly valuable for fast screening of a spe-cific property (e.g., binding of a compound to a membranereceptor), but further tests on cell cultures to obtain athorough understanding of the cellular response includingmetabolism or cell signaling would be indispensible.

6. Tissue engineering and drug testing

Unsuccessful identification of new environmental toxins isone of the causes of the high costs of pharmaceutical devel-opement. One of the major obstacles to rapidly screen thetoxicity of drug candidate is the lack of experimental modelsystems that can replace costly and time-consuming animalstudies. Even though considerable advances were made inthe development of cell culture models as surrogates of tis-sues, it is still difficult to maintain stable differentiationand expression of tissue-specific functions with culturedcells [14,141].

Microscale engineering technologies, microfabrication andmicrofluidics, enable unprecedented capabilities to controlthe cellular microenvironment with high spatiotemporal pre-cision and are able to stimulate cells with mechanical and bio-chemical signals in a more physiologically fashion [11,142-145].We would like to emphasize here research focusing on tissueand organ engineering rather than work on cell cultures or

Precipitant

Buffer

Protein

Fluorocarbon

A.

B.

C.

Figure 5. Plug-based protein crystallization using MPCS. A. A

microphotograph of crystallization experiments in plugs

being formed in the CrystalCard. B. Dyed protein crystals in

the MPCS CrystalCard. C. A schematic drawing (left) of a

channel containing a smooth pH gradient (right) generated

using a low-pH buffer, a high-pH buffer and a pH indicator.Adapted from [78]. Image adapted with permission from IUCR.

Copyright 2008.

MPCS: Microcapillary protein crystallization system.

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single cells, as we consider it particularly interesting for drugtesting. Up to now, based on microfluidic technology, severalmodels have been fabricated: blood vessels [146,147],muscles [148], bones [149], airways [150], liver [151-154], brain [155],gut [156] and kidney [157,158].Recently, Huh et al. [14] provided the proof of principle for

a biomimetic microsystems approach that replicated the keystructural, functional and mechanical properties of the humanalveolar--capillary interface. It incorporated multiple tissues,including active vascular conduits, in a dynamic and mechan-ically relevant organ-specific microenvironment. They micro-fabricated a microfluidic system containing two closelyapposed microchannels separated by a 10 µm, porous, flexiblemembrane made of PDMS coated with collagen (extracellularmatrix). The membrane provided the support to culture, onopposite sides, human alveolar epithelial cells and human pul-monary microvascular endothelial cells (Figure 6). This ver-satile system enabled direct visualization and quantitativeanalysis of diverse biological processes of the intact lungorgan in ways that have not been possible in traditionalcell culture or animal models. After the cells were grown toconfluence, air is introduced into the epithelial compart-ment to create an air--liquid interface mimicking the alveolarair space. Then, vacuum was applied to the side chambersproducing an elastic deformation that causes stretching ofthe porous PDMS membrane and the adherent tissue layers(Figure 6A right versus left). When the vacuum was released,elastic recoil of PDMS causes the membrane and adherentcells to relax to their original size. This design replicateddynamic mechanical distortion of the alveolar--capillaryinterface caused by breathing movements. Additionally, themicrofluidic configuration enabled manipulation of fluidflows, as well as delivery of cells and nutrients, to theepithelium and endothelium independently.A further step into the replacement of animal studies con-

sidering transport of metabolites and consecutive metabolismpathways are the microchip platforms designed by Sung andShuler that comprise tissue from multiple organs, termedmicro cell culture analog. In a recent study, they designed a3D hydrogel cell culture platform to study the cytotoxicityof an anticancer drug [159]. On a single device, colon cancercells, a hepatomatic cell line (mimicking the liver) and a mye-loblast cell line (mimicking marrow) were cultured in sepa-rated chambers, and connected via microchannels. The chipfacilitated mimicking the pharmacokinetic profile of a pro-drug (Tegafur) that was first metabolized in the liver tissueto the active drug 5-fluorouracil, which caused finally thedeath of the tumor cells. Such studies could provide valuableinformation on the efficacy and toxicity of a drug in a fairlyrealistic environment and at an early stage of drug testing.

7. Expert opinion

Nowadays, microfluidic technology can be considered as apowerful tool for various applications in the drug discovery

and development process. Many fields of chemical synthesis,protein crystallization, high-throughput drug screening anddrug delivery can benefit from microfluidic-based approachesto address a number of limitations imposed by conventionalmacroscopic methods including expensive processes and largevolumes of reagents. Microfluidic technology has a greatpotential to improve industrial processes and reduce costs atthe same time. Additionally, further economic interest couldbe triggered by the many unexplored advantages that micro-fluidics offer in related fields such as nanotechnology. Inthese fields, the fluidic platforms are a microsized tool toform nanowires and nanoparticles, and to align, positionand functionalize such structures for further uses [160,161].

Despite the fact that many approaches and microfluidic-based methods have reached an advanced level to be transferredinto standard routine operations, some significant weaknesseshave not been overcome. Processes that are simple to achievein conventional formats remain still difficult on a microfluidicplatform; solutions and chip operations are often too sophisti-cated, neither robust and reliable nor flexible enough for appli-cations beyond proof-of-concept assays. These processesinclude washing and purification steps, extraction of com-pounds and solvent evaporation or distillation. With regardto this, some interesting solutions on how to perform on-chip continuous distillation processes at a low temperature byutilizing carrier gases in the laminar flow regime [162] and inthe droplet regime have been presented [163]. Moreover, micro-fluidic approaches are prototype devices, seldom automated oroptimized for daily utilization by non-expert users, in contrastto conventional commercialized equipment that has been fullydeveloped and customized.

Another aspect that hinders the development of commonstandards for microfluidic approaches is the variety of materi-als that can be possibly used for chip fabrication. Up to date,there is no material that perfectly fulfils all desired propertiesconcerning availability, ease of fabrication, price, optical orelectromechanical properties, thermal conductivity and stabil-ity. Most commonly, the polymer PDMS is utilized, which isvery useful in academic research as it allows reasonably cheapand fast fabrication of prototypes. However, some limitationsof the material, including poorly defined and unstable surfaceproperties and instability against most organic solvents, hin-der the use of PDMS for many routine applications. Alterna-tive materials such as silicon, glass, and other polymers orhydrogels are available, but their use involves other fabricationprocesses, and implementation of electrodes or active modulessuch as valves and pumps is more difficult [164].

Among the great benefits of microfluidic solutions is theimplementation of different functional units on a singledevice, or the serial coupling of devices, which is, however,still problematic. Depending on the individual processes,each module may require a specific material or surface coat-ing, different solvents or buffer composition, different flowrates or residence times (e.g., long and short incubationtimes), so that a fully continuous operation is usually not



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achievable and not practicable. In this regard, further pro-gresses are required as well on the aspect of assay develop-ment, where conventional chemical reactions or testingprocedures have to be modified and optimized accordingly.

Nevertheless, excellent work and much effort of researchersfrom various fields has lead to many robust microfluidic solu-tions that are not any more in infancy stages, but can bealready considered as standard methods. A good example inthis respect is the development of microdroplet reactors that,a few years ago, was the subject of fundamental and proof-of-concept studies only, and is nowadays a standard tool inmicrofluidics. Likewise, semi-continuously operating plat-forms with implemented valves have been designed, havingcomplex geometries, and enable fully automated processing.Furthermore, the exponential growth of microfluidic researchin the past years and, frequently, new demonstration ofunprecedented capabilities of microreactors as a result of thecreativity of researchers underline that many of the potentialapplications are largely unexploited even in research labs sofar. With respect to drug discovery, lead optimization andpreclinical safety evaluations, which are critical bottlenecks

in pharmaceutical development programs, could highlybenefit from the advantages promised with microfluidicapproach. An ideal ADME/TOX chip able to mimichuman physiology under drug treatment for the extrapola-tion of specific pharmacokinetic and pharmacodynamicinformation in a easy ready-to-use kit with minimal periph-erals for operation and without the need of specializedhuman work could be extremely precious for several stepsof the drug discovery process and, therefore, of undoubtedcommercial value.

Acknowledgments

The authors thank A Jahn and F Kurth for proof-readingthe manuscript.

Declaration of interest

P Dittrich and D Lombardi have received funding from theEuropean Research Council under the 7th FrameworkProgramme (ERC Starting Grant No. 203428 nµ-LIPIDS).

A.

B.

C. D.

E.

Epithelium

Endothelium

Air

Air

Air

Membrane

Side chambers

Capillaries

Alveolus

Vacuum

Vacuum

Stretch

Upper

PDMSetchant

Side chambers

Layer

LayerLower

Porousmembrane

Diaphragm Pip

Figure 6. Biologically inspired design of a human breathing lung-on-a-chip microdevice. A. The microfabricated lung mimic

device uses compartmentalized PDMS microchannels to form an alveolar--capillary barrier on a thin, porous, flexible PDMS

membrane coated with ECM. The device induces strain in the cell to emulate physiological breathing movements by applying

vacuum to the side chambers and causing mechanical stretching of the PDMS membrane forming the alveolar--capillary

barrier. B. During inhalation in the living lung, contraction of the diaphragm causes a reduction in intrapleural pressure (Pip),

leading to distension of the alveoli and physical stretching of the alveolar--capillary interface. C. Three PDMS layers are

aligned and irreversibly bonded to form two sets of three parallel microchannels separated by a 10-µm-thick PDMS membrane

containing an array of through-holes with an effective diameter of 10 µm. Scale bar, 200 µm. D. After permanent bonding,

PDMS etchant is flown through the side channels. Selective etching of the membrane layers in these channels produces two

large side chambers, to which vacuum is applied to cause mechanical stretching. Scale bar, 200 µm. E. Images of an actual

lung-on-a-chip microfluidic device viewed from above.Adapted from [14] with permission from AAAS. Copyright 2010.

ECM: Extracellular matrix; PDMS: Polydimethylsiloxane.

Lombardi & Dittrich


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Unravelling single cell genomics: RSC;

2010

AffiliationDario Lombardi1 & Petra S Dittrich†2

†Author for correspondence1Department of Chemistry and

Applied Biosciences,

ETH Zurich,

Wolfgang-Pauli-Str. 10,

CH-8093 Zurich,

Switzerland2Assistant Professor for Bioanalytics,

Department of Chemistry and

Applied Biosciences,

ETH Zurich,

Wolfgang-Pauli-Str. 10,

CH-8093 Zurich,

Switzerland

Tel: +41 44 633 68 93;

E-mail: [email protected]



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Documents

Advances in microfluidics for drug discovery