Exploring Living Multicellular Organisms, Organs, and Tissues Using

Review pubs.acs.org/CR

Exploring Living Multicellular Organisms, Organs, and Tissues Using Microfluidic Systems Venkataragavalu Sivagnanam† and Martin A. M. Gijs* Laboratory of Microsystems, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland 1. INTRODUCTION 1.1. Microfluidic Systems for Biology

Lab-on-a-chip (LoC) systems allow the implementation of benchtop analytical methods/protocols using low reagent volumes, short processing times, and this potentially at lowcost and with high sensitivity.1−3 During the past decade, microfluidics-enabled systems have contributed to the development of many clinical and molecular diagnostic instruments indeed. As the dimensions of the microfluidic channels of a chip are typically of the size of single cells, LoC systems have also emerged as a front-runner choice for performing cell-based analysis and studies.4−8 LoC devices allow easy handling and positioning of individual cells, as well as studying the latter on a one-to-one basis and in a parallel fashion. High-throughput single-cell analysis offers the possibility of studying a large quantity of individual cells from the same population, thereby providing detailed statistical information on the biological variance within the population. Microfluidics-based research on cells has been reviewed a number of times focusing on different themes. Microfluidic chemical gradient devices and their application in the study of cell chemotaxis and morphogenesis were reviewed by Kim et al.9 Others have focused on cell-based vascular research,10 neurobiology,11,12 materials and technologies and three-dimensional (3D) cell cultures,13 or cell biology in general.14,15 With the advancement of technology, scientists have shown increased interest in applying LoC concepts for performing studies on 3D ensembles of cells, rather than on single cells or cells cultured on two-dimensional (2D) surfaces. Under the umbrella of microsystems-based tissue-research, a lot of work was focused on tissue engineering starting from cells.16,17 Microfluidic systems have been shown to be very useful for the growth of spheroids (spherically shaped agglomerates of cells)18−20 and utilization of the latter as tissue sample models for performing drug discovery experiments and pharmaceutical analysis.21 Tissue engineering literally means creating tissues from single or multiple cells by helping them to grow together in a directed 3D fashion, either by seeding them on a scaffold structure made of biodegradable materials or by embedding and culturing them inside a porous gel matrix.22−24 However, a “bottom-up” single cell-based approach can hardly mimic an in vivo biological system that has a complex structure and exhibits unique functionalities. Thanks to the advances in microfabrication technologies and using the full potential of microfluidics, more complex 3D microenvironments have been created, in which multiple cell lines were cultured in

CONTENTS 1. Introduction 1.1. Microfluidic Systems for Biology 1.2. Scope of This Review 1.3. Synopsis of the Reviewed Work 2. Living Multicellular Organisms 2.1. Mammalian Embryos 2.2. Danio rerio (Zebrafish) Embryos and Larvae 2.3. Drosophila melanogaster (Fruitfly) Embryos 2.4. Caenorhabditis elegans (Roundworm) Embryos, Larvae, and Adults 2.4.1. Worm Culture and Imaging 2.4.2. Behaviorial Studies 2.4.3. Neurobiology Studies 3. Organs 4. Tissue Slices 4.1. Per(i)fusion of ex Vivo Tissue Slices 4.2. Immunohistochemical Analysis of Biopsied Tissue Slices 5. Conclusions and Outlook 5.1. General 5.2. Multicellular Organisms 5.3. Organs 5.4. Tissues Author Information Corresponding Author Present Address Notes Biographies Acknowledgments References

A A E E G G H I K K O R U W W AB AB AB AC AC AC AD AD AD AD AD AE AE

Received: November 16, 2011

© XXXX American Chemical Society

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dx.doi.org/10.1021/cr200432q | Chem. Rev. XXXX, XXX, XXX−XXX

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Table 1. Summary of Other Reviews Mentioned in This Review and Their Point of Focus review topic microfluidic devices microfluidic chemical gradient devices microfluidic technologies for vascular cell biology microfluidic technologies for neurobiology microfluidic devices for mimicking the in vivo environment microfluidic devices 3D in vitro tissue physiology microfluidics-based “organs-on-chip” microfluidic technologies for high-throughput in vivo applications microfluidic chips for biological studies microsystems for controlled genetic perturbation of live Drosophila embryos high-throughput screening mammalian embryo culture methods C. elegans’s biology and its interest for chemical and biological research laser microsurgery of C. elegans brain slices studied on chips experimental microfluidic in vitro systems to study liver metabolism and/or toxicity microfluidic technologies for use in drug discovery optical imaging techniques and microfluidics microfluidic probes

focus

ref

microfabrication technologies applications in the study of cell chemotaxis and morphogenesis studying cell growth and behavior in microfluidic channels neuron-based studies cell culture and engineering cell biology in general cell-based studies cell coculture and differentiation in on-chip microcompartments; microenvironment engineering phenotyping, imaging, and screening of multicellular organisms C. elegans and zebrafish40,41 RNA interference, development studies, and drug screening

2 9 10 11,12,36,37 13 14,15,38 23 27−29,39

small animal models comparison of microwell, microdrop, and microchannel platforms discussion of worm tools and comparison of C. elegans with other model organisms laser−cell interaction and instrumentation options offered by microfluidic technology for neuroscience predominantly cell-based studies

35 44 45,46

cells, organs, and organisms comparison of “bulky” imaging techniques and on-chip methods; applied to C. elegans local processing of tissue slices and biochemical concentration gradient generation

50 51−53

separated chambers, representing liver, marrow, or a tumor, which could communicate via microchannels mimicking blood vessels.25 This platform has been used to evaluate cytotoxic effects of anticancer drugs on the different cell types. Another biomimetic microfluidic system has reconstituted a functional alveolar−capillary interface of the human lung, by culturing alveolar epithelial cells and microvascular endothelial cells, respectively, on one side of a porous polydimethylsiloxane (PDMS) membrane coated with fibronectin or collagen. The elastomeric device mimicked physiological breathing movements when vacuum was applied to special on-chip chambers. Also, by culturing a confluent layer of human intestinal epithelial cells on one side of the membrane and coculturing a normal intestinal microbe on top of the luminal surface of the epithelium, a human “gut-on-a-chip” was demonstrated, which exhibited intestinal peristalsis-like motion.26 “Organs-on-a-chip” systems, which are characterized by microcompartments that allow on-chip cell coculture, differentiation of the cells into tissue layers, and reconstitution of tissue−tissue interfaces, have also been reviewed.27−29 Such systems have potential to be used in drug discovery or toxicity testing under more physiologically realistic conditions than when using a 2D layer of a single type of cell. On the other hand, instead of using a “bottom-up” cell (co)culture approach to reconstitute organ function, also a “top-down” approach can be chosen, in which living organisms or explanted tissues can be arranged in a well-controlled spatiotemporal configuration on a microfluidic chip, allowing perfusion experiments, electrophysiology studies, and evaluation of the impact of drugs and toxins, mimicking even more closely the in vivo situation. It is useful to point out here that the term in vivo refers to biological experiments that are performed with intact living organisms in their usual state, while ex vivo refers to studies on a functional biological component that has been isolated (explanted) from the intact

31,32 33,37,40−43 34

47 48 49

54,55

organism and in vitro generally refers to studies that are conducted using components of an organism that have been isolated from their usual biological context. One of the most commonly used animal models in biology is the murine model Mus musculus. However, the roundworm Caenorhabditis elegans, the fruitfly Drosophila melanogaster, and the zebrafish Danio rerio are gaining momentum as organisms for screening tests, as they combine genetic amenability, low-cost, and culture conditions that are compatible with large-scale screens. Their main advantage is that they allow high-throughput screening in a whole-animal context; a trend of increased use of microfluidic environments for this purpose is observed. The promise of microfluidic technology for systems biology, encompassing biological research on the molecular level, cellular level, and organism level, was reviewed by Feng et al.30 Excellent reviews of microfluidic technologies and their potential for the phenotyping, imaging, and screening of multicellular organisms were presented by Crane et al.31 and Wlodkowic et al.,32 while the review of Chronis focused on microfluidic chips for biological studies of C. elegans.33 Another dedicated review focused on microsystems for controlled genetic perturbation of live D. melanogaster embryos.34 The importance of these animal models in biology research and more particularly in highthroughput screening methods for drug discovery was reviewed by Giacomotto and Ségalat.35 We noticed here and in the following that a number of reviews already exist, which are linked to particular topics discussed in the actual review paper. For the interested reader, Table 1 shows an overview of other review papers that are related to this Review. Many different technologies for the realization of fluidic microsystems have been developed.2,56 The microfabrication sequence of a basic chip with a single microfluidic channel layer involves the patterning of the microchannel layout, a bonding operation to seal the open channels, eventually followed by surface coating or functionalization steps. Glass and silicon have B


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of processing steps, by which various features can be implemented in a PDMS device.56,61 Usually the process starts by the microfabrication in a clean room of a microstructure made in a resin like SU-8 or in silicon (Figure 1a), which is subsequently used as a master for application of liquid PDMS (the stringent conditions of a clean room are less required for this replication step). After polymerization, the PDMS sheet can be peeled off from the substrate containing the master (Figure 1b), and the latter can be reused. Thin PDMS membranes (several 10 to several 100 μm in thickness) can be made by spinning liquid PDMS on a substrate put on a turning table, after which the polymerized membrane can be peeled off from the substrate. PDMS sheets and membranes can be combined via irreversible bonding, after a plasma-activation step of the different parts, resulting in hydroxylated surfaces that, after being brought in contact, develop permanent bonds. In this way, PDMS chips with various features can be realized, such as microfluidic chambers, multiple level microchannels, and membranes (Figure 1c). The latter can be deformed (actuated) by external pressure sources and can be used, for example, for pumping, as valves, or for immobilizing temporarily a living worm by deforming the membrane around it (Figure 1d). PDMS “microplumbing” technology represents until now the preferred option for many scientists working with microfluidic applications and is particularly attractive, when a low number of microfluidic chips are sufficient for the biological study of interest. However, when large numbers of chips (>10− 100) are needed, microfabrication of the latter can become a tedious and labor-intensive process. Polymer mass-production techniques, like injection molding and hot embossing, form here an interesting option.56,61 Such-produced parts can also be combined with thin membranes by thermal bonding processes, so that complex microfluidic chips can be realized. The technique is however less exploited until now, due to the relatively high costs of the machines and of the master tools needed for replication. However, this may change in the future as specialized companies and dedicated foundries62,63 offer now their services to potential users of these microfluidic chips. The latter are becoming more often available in laboratory standard formats, like that of a microscope slide or a microtiter plate. There are different ways to load a biological sample into a microfluidic chip in a reproducible way. For investigation of a unique sample or a low number of discrete samples, a frequently used technique is to place the sample or sample substrate manually in the microfluidic system, after which microsystem sealing is achieved and automated microfluidic protocols are implemented. This is the case, for example, when culturing a living tissue slice64 or detecting biomarkers on a sliced tissue biopsy sample,65,66 or when culturing organs like a plant root67 on a chip. When a high number of identical or

been dominating materials for the realization of microfluidic chips at the beginning,57 but have been more often replaced by polymers. Microfabrication of glass56 is relatively expensive due to the requirement of clean room-based technologies such as deep plasma etching or hydrofluoric acid-based wet etching. This partly explains the interest in the replication of microchannel master structures, which are mostly realized in a clean room, into an elastomeric material like PDMS,58,59 which has become a leading microfluidic technology that is frequently used by the LoC research community. Bonding different molded PDMS sheets in a stack allows the realization of more complex chips, where, for example, microfluidic control functions, like valving or pumping, can be combined with the primary microfluidic circuit.60 Figure 1 shows a basic sequence

Figure 1. Typical processing steps of a PDMS device. (a) Microfabrication of a microstructure to be used as a master for application of liquid PDMS. (b) After polymerization, the PDMS sheet is peeled off from the substrate. (c) PDMS sheets and membranes can be combined via irreversible bonding into chips with various features, such as microfluidic chambers, multiple level microchannels, and membranes. (d) External pressure-induced deflection of a membrane allows immobilizing temporarily a living worm, for example.

Figure 2. This Review focuses on “top-down” microfluidics-enabled studies of biological objects, like tissues, organs, and whole living organisms. Parts (a)−(c) are representative micrographs showing (a) a dorsal view of a fruitfly’s embryo (D. melanogaster), (b) a fully developed nematode worm (C. elegans), and (c) a fluorescent micrograph of a biopsied breast cancer tissue slice stained with different markers. C


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Table 2. Summary of Microfluidics-Based Research With Respect To the Organism, Organ, or Tissue Studied organism/organ/tissue focus of research

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Mouse/Bovine Oocytes and Embryos mouse embryo development in microchannels use of a partially constricted channel in combination with mechanical stimulation for bovine embryo development mouse embryo development in microfunnels under exposure to a pulsatile flow removal of the zona pellucida and cumulus cells from around bovine oocytes/embryos integrated multiple processing steps of in vitro fertilization protocols on mouse oocytes/embryos microfluidic platform for mouse embryo culture in 5−500 nL volumes electrochemically monitoring oxygen consumption of a mouse embryo using electrodes integrated in a microwell Danio rerio (Zebrafish) Embryos development of fish embryos inside microchannel-based aqueous compartments in a continuous oil phase programmed droplet transport in an electrowetting-on-dielectric-type actuation device hatching and development under continuous flow of buffer in culture wells high-throughput platform for in vivo chemical exposure and genetic screens at cell resolution; laser microsurgery; microfluidic chemical gradient generator phenotype-based evaluation of toxic and teratogenic potentials of drugs microrobotic system for fully automated injection of biomolecules in embryos electroporators for the patterned delivery of nucleic acid molecules into embryos Drosophila Melanogaster (Fruitfly) Embryos spatiotemporal control of embryo’s temperature environment using laminar flow streams; study of effect on embryo development self-assembly using surface tension-induced forces of the embryos onto oil adhesive pads; thermal perturbation; drug screening application hydrodynamic forces to orient embryos in an upright position, exploiting their anisotropic embryonic shape; study of morphogen gradients in the embryo’s dorsoventral patterning system automated system for mass-injection of embryos based on injection microneedles made of silicon nitride for application in high-throughput RNA interference screens in vivo imaging of cellular response to neural injury in Drosophila larvae Drosophila larvae navigation in controlled airborne cues Caenorhabditis elegans (Roundworm) Embryos, Larvae, and Adults “behavior” and “olfactory” chips for trapping of single worms, and monitoring of their behavior and neural function high-speed microfluidic sorter for screening phenotypic features at subcellular resolution in physiologically active animals “worm clamp” microfluidic system for immobilizing animals; lifespan and imaging studies flow-cytometer adapted for worm profiling to generate “chronograms”, 2D representations of fluorescence intensity along the body axis at a rate of up to ∼100 animals/s fluorescence-based worm sorting at rates up to 2500 animals per hour using a software control interface parallel behavior tracking of single worms in microchamber arrays and delivery of stimuli with precise temporal control lens-free fluorescent or tomographic imaging using a video camera chip in CMOS or CCD technology; no optical microscope needed encapsulation of eggs, along with E. coli cells as animal feed, inside aqueous droplets; larval development study encapsulating worms in droplets and arraying them using hydrodynamic immobilization traps study of sleep- and wake-like behavior and neuron rewiring of developing C. elegans larvae in agarose hydrogel compartments reversible immobilization of worms using a thermo-sensitive sol−gel transition for high-resolution imaging and development studies injection of transfected eggs in clusters into a channel on a microfluidic chip temperature stimulation of embryos study of the effect of on-chip immobilization approaches on postimmobilization locomotion behavior microfluidic devices containing a particle medium, arrays of microposts, or sinusoidally shaped microchannels for studying C. elegans’s locomotion behavior application of electric fields for controlling the movement and sorting of worms high-throughput droplet-based analysis/screening by studying the effect of a neurotoxin at single-animal level study of locomotion patterns of larvae of the pig parasitic nematode Esophagostomum dentatum as a function of the concentration of drugs measurement of the muscular force developed by worms PDMS chip clamped against an agar plate on which the animals move to perform an aerotaxis assay study of information processing by C. elegans olfactory neurons and interneurons that control food- and odor-evoked behaviors microfluidic maze for investigating the exploratory and reward (e.g., food)-associated learning behavior passive synchronization of worm populations based on their age and size in different microfluidic maze architectures complete automation of handling, high-resolution microscopy, phenotyping, and sorting of C. elegans platform for performing automated neuronal functional (calcium) imaging in worms precision on-chip femtosecond laser nanoaxotomy in C. elegans and nerve regeneration studies programmable microvalve-based microfluidic array for real-time and long-term monitoring of the neurotoxin-induced responses of individual worms laterally orienting and immobilizing worms for dorso-ventral visualization of D-type motor neurons synaptic transmission at the neuromuscular junction enabled by optogenetics; change in body length by muscular contraction and stimulation by blue light electrophysiological measurements on the pharynx of C. elegans; effect of antinematode drugs large-scale in vivo screens for identification of compounds that affect neurite regeneration in C. elegans after microsurgery effects of ethanol on neuron function D

71 72,73 74 75,76 77,78 79 80 81 82 83 84−87 88,89 90 91 92,93 34,94−96 97,98 99−102 103 104 105,106 107 69,108−111 112 113 114 115−119 120 121 122 123 124 125 126 127−131 132−137 121 138,139 140−142 143,144 145,146 147 148 68 149 150−154 155 156 157 158 159 160


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Table 2. continued organism/organ/tissue focus of research

ref

Caenorhabditis elegans (Roundworm) Embryos, Larvae, and Adults compact disc system for cultivating C. elegans, enabling automated feeding, waste removal, microscopic observation, and tests under hypergravity conditions Organs generation of molecular gradients in 3D to create an assay for precise induction and guidance of growing blood vessels fixing and probing small arteries, in particular resistance arteries obtained by microdissection from wild-type mice, inside PDMS chips PDMS chips to hold and cultivate live roots of Arabidopsis thaliana and expose them to a locally changing chemical environments with high spatial resolution Tissues Rrodent hippocampus brain slice studies in PDMS mini-wells integrated with microfluidic channels using standard microelectrode arrays perfusion and culture chamber for organotypic brain slices obtained from rodents using a platform made of microposts; insertion of recording electrodes in the tissue perfusion chamber with an array of microfluidic channels and vias or with a semipermeable membrane incorporated into the bottom surface of the chamber precise spatiotemporal control of oxygen provision to mice brain slices microfluidic probe for the localized exposure of brain slices to chemicals and for local immunohistochemical analysis microfluidic chamber that incorporated fluid ports with active suction to achieve localized chemical stimulation of brain slices microneedles to perfuse the interior of 0.4−1 mm thick rat brain tissue slices microfluidic device in glass for investigating rat liver tissues and human colorectal tissue biopsies under continuous perifusion microfluidic device in glass for maintenance and interrogation of head and neck squamous cell carcinoma tumor biopsies microfluidic chip for the perifusion of rat liver slices and intestinal slices for metabolism and toxicology studies sequential perifusion of two tissue slices, either from the same organ or from two different organs, intestine and liver; combination with automated metabolite detection microfluidic half-chamber put on a Petri dish surface for accommodating a rat/human heart tissue slice; in situ electrochemical monitoring of the reactive oxygen species released from the tissue slice migration studies of liver and kidney cells originating from explants placed in microchannels spatiotemporal gradients of hormone applied to animal cap tissues isolated from Xenopus laevis (clawed frog) embryos PDMS-based microfluidic devices for multiplexed immunohistochemical detection of different cancer biomarkers expressed on clinical breast tissue samples microfluidic in situ hybridization to detect microRNA on formalin-fixed paraffin-embedded sections of a mouse brain

161,162

163 164 67,165,166

167,168 169−171 172,173 174 175,176 177,178 179,180 181,182 183 184−187 185,188 189,190 191,192 65 66,193−196 197

biopsies were immobilized and cultured in a microfluidic environment. Tissue slices are very thin and by that naturally compatible with planar microfluidic technology. Once positioned on a chip, they were perfused and cultured for physiological and metabolomic research purposes, as well as for drug screening applications. We finally review the work on microfluidic chips for in vitro immuno-histochemical imaging of fixed clinical tissue slice samples (Figure 2c). These systems allow the multiplexed detection of biomarkers on cancerous tissues for clinical diagnostic applications. In many of the reviewed research articles, the easy integration of microfluidic control and detection modules was a key factor in helping to bridge the gap between in vitro and ex vivo experimental investigations. This certainly will promote more automated and high-throughput applications further in the future.

similar biological samples are available and/or need to be screened in a high-throughput fashion, a reservoir holding all biological objects is put in fluidic contact with the chip, after which a flow (perfusion) of the sample liquid into the chip is induced via an external pumping mechanism. A highthroughput chip can be designed such that its natural flow patterns allow positioning single biological objects into the different trapping and analysis sites of the device. This principle was used, for example, for the positioning of D. melanogaster embryos in an upright position for study of their anisotropic embryonic shape68 or for the arraying of C. elegans roundworms via tapered channel-induced immobilization.69 When multilayer PDMS chips with more complex microfluidic control options are used, these “flow-induced” trapping procedures can be combined with active and automated local manipulation and chemical exposure sequences of the individual worms.70 1.2. Scope of This Review

1.3. Synopsis of the Reviewed Work

We review the use of microfluidic chips for investigating under controlled physical and chemical conditions: (i) in vivo whole living organisms, (ii) ex vivo organs, and (iii) ex vivo tissue slices and in vitro tissue slices obtained from human tumors. Organisms, like roundworms, zebrafish embryos and larvae, and fruitfly embryos and larvae, have been advantageously used as integrated biosensors for toxicological experiments and drug screening. For example, D. melanogaster embryos (Figure 2a) were investigated under extreme chemical conditions on-chip, and developmental biology studies were performed using C. elegans (Figure 2b). Moreover, C. elegans laser microsurgery followed by tissue or organ regeneration studies have been achieved on-chip. We also discuss how organs taken from biological specimens and living tissue slices obtained from

To give the reader a quick overview of the literature, Table 2 summarizes the reviewed research articles with respect to the organism, organ, or tissue studied, mentioning the points of focus of the research. In addition, Table 3 gives a quick overview of the technologies and innovations implemented in the different microfluidic systems for multicellular organism/organ/tissuebased research. We shortly highlight the key advantage of each design, its limitation, and aspects regarding the ease of operation and fabrication of the microfluidic devices, and mention representative references. E


F

large area (2 cm2), thin and rigid microfluidic chamber for immunohistochemical analysis

tapered microfluidic channel for C. elegans clamping C. elegans immobilization by deflection of a thin PDMS membrane hydrodynamic trapping and arraying of droplets C. elegans immobilization by inducing sol−gel transition in worm chamber microfluidic probe

very local perifusion of tissues; cellular resolution immunno-histochemical assay times within a few minutes

controlled manipulation of individual droplets liquid actuation by rotation; no pump needed “self-trapping” of individual worms transported in a liquid stream high-resolution time-lapse imaging of animals without the need for anesthesia trapping based on fluidic resistances circuit design reversible, biocompatible, no membrane deflection-based technology needed

electrowetting-on-dielectric actuation-based droplet transport microfluidic compact disc system

droplet microfluidics

microfluidic stickers

rapid prototyping, ease of fabrication; biocompatible; integration of thin membranes with bulk parts generic approach to position biological objects into closed microchannels controlled liquid compartments; highthroughput manipulation possible

multilayer soft lithography

key advantage

rapid prototyping, biocompatible

microfluidic systems in PDMS

technology

precise and uniform height of the microfluidic chamber over the tissue surface needed; high pressure-resistant microfluidics

plasma etching techniques needed to realize the shallow chambers

careful microfluidic design needed

simple PDMS technology

special polymer to be added in the culture media

low-throughput technique

simple PDMS technology

more complex multilayer PDMS technology

scanning probe-like technique; accurate xyz displacement control required easy clamping of the microfluidic halfchamber against the tissue slice

easy, automated arraying of droplets at fixed positions exchange of sol−gel and normal medium required; extra manipulations

automated protocols and high number of analyses possible operation in remote environment (space); stroboscopic observation needed analysis of worm locomotion patterns and local chemical stimulation possible individually addressable screening-chambers, using valve technology

once optimum conditions achieved, high number of analyses possible

easy assembly

polymeric “half-channels” that adhere on wet flat substrates careful design of microfluidic circuits needed, as well as precise flow control of different liquids cleanroom microstructuring techniques required polymer molding technique; hydrophobic/hydrophilic disc patterning simple PDMS-based technology

ease of operation elastomeric devices; bubbles can be removed, as PDMS is permeable to gas; suitable for living animal experiments active microfluidic systems containing valves and pumps; automated protocols

ease of fabrication replication from master structures; PDMS bonding; minimum infrastructure needed PDMS replication and bonding; alignment between layers needed

careful microfluidic design needed

more complex microfluidic chip with transparent actuation electrodes needed single assay, liquid flow always from inside to outside of the disc

need for continuous (oil) phase; extra liquid handling

manual technique, low-throughput operation

multiple fluidic inputs can lead to complex interfacing to the outer world

not true batch process; not easy to upscale

limitation

Table 3. Summary of Microfluidic Techniques and Innovations Exploited for Animal/Organ/Tissue Research ref

195

175

123

121

107,110,111

69,105,108,109

161,162

82

81,120,121

198

60

58,59

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2. LIVING MULTICELLULAR ORGANISMS

mechanical stimulation, realized by simply placing the microfluidic chip on a tilting machine that provided embryo movement via gravity, mimicking peristaltic muscle contraction. Conventional photolithography and a PDMS replica molding process were used to make straight or constricted microchannels. The fertilized embryos were cultivated on the microfluidic in vitro cultivation system until the blastocyst, hatching, or hatched blastocyst stages. The system was designed for transferring cleaved embryos in a new culture device filled with CR1aa medium supplemented with 10% fetal bovine serum. The quality of blastocysts in the microfluidic channel was compared to bovine embryos cultivated by the conventional droplet method. The proportion of eight-cell embryo development among total embryos in the constricted channel (57 ± 14%; mean ± standard deviation) was superior to that in the straight channel (24 ± 11%). This suggested that the effect of the constriction was vital for the early development of the bovine embryos. To test the quality of the embryos, a double staining method was performed: a blue stain was used for labeling the trophoblasts, which are cells forming the outer layer of a blastocyst, that serve to provide nutrients to the embryo, and a red stain for labeling the inner cell mass. A high proportion of inner cell mass over trophoblast mass and more staining is indicative for a higher quality of the blastocysts.72,200 The double-staining method revealed that embryos cultivated in the microfluidic device had quality similar to those cultivated according to the conventional method. Other work of the same group reported the mechanical stimulation via deflection of a PDMS membrane of bovine embryos using a micromodulated syringe pump.73 Heo et al.74 have compared the culture of mouse embryos in microdrop static control experiments, in microfunnels under static liquid conditions, and in microfunnels under dynamic microfluidic conditions. Blastocyst cell numbers in the dynamic microfunnel cultures more closely matched the numbers obtained from in vivo experiments than the other types of culture. This may be because of the fact that a dynamic microfunnel embryo culture system better mimics the fluid-mechanical stimulation embryos experience in vivo. Microfluidic methods also proved their utility for the removal of the zona pellucida75 and cumulus cells76 around the embryo. The zona pellucida is a passive glycoprotein membrane encasing the embryonic cells during the first 5−8 days of development, while cumulus cells surround the oocyte when it is retrieved from a donor. Manipulation of the zona pellucida is common to many in vitro production procedures. Zona pellucida removal was achieved by briefly washing the embryo by flowing a plug of lysing agent over it, while cumulus cells were gently sucked from a cavity hosting the embryo via a dedicated microfluidic channel. Another microwell-structured microfluidic device demonstrated integrated multiple processing steps of in vitro fertilization protocols, like single mouse oocyte trapping, fertilization, and subsequent embryo culture.77 A microwell array was used to capture and hold individual oocytes during the flow-through process of oocyte and sperm loading, medium substitution, and debris cleaning. Use of the microfluidic device allowed simplifying oocyte handling and manipulation, as well as rapid and convenient medium changing. Fertilization was achieved in the microfluidic devices with fertilization rates similar to those of standard oil-covered drops in Petri dishes. Embryos could be cultured to blastocyst stages with developmental status individually monitored and tracked. Ma et al. proposed a microfluidic device that integrated each step of the in vitro fertilization process, including the positioning of

2.1. Mammalian Embryos

The word embryo (Greek: swelling within) refers to a growing organism in its earliest stage of development, from the first cell division until birth, hatching, or germination. In humans, it is called an embryo until about 8 weeks after fertilization, and from then it is called a fetus. However, according to biology, a living being or organism grows, takes in nutrients, and expels metabolic waste, which is the case for an ovum starting immediately after conception. Animals that develop in eggs outside the mother’s body are usually called embryos throughout their development. Embryos studied in chips are mostly of submillimeter size and therefore have intrinsic geometrical compatibility with microfluidic systems, just like single cells or spheroids. In vitro production of human embryos has become an important factor in the treatment of infertility. In traditional embryo culture, different culture systems and media are used, but the temperature is always at 37 °C, and the presence of 5% CO2 is needed to maintain the pH of the medium at 7.2−7.4. Culture can proceed in small droplet volumes (10−50 μL) of medium under a layer of paraffin oil or in a large volume (1 mL) without oil. Simple media contain salts, bicarbonate, energy substates (pyruvate, lactate, glucose), and proteins, while in complex media amino acids and vitamines are added. Because the nutritive requirement of the embryos changes in function of their stage of development, culture is performed in different media according to their cleavage stage.199 Traditional in vitro culture hence is in relatively large volume droplets (larger than 10 μL) of medium, when compared to the submicroliter amounts of liquid present within the crypts inside the lumen of the female reproductive tract. Microfluidics allows for the creation of culture systems of a scale similar to the characteristic diameters of the embryo’s natural in vivo environment. Advances in embryo culture platforms for preimplantation embryo development have been reviewed.44 Microwell, microdrop, and microchannel platforms were compared and the role of static/dynamic fluid conditions and special surface treatments highlighted. A study demonstrated the embryonic development of twocell mouse embryos to the blastocyst stage (up to 96 h of development) in a microfluidic channel structure.71 Three different microfabrication materials, silicon, PDMS, and borosilicate glass, were used to fabricate microchannels that were operated under static medium conditions, that is, without application of flow. Control embryos were cultured in tissue culture dishes in 30 μL drops of Mouse Embryo Culture medium under 5 mL of light mineral oil. Embryos cultured in the silicon/borosilicate and PDMS/borosilicate microchannels exhibited a faster rate of cleavage and produced more blastocysts than did embryos in control microdrops. Furthermore, microchannels had a lower percentage of degenerated embryos than control embryos, suggesting that the small volumes offered by microchannel culture systems may provide a culture environment that more closely mimics the in vivo environment. Another study reported a microfluidic platform that enabled mouse embryo culture in 5−500 nL volumes, showing that groups of two embryos per microfluidic well successfully developed to the blastocyst stage, at a rate comparable to those cultured in 20 μL drops.79 Kim et al.72 demonstrated a microfluidic in vitro culture system for bovine embryos. Improved embryo development resulted from the use of a partially constricted channel in combination with G


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Figure 3. A microfluidic device for in vitro fertilization. (a) Photograph showing the three regions of the device: 1, sperm inlet pools, 4 mm in diameter; 2, sperm screening channel, 1.5 mm in width, 9 mm in length, and 80 μm in depth; 3, oocyte-embryo positioning well, 6 mm in diameter. (b) SEM image of the central 4 × 4 array of octa-column units used for hosting oocytes and embryos. (c) Microphotograph of a representative mouse embryo at 104 h after fertilization in the microstructure. Reprinted with permission from ref 78. Copyright 2011 American Chemical Society.

Figure 4. (a,b) The movement of a 5 mm diameter droplet containing a zebrafish embryo (24 h post fertilization) in an electrowetting-on-dielectrictype of device. The voltage applied to the electrode was 95−105 Vrms at 8 kHz. It took 1−2 s for the embryo to move to the next electrode. (c) The embryo 48 h after transport, maintained in room temperature fresh water. (d) Schematic of an integrated microfluidic chip for an embryonic zebrafish assay, consisting of a top plate with a concentration gradient generator and embryonic inlet array, a middle plate with a set of independent array chambers for embryo culturing, and a bottom layer. (e) The assembled microfluidic device, with two inlets of solution (medium and drug +medium), used to generate drug gradients, in order from chambers 1 (high) to 7 (low), for exposure to the embryos trapped in the culture rooms. (f) Magnified section of a single chamber structure including an embryo inlet from the device upper layer, and a culture compartment containing several embryos, sandwiched between inlet and bottom plate, with connection to a flow microchannel. (g) Photograph of the fabricated microfluidic chip. (a−c) Reprinted with permission from ref 82. Copyright 2009 The Royal Society of Chemistry. (d−g) Reprinted with permission from ref 88. Copyright 2011 American Institute of Physics.

understanding of hematopoiesis, that is, the formation of blood cells from hematopoietic stem cells, in this animal model.201 The standard culture protocols for zebrafish embryos and larvae have been derived from cell culture, meaning that the animals are commonly kept and raised on microtiter plates or in Petri dishes.202 Refreshment of buffer solutions in such situation is invasive and causes stress to the larvae and embryos, as it requires periodic aspiration and replacement of the old buffer by a fresh solution. Another problem is that, for toxicity testing, the concentration of the active compounds in the exposing solution might not be precisely controlled because of evaporation and nonselective adsorption effects on the wall of the wells. Microfluidic technology offers a straightforward solution to all of the problems sketched above. Eggs from D. rerio have been introduced inside Teflon tubes of 1.2 mm inner

oocytes, sperm screening, fertilization, medium replacement, and embryo culture (see Figure 3).78 The embryo growth rate and blastocyst formation were similar for Petri dish control and microfluidic device experiments. Date et al.80 reported the measurement of the oxygen consumption of a single mouse embryo that was positioned in a microwell with integrated microelectrodes. The oxygen consumption in the liquid measurement medium was electrochemically recorded using the electrodes. 2.2. Danio rerio (Zebrafish) Embryos and Larvae

Small size, optical transparency of complex organs, and ease of culture make zebrafish embryos and larvae attractive organisms for in vivo genetic and toxicology studies. The biology of D. rerio is well-known, and, for example, a number of zebrafish studies in well plates have led to major breakthroughs in the H


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Another article described the development of a microfluidic array system for phenotype-based evaluation of toxic and teratogenic potentials of clinical drugs on zebrafish.88 The microfluidic chip consisted of a concentration gradient generator and an array of open embryonic culture sites (see Figure 4d−g). Developmental toxicity effects of doxorubicin exposure to the eggs were assessed via monitoring of the embryo’s heart rate, and its teratological effects were characterized by precise evaluation of a number of morphological parameters of the developing embryos. Moreover, the potential toxicity of antiproliferating drugs on zebrafish embryos was evaluated. Another group developed a microfluidic device in silicon and glass that incorporated a microfluidic gradient generator, a row of fish tanks, and output channels.89 The gradient generator allowed dose-dependent drug and chemical studies. In particular, embryo development abnormalities were observed under the influence of the drug valproic acid. Also, the injection of foreign materials, like deoxyribonucleic acid (DNA) or drug compounds, in living cells or embryos is a widely used technique that has significant implications in genetics, drug discovery, and other biological applications. While using a single glass micropipet for microinjection remains a very efficient technique in terms of cell or embryo damage and viability, such type of manual operation is very slow. Therefore, a microrobotic system was proposed for fully automated zebrafish embryo injection, which overcame many of the inherent problems of manual operation; operational injection speeds of 15 zebrafish embryos per minute were reported.90 Other authors used microfluidics and microfabrication techniques to produce electroporators for the patterned delivery of foreign molecules into zebrafish embryos.91 Applying 10−20 V and 50−100 ms electrical pulses via micropatterned platinum electrodes created transient pores in the cell membrane, which allowed the entering of extracellular compounds that were present in the vicinity of the pore into the interior of the cell. Fluorescently labeled DNA and messenger ribonucleic acid (mRNA) were successfully delivered at high survival rates (90%) of the embryos. All of these results demonstrate the potential of using active microfluidic devices as an alternative to microwell plates for zebrafish-based assays.

diameter in microfluidic segments of embryo media separated by an oil phase.81 The volume of the microsegements varied between 2 and 11 μL, and the development processes of fish embryos inside these fluidic compartments were observed over a time period of 80 h, until hatching time. After 5 days, the fish larvae were brought out of the microfluid segments and transferred into breeding reservoirs. Effects of the membranedamaging anionic surfactant sodium dodecyl sulfate (SDS) alone and SDS with the addition of CuCl2 were investigated. Low SDS concentrations with and without copper(II) ions were supportive, while higher SDS concentrations led to negative impacts on the development of the embryos. In other work, programmed transport of zebrafish embryos within droplets in an electrowetting-on-dielectric-type device was demonstrated (see Figure 4a−c).82 A two-plate droplet microfluidic device was used for these experiments. The bottom plate had an array of electrodes beneath a dielectric layer for addressing droplet movement, and the top plate had a common ground electrode. Advantages over a one-plate device are that droplet evaporation is slow and that it is simple to control the velocity of the droplet by changing the gap between the plates. Upon applying voltage on a bottom electrode, a droplet situated on a nonactuated electrode with some overlap with the actuated electrode moved to the actuated one. The electrowetting-on-dielectric force is enhanced when the droplet, which is originally over the nonactuated electrode, has a larger area of overlap with the neighboring electrode to which the actuation voltage is applied. Keeping the droplet volume the same and squeezing the droplet by reducing the gap between the plates leads to a larger droplet overlap with the neighboring electrode, and hence to a larger force. A zebrafish embryo transported 2 h after fertilization developed normally and hatched. Also, dechorionation was demonstrated by mixing a droplet of digestive reagent with a droplet containing the embryo. Another LoC device made from borosilicate glass demonstrated under controlled temperature conditions the hatching and development of early D. rerio embryos under continuous flow of buffer in the culture wells (a flow of 2 μL per minute was applied to an embryo-containing well during 5 days).83 Other authors presented a highthroughput platform for cellular-resolution in vivo chemical and genetic screens on zebrafish larvae.84 The system automatically pumped zebrafish larvae from reservoirs or multiwell plates, and positioned and rotated them for highspeed confocal imaging and laser manipulation of both superficial and deep organs without damage. Small-scale test screening of retinal axon guidance mutants and neuronal regeneration assays was performed in combination with femtosecond laser microsurgery. The same research group developed a multichannel microfluidic perfusion platform for culturing zebrafish embryos and capturing live images of various tissues and organs inside the embryo.85 Multiple animals could be processed at the same time, and image recognition algorithms were developed that allowed fully automated manipulation of the animals, including orienting and positioning into regions of interest within the microscope’s field of view. Others presented a miniaturized array system for automated trapping, immobilization, and microperfusion of zebrafish embryos.86,87 Passive docking of the embryos using hydrodynamic positioning structures was combined with timelapse imaging to provide developmental analytical data. The PDMS-based system was also used to test an antiangiogenic compound using transgenic animals.

2.3. Drosophila melanogaster (Fruitfly) Embryos

The fruitfly is of particular biological interest because it serves as a model organism for developmental and cellular processes common to higher eukaryotes, including humans. Fruitflies are typically bred and kept with food in a closed vial of 30−50 mL volume. Because adults can reproduce throughout their life, a strategy to maintain a stock of age-synchronized embryos or larvae is to move groups of adults to fresh vials where they lay eggs overnight, which are subsequently manually harvested.203 To investigate the role of the different genes, a powerful method is to work with mutants, which, for example, can be obtained by ribonucleic acid (RNA) interference, a biological technique that moderates the activity of the genes in cells via injection of certain small RNAs that can bind to other specific mRNA molecules and thereby either increase or decrease their activity. To produce mutants by injection of genetic material, a critical part of the procedure is to position the embryos prior to injection. While this is very labor-intensive with the classical technique, microfluidics can play an important role in positioning the embryos at well-defined positions and with a I


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Figure 5. (a,b) The rate of development in each half of a D. melanogaster fruitfly embryo exposed to a temperature step. Each half of the embryo is in a different cell cycle, as demonstrated by the difference in nuclear density. The number of nuclei in enlarged areas are shown underneath in yellow numbering. Embryos are exposed to a temperature step of 20 °C/27 °C for 140 min. (a) Anterior half 20 °C, posterior half 27 °C; (b) anterior half 27 °C, posterior half 20 °C. In all images, higher nuclear density was observed in the warmer half of the embryo. (c−e) Microfluidic embryo-trap array for high-throughput arraying of vertically oriented D. melanogaster embryos. (c) Photograph of the device (left) and a micrograph detailing the trapping region (right). Scale bar, 1 cm (left) and 500 μm (right). (d) Schematic showing the embryo trapping process: an embryo is guided into the trap (left); the flow around the embryo orients it vertically (right), after which the trap contracts and secures the embryo. The large arrows show the direction of bulk flow in the serpentine channel. (e) A section of the array with trapped embryos (dark circular object in each trap). Scale bar, 500 μm. (f) Image of D. melanogaster embryos immobilized on a patterned array of oil-coated immobilization sites with 800 μm × 800 μm pitch. (a,b) Reprinted with permission from ref 92. Copyright 2005 Macmillan Publishers Ltd. (c−e) Reprinted with permission from ref 98. Copyright 2011 Macmillan Publishers Ltd. (f) Reprinted with permission from ref 95. Copyright 2004 Elsevier BV.

differential interference microscopy.94 A particular feature of this work was the self-assembly using surface tension-induced forces of the embryos onto oil adhesive pads, when using an alcohol surfactant carrier fluid. The same group also reported drug screening experiments on live Drosophila embryos in both 96-well plates and microfluidic channels: effects of colchicine and cytochalasin D on cellularization and embryogenesis were studied.34 To determine the functions of genes in a high-throughput genome-wide fashion, it is advantageous to use microfluidic technology for the positioning of a multitude of embryos at regular positions on a substrate. Hydrodynamic forces in a microfluidic chip were used to orient Drosophila embryos in an upright position, exploiting their anisotropic embryonic shape (see Figure 5c−e).97,98 Using this technique, morphogen gradients in the dorsoventral patterning system of the embryo could be quantified, a task that was before complicated by the need to manually orient individual embryos. In the Drosophila embryo, dorsoventral patterning is initiated by the nuclear localization gradient of Dorsal (Dl). D1 is one of the maternally active dorsal-ventral polarity genes of Drosophila. The microfluidic device enabled high-throughput analysis of the dorsoventral patterning system at the level of the inductive cues and their signaling and transcriptional targets in multiple genetic backgrounds. Data for dozens of embryos were sufficient for statistical analysis of spatial patterns in both

certain orientation on a chip in a semiautomated way. Moreover, precision injection tools can be provided using microfluidic technology, and microfluidic circuits can be used for extremely well controlling physicochemical environmental parameters during embryo development. A PDMS-based microfluidic device was realized that allowed the temperature environment of Drosophila embryos to be spatiotemporally controlled.92,93 Using a simple Y-junction device, the temperature of different regions of a live Drosophila embryo could be controlled in space and time by flowing two laminar aqueous streams of different temperature over the embryo that was positioned in the flow on a thin adhesive tape. It was shown that a chemical reaction compensation system of the embryo could counteract the effects of extremely unnatural environmental conditions, the temperature step, in which the anterior and posterior halves of the embryo were developing at different temperatures and thus at different rates. Figure 5a,b shows the effect of the temperature step on the development of the embryo, as evidenced by the different number of nuclei, which were immunostained using standard methods. Perturbing the physical environment was shown to be a complementary approach to perturbing the molecular components of an embryonic chemical reaction network. Another study reported how thermal perturbation of Drosophila embryos in a microfluidic device resulted in abnormal morphogenetic movements in live embryos, as evidenced using time-lapse J


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chemical gradient generator chips, and to expose these to freely moving larvae. In combination with machine-vision algorithms, navigational decision making of the larvae in response to ethyl acetate as a volatile attractant and carbon dioxide as a repellant was studied.

wild-type and mutant backgrounds. Using this device to image a large number of embryos, an outstanding issue regarding the spatial extent of the Dl morphogen gradient could be resolved. Positioning and immobilization of Drosophila embryos in 2D arrays via self-assembly was already presented in another study (see Figure 5f).95,96 The method was based on fluidic microfixation of the embryos via an oil droplet onto thiolated monolayers that self-assembled onto gold pads. Immobilization yield, the number of misplaced embryos, alignment properties, and adhesion force of the embryos were measured for samples with four different pad geometries. For samples with 250 μm × 400 μm sized rectangular pads, an immobilization yield of 85% was achieved, but a substantial amount of clustering was observed. By reducing the pad size to 200 μm × 200 μm and changing the pad pitch and shape, the number of misplaced embryos was reduced to less than 5%, but the immobilization yield was lower (72%) for these samples. The same research group also presented the design and characterization of a pressure-driven microfluidic sorter designed for Drosophila embryos.204 For studying multicellular organisms in a microchannel, microfluidic stickers were proposed as a generic approach to position biological objects into closed microdevices.198 Stickers were molded from a biocompatible UVpolymerizable resin forming “half-channels” that tightly adhered on wet glass coverslips. In this way, cells or wing imaginal disks that were dissected from D. melanogaster pupae were studied. Microfluidic chips in PDMS were used to immobilize unanaesthetized Drosophila larvae by clamping over periods of up to 10 h.103 These chips were used to characterize several subcellular responses to axotomy by laser microsurgery, and allowed monitoring axonal regeneration in vivo. In other work, an automated system was developed for reliable mass-injection of Drosophila embryos based on injection microneedles made of silicon nitride.99,100,102 Targeted applications were high-throughput RNA interference (RNAi) screens. The method consists of exposing cells to specifically designed double stranded (ds) RNA corresponding to a gene of interest. Inside cells, dsRNA is used by endogenous enzymes to recognize and destroy the corresponding mRNA, thus inactivating the gene function. A typical volume of 60 pL per embryo could be reliably and rapidly delivered within tens of milliseconds to embryos that were attached to a glass slide surface and covered with oil. The system automatically screened the glass slide for embryos and reliably detected and injected more than 98% of all embryos. A first RNAi experiment was successfully performed with dsRNA corresponding to the segment polarity gene armadillo at a concentration of 0.01 mM. Almost 80% of the injected embryos expressed an expected strong loss-of-function phenotype. Another group reported an automated instrument for high-throughput injection of Drosophila embryos, based on an inverted microscope and image recognition software.101 To understand gene function in the control of early development of the fruitfly, DNA was injected in very young embryos. In a fraction of these embryos, the injected DNA was incorporated in the chromosomes, becoming part of the next generation of the fruitfly. Many hundreds of embryos could be robotically injected in a matter of hours. Other work reported the navigation of Drosophila larvae in a 30 cm × 30 cm “arena”, in which a controlled gradient of airborne chemical cues could be applied.104 This system was used to deliver gaseous stimuli in defined spatial and temporal patterns, analogous to what was demonstrated in liquid

2.4. Caenorhabditis elegans (Roundworm) Embryos, Larvae, and Adults

C. elegans is the multicellular organism most frequently studied in microfluidic chips and forms an attractive animal model for biological and medical research, because of its relatively small size, a well-mapped neuronal system, a good transparency, and a relatively short life cycle of around 2 weeks. The life cycle is temperature-dependent: C. elegans goes through a reproductive life cycle (egg to egg-laying parent) in 5.5 days at 15 °C, 3.5 days at 20 °C, and 2.5 days at 25 °C. C. elegans eggs are fertilized within the adult hermaphrodite and laid a few hours afterward, at about the 40 cell stage. Eggs hatch and animals proceed through four larval stages (L1−L4), each of which ends in a molt. When animals reach adulthood, they produce about 300 progeny each. Unlike single cell arrays, realization of single-animal arrays can be challenging due to the animal’s size and motility. Traditionally, C. elegans culture is performed in the laboratory on the surface of nematode growth medium agar plates that have been coated with Escherichia coli bacteria for feeding.205 Large quantities of worms can be transported between different plates by displacing chunks of the agar, while individual worms are transported with the help of a small handling wire. To obtain age-synchronized worm populations, embryos are isolated from actively growing cultures by exposing the complete population a few minutes to a hypochlorite solution. The embryos, protected by a tough eggshell, survive this treatment and can be removed from their parents by centrifugation. C. elegans mutants can be obtained using the RNA interference technique by feeding worms with special bacteria expressing double stranded RNA. It is clear that microfluidics has enormous potential for replacing the laborintensive manual procedures sketched above, which are especially tedious if large numbers of animals need to be processed. All aspects of worm research can be dramatically improved using automation-prone microfluidic protocols, ranging from observation of worm behavior, embryo selection, to exposure to toxicological or pharmaceutical compounds. Different novel approaches have been reported for arraying and immobilizing motile C. elegans in microfluidic networks, allowing long-term imaging and highly precise manipulation. Two short reviews highlighted advances in microfluidic technologies for the manipulation of C. elegans.33,37 Distinction was made between microfluidic chips that were capable of handling either single worms or populations of worms in a high-throughput fashion. Such “worm chips” were mainly used to study neural circuits and behavior, to perform large-scale phenotyping and morphology-based screens, as well as to understand axon regeneration after injury. Another review discussed C. elegans’s biology and its particular attractiveness for chemical and biological research, in comparison with other model organisms.45 That review also provided examples of chemically relevant research and discussed the main microfluidic tools that have become available to study the worms. 2.4.1. Worm Culture and Imaging. Before the era of microfluidic worm chips, studies of C. elegans were laborintensive, as the methods used for confining and stimulating the worms during imaging were far from ideal. For example, a K


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Figure 6. (A,B) Immobilization and subcellular imaging using a worm sorting chip. (A) Image of the on-chip sorter (scale bar: 500 μm). The sorter consists of control channels and valves that direct the flow of worms inside flow channels in different directions. A worm is captured in the chamber by first suction against the right channel, after which it is released and restrained by the multiple suction channels on the left. (B) A single worm is shown trapped by the multiple suction channels. A combined white-light and fluorescence image is taken by a cooled CCD camera. mec-4::GFPexpressing touch neurons and their processes are clearly visible (scale bar: 10 μm). (C,D) Schematic of another device used for C. elegans immobilization. A PDMS membrane is deflected using 14 psi nitrogen gas in the control channel, which was realized in the PDMS2 layer. This membrane immobilizes C. elegans present in a channel realized in the PDMS1 layer. (E) Design of an array of four worm clamps. The array is designed such that, on average, one worm is sorted into each clamp. (A,B) Reprinted with permission from ref 107. Copyright 2007 The National Academy of Sciences of the USA. (C,D) Reprinted with permission from ref 111. Copyright 2011 John Wiley & Sons. (E) Reprinted with permission from ref 69. Copyright 2007 The Royal Society of Chemistry.

mm from a width of 100 μm to 10 μm (see Figure 6E). Their PDMS-based device was made of bifurcated microchannels, all merging into a common inlet on one side and a common outlet on the other side. As the animals were sucked into the microchannel network, they were pushed through the tapered channels up to the point where they were stuck. Once a worm clamp was loaded, the hydrodynamic resistance of that particular part of the microfluidic network became high, and this automatically redirected the other animals toward the vacant worm clamps. Thanks to the tapered design of the worm clamp, worms of different sizes could be easily accommodated. Their results showed that about 90% of the worm clamps were filled with single worms, while the remaining 10% of worm clamps had either no or multiple worms. The worms were seen to be randomly oriented with either head or tail at the front. The authors also showed that almost all of the trapped worms could be removed from the clamps by simply pushing a buffer solution from the outlet toward the inlet of the device. The worms recovered after immobilization did not show any significant internal or external damage or change in their typical behavior. In yet another study, the same group demonstrated the possibility to culture and clamp (immobilize) individual worms using a PDMS microfluidic array, also using a bifurcated channel network design, with a branched inlet and a common outlet. Each branch of the microfluidic array was comprised of a circular confinement chamber for culturing the worms and an adjacent worm clamp for immobilizing the adult worms. The former could be selectively addressed using a prefabricated screw valve.108,109 To demonstrate the applicability of this device for performing longitudinal studies/measurements, the individual worms were loaded via suction at the fourth larval stage into each confinement chamber and investigated over their lifespan. The authors noticed that the average lifespan of worms cultured inside the confined culture chambers was around 10 days, whereas it was around 12 days for those cultured on conventional culture plates. They successfully used the device to monitor and measure the changes in the two phenotypes, body size and locomotion, as the worm ages to death. A flow-cytometer adapted for nematode profiling was used to generate “chronograms”, 2D representations of fluorescence intensity along the body axis and throughout development from early larvae to adult, and this at a rate of up to ∼100 animals/ s.112 Although this instrument did not rely on a microfluidic

typical experimental setup involved application of biocompatible glue on specific segments of the worm to achieve permanent immobilization on a hydrated agar pad, or biologists manually moved around worms one-by-one with help of a small wire. In 2007, several groups demonstrated microfluidic-type worm chips that were used for in vivo C. elegans manipulation and imaging. Chronis et al. presented so-called “behavior” and “olfactory” chips for trapping of single worms, and monitoring their behavior and neural function.105 The PDMS-made chips were worm traps, which were optimized for the size of young adults (approximately 1 mm long and 40 μm in diameter) and contained a tapered microfluidic channel with a width gradually decreasing to 40 μm at one end, which effectively blocked the worm, while still permitting analysis of its locomotion patterns in the wider section of the microfluidic channel, although one could argue that the size and shape of the trap hindered the normal locomotion pattern.206 The olfactory chip had a similar tapered trap design, but allowed, besides the trapping, controlled exposure of the “nose” of the worm to chemical stimuli. The group of Yanik demonstrated a high-speed microfluidic sorter that could isolate and immobilize C. elegans in a well-defined geometry (see Figure 6A,B) for screening phenotypic features at subcellular resolution in physiologically active animals.107 These microfluidic devices consisted of flow and control layers made from flexible polymers. The flow layers contained microchannels for manipulating C. elegans, immobilizing them for imaging, and delivering media and reagents. The control layers consisted of microchannels that, when pressurized, provided a valving function by deflection of a membrane into the flow channel for blocking or redirecting the flow. The integrated chip contained individually addressable screening-chamber devices for incubation and exposure of individual animals to biochemical compounds and highresolution time-lapse imaging of many animals without the need for anesthesia. In another study, fast time-lapse images of green fluorescent protein (GFP)-tagged moving vesicles were obtained in mechanoreceptor neurons thanks to immobilization of C. elegans using a microfluidic device with a deflecting membrane (see Figure 6C,D).111 Also, Q neuroblast divisions and mitochondrial transport could be observed. The Whitesides group proposed a “worm clamp” microfluidic system for arraying and immobilizing C. elegans.69 Their system comprised an array of worm clamps, which each consisted of a microfluidic channel that gradually tapered over 5 L


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Figure 7. (a,b) Photograph and schematic diagram of a worm imager. Worms in a 500 μm high microchamber are illuminated with an LED and cast a shadow onto a CMOS video camera chip attached at the bottom of the chamber. (c) Shadow image of worms in a rectangular microchamber. Reprinted with permission from ref 117. Copyright 2005 Elsevier BV.

Figure 8. Optofluidic microscope device for observation of C. elegans. (a) Schematic illustration of the upright operation mode of the device. The apertures (white circles) are defined on an Al-coated 2D CMOS image sensor and span across the whole microfluidic flow channel. (b) The actual device as compared to a U.S. quarter. (c) Images of wild-type C. elegans L1 larvae: (i) duplicate images acquired by two optofluidic microscope arrays for the same C. elegans; (ii) direct projection image on an unprocessed CMOS sensor (without apertures) with 9.9 μm pixel size; and (iii) conventional microscope image acquired with a ×20 objective. Reprinted with permission from ref 116. Copyright 2008 The National Academy of Sciences of the USA.

a worm could be loaded and inspected; fluorescence-based sorting was achieved at rates up to 2500 animals per hour. Using this software control interface, preconfigured image processing modules could be selected to help clarify and accentuate phenotypical characteristics. When a worm was in the field of view of the microscope, one of over 40 combinations of image processing options could be selected and subtle phenotypes emphasized. For markers that are out of focus, one option was, for example, to acquire a small z-stack of images at different focal planes. An integrated circuit (IC) video camera chip made using complementary metal-oxide-semiconductor (CMOS) technology has been used for imaging and investigating the behavior of C. elegans during spaceflight.117 CMOS refers to both a particular style of digital circuitry design and the processes used to implement that circuitry on ICs. As CMOS circuitry dissipates less power than other types of circuits, the vast

chip, its functioning may be called truly microfluidic. The purpose of this study was to characterize the transcriptional activity of gene promoters, in time and in space, which is seen as a critical step toward understanding complex biological systems. An in vivo spatiotemporal analysis for ∼900 predicted C. elegans promoters (∼5% of the predicted protein-coding genes), each driving the expression of GFP, was provided. Automated comparison and clustering of the obtained in vivo expression patterns showed that genes coexpressed in space and time tend to belong to common functional categories. By analyzing large numbers of animals of all sizes and ages at high throughput, a digitized overview of the promoter activity throughout postembryonic development was obtained. A computer-assisted methodology was developed to allow an expert to determine in real-time whether C. elegans animals that were screened on a microfluidic chip exhibited a behavior that was of interest for the specific study.113 In a matter of seconds, M


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Figure 9. Principle of droplets capturing in a trap array. The successive generated droplets are numbered sequentially. The two paths from junction I to II are named as Path 1 and Path 2, and the flow resistances along them are defined as R1 and R2, respectively. The process for trapping one droplet is described as follows: (a) R1 > R2, droplet 1 at junction I will flow into Path 2; (b) R2 > R1, droplet 2 at junction I will flow into Path 1 and be trapped; and (c) R1 > R2, droplet 3 at junction I will flow into Path 2 and enter the next trapping process. (d) Image of 24 arrayed droplets with encapsulated worms. The white arrows indicate the positions of the worms. (e) Representative images of mobility shapes of a single worm in response to the neurotoxin metabolite MPP+. The worms were incubated with 3 mM MPP+ for 70 min: (I) C-shape; (II) sine wave-shape; (III) omega-shape; (IV) tetanic. Reprinted with permission from ref 121. Copyright 2008 The Royal Society of Chemistry.

majority of modern IC manufacturing is based on CMOS processes. The camera system encompassed a polycarbonate microculture chamber with a volume of 4 μL that supported worm cultures in liquid media. Gas exchange was enabled through a gas-permeable membrane forming the top of the chamber. By sandwiching the microculture chamber by the CMOS video camera chip on one side and a light source on the other side, shadow images of the worms were acquired by the CMOS sensor (see Figure 7). This system abandoned the conventional microscope design, which is heavy, and requires expensive lenses and large space to magnify images. Image artifacts due to diffraction at the worm bodies could be reduced by decreasing illumination wavelength and object/camera distance. As an alternative to video acquisition, the filtered video output signal was used to determine worm activity, yielding a system that allowed image acquisition in combination with a low-bandwidth activity measurement. When placing a specimen directly on a CMOS image sensor, the resolution of the projection image is given by the sensor pixel size, and, because the typical pixel size of a commercial CMOS sensor is a few micrometers, it is difficult to conceive or develop a direct projection imaging strategy by which single-time-point images can be acquired at high resolution. A high-resolution (∼0.9 μm), lens-less, and fully on-chip microscope based on the optofluidic microscopy method has been used to visualize C. elegans larvae (see Figure 8).116 This system utilized microfluidic flow to deliver animals across arrays of micrometer-size apertures defined on a metal-coated CMOS sensor to generate direct projection images. However, the CMOS sensor grid was covered with a thin metal layer, followed by the etching of a small aperture at the center of each sensor pixel. By exploiting the time dimension during the image acquisition process of a transiting animal, it was possible to develop viable highresolution direct projection imaging strategies, in which

resolution and sensor pixel size were independent. Such highresolution on-chip microscopes are essential for improving efficiency of low-cost and portable bioanalytical applications. Another study reported the lens-free on-chip fluorescent imaging of transgenic C. elegans.115 The animals were excited using a prism interface placed on top of a microfluidic chip, and their emitted fluorescent signal was recorded with a chargecoupled device (CCD) placed underneath the microfluidic chip without the use of a lens. As the raw lens-free microfluidic images looked blurred, a compressive sampling algorithm was proposed to decode the raw images into higher resolution (∼10 μm) images. The same research group reported lens-free optical tomographic imaging of C. elegans118,119 in a volume as large as 15 mm3 on a chip, by exposing the animal to a partially coherent light source and by recording lens-free in-line holograms for different illumination angles. Multiple holograms obtained for each angle were then digitally processed using a pixel superresolution technique to create a single highresolution hologram of each angular projection; these were subsequently used to calculate tomograms of the animal. Applicability of droplet-based microfluidic devices for creating and screening an array of droplets containing C. elegans was also demonstrated. Droplet microfluidics gained a lot of attention due to its possibility to not only create several thousands of picoliter−nanoliter volume aqueous droplets in a continuous biocompatible oil phase, but also to manipulate them, for example by merging, splitting, and immobilization of droplets inside a microfluidic channel. Clausell-Tormos et al. demonstrated the encapsulation of C. elegans eggs, along with E. coli cells as animal feed, inside 660 nL aqueous plugs and thereafter tracked the larvae during their various phases of development.120 They noticed that 4 days-old adult worms gave birth to progeny, and droplets were seen accommodating about 20 worms at a time. While the droplet-based system is easy to N


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Figure 10. (a) Diagram of an “artificial soil” device. The broken line indicates the molded PDMS component. Within this region, the color black indicates spaces that are open to fluid flow. The worm is injected via one of four injection ports and crawls among microscopic posts in the central region, which measures 1 × 1 cm2. The device is fed by three fluid reservoirs (A−C). Gray lines indicate polyethylene tubing. (b) C. elegans crawling behavior in nine artificial soil devices. (A−I) Each device has a unique topography, defined by post diameter and the minimum gap between posts. Rows show devices with the same post diameter; columns show devices with the same gap. Post diameters and gaps are indicated in the margins. Reprinted with permission from ref 127. Copyright 2008 The American Physiological Society.

put in place and does not require complex microfluidic architecture, it lacks the flexibility to maintain the mother animal isolated from its own progeny. In other work, Shi et al. demonstrated a microfluidic device that not only generated droplets encapsulating animals but also arrayed them using immobilization traps (see Figure 9).121 The microfluidic device was comprised of a T-junction droplet generator combined with a long square-waveform serpentine microchannel. The main flow channel was short-circuited via an array of cavities, which trapped a droplet by virtue of the difference in fluidic resistances between the main flow path and the cavity-based short-circuited flow path. Around 60% of the trapped droplets contained a single worm, and the motility of worms inside the trapped droplets was in good comparison to those cultured in well plates. The Lu group207 developed a gel-based technique for reversible immobilization and time-lapse imaging, from the L1 stage to adulthood, of worms that were cultured inside a microfluidic chip. Their PDMS microfluidic device consisted of a bottom flow network comprising culture chambers, each having an inlet and outlet valve, for selectively retaining and culturing the animals inside the chip. The top flow network of the device comprised pneumatic circuitry for valve control and channels for flowing a preheated solution for precise control of the temperature of the culture media. They used a temperaturesensitive Pluronic F127 block copolymer in the culture media for selective immobilization of the worms at different time intervals. They showed that, when the temperature of the

culture media was increased by a few degrees (around room temperature), the latter turned into a gel state and, as the temperature dropped, it returned to the sol state. Their results showed that the Pluronic F127 gel-based immobilization and imaging did produce results comparable with conventional methods. The worms were unharmed by the gel-based immobilization (typically during 10 s) and small-range temperature oscillations of culture media. Also, the authors showed that their immobilization process did not pose any problem for imaging, even at subcellular resolution. Pressure pulse-induced hydrodynamic gating was proposed for the selective injection of GFP-transfected C. elegans eggs into a channel on a microfluidic chip.124 Egg-containing plugs of various volumes could be injected in the channel starting from a continuous sample stream. Another microfluidic approach has been proposed for temperature stimulation of C. elegans embryos.125 Microelectrodes were embedded into a microfluidic channel to generate either a temperature gradient through the culture chamber or a local heat spot under specific embryos that were trapped in the channel. First results of the temperature difference-induced synchronization of cell division in two-cell stage embryos were obtained. 2.4.2. Behaviorial Studies. The effect of two on-chip immobilization approaches on postimmobilization locomotion behavior of C. elegans was studied.126 The first approach utilized a deformable PDMS membrane to mechanically restrict the worm’s movement, while the second one created a CO2 microenvironment by passing pure CO2 through the membrane O


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control layer, which diffused through the PDMS in the flow layer that contained the animals. Both methods were appropriate for the short-term (minutes) worm immobilization. The CO2 method offered the additional advantages of longterm immobilization (1−2 h) and resulted in worms with higher locomotion speed when placed on food-free agar plates after immobilization. Microfluidic devices in PDMS containing arrays of microposts were proposed for studying C. elegans’s locomotion behavior (see Figure 10): agarose-free, micrometerscale chambers and channels were made that allowed the animals to crawl, as they would on agarose.127 One micropost device was aimed to mimic a moist soil matrix (the worm’s natural environment) and facilitated rapid delivery of fluidborne stimuli. A second type of device consisted of sinusoidal channels (each of a fixed amplitude and wavelength) in PDMS, which were used to regulate or impose the waveform and trajectory to the crawling worms. It was shown that wild-type C. elegans were able to pass through several different channels, suggesting that the mechanisms for generating and propagating undulations were largely independent of the channel amplitude and wavelength. The worms, however, faced difficulty in passing through sinusoidal channels whose amplitude and wavelength were larger (2 times or more) than the dimensions of their natural undulatory movement. Another study was based on a locomotion experiment in two modulated sinusoidal channels,128 one with gradually increasing amplitude and the other with gradually decreasing amplitude. It was observed that worms moved vigorously through channel sections whose amplitude and wavelength matched that of the worm’s natural undulations. Beyond this range, the worms had problems in moving forward and eventually stopped. Related to this, an interesting study reported the locomotive behavior of C. elegans swimming in a fluid with particles of various sizes and found that the nematode swims a greater distance per undulation than it does in a fluid without particles.129 This result was unexpected due to the generally low performance of a body moving in a high drag medium. A model was proposed, in which the saturated granular system was approximated as a porous medium where only the hydrodynamic forces on the body were considered. Another group presented molded micropost microfluidic structures fabricated from agar.130 In such chips, worms were shown to combine the fast gait of swimming with the more efficient movements of crawling. When the wavelength of the worms matched the periodicity of the post array, the microstructure directed the swimming and increased the speed of C. elegans 10-fold. Mutants defective in mechanosensation (mec-4, mec-10) or mutants with abnormal waveforms (unc-29) did not perform this enhanced locomotion and moved much slower in the microstructure than wild-type worms. Microfluidic micropost “arenas”, consisting of fluidfilled channels between cylindrical microposts, were proposed for studying normal C. elegans crawling behaviors and for delivery of well-controlled pulses, stripes, and linear spatial gradients of chemical stimuli.131 These spatiotemporal stimulus patterns were quantified and modeled, and automated behavioral classification software was used to analyze wildtype and mutant worms in various olfactory environments. Different stimulus configurations preferentially revealed turning dynamics in a biased random walk, directed orientation into an odor stripe and speed regulation by odor. Both expected and unexpected responses in wild-type worms and sensory mutants were identified by quantifying dozens of behavioral parameters.

Sleep- and wake-like behavior during the development of C. elegans larvae, quantified by the rate of pharyngeal pumping, was studied in agarose hydrogel microcompartments.122 Arrays of these microcompartments were cast on PDMS molds; the 10 μm high compartments were, after demolding, filled with an E. coli bacterial suspension and C. elegans eggs, and thereafter sealed with glass coverslips. Individual animal development was studied using differential interference contrast and fluorescence microscopy. Besides a behavioral study, also the nervous system rewiring was imaged in individual young larvae of transgenic C. elegans that expressed the synaptobrevin GFP fusion reporter SNB::GFP in the six DD neurons to visualize synapses. An integrated device was proposed for sorting C. elegans and for measuring forces during locomotion. To measure the force developed by a worm, the latter was put inside a closed microchannel with parallel arrays of elastic PDMS pillars.141,142 When moving in a sinusoidal manner, the worm bended the pillars, whose deflection was sensed by a camera and converted to force knowing the pillar’s elastic spring constant. Pillar diameters were between 40 and 60 μm, and their maximum height was 100 μm. Maximum developed forces by the worms were in the 30−35 μN range. Another approach involved the use of an integrated fiber-optic microfluidic device capable of measuring the muscular force of worms normal to their translational movement direction.140 A thinned single mode fiber cantilever was placed adjacent to a sine wave-shaped channel with multiple open throughs. A physical contact between a moving worm and the cantilever bent the latter, and the deflection (and associated force) was quantified by measuring the decreased light coupling in a static receiving multimode fiber. In this way, O. dentatum L3 larvae were found to generate normal forces in the 10 μN range. Also, the device was used to measure force responses of levamisole-sensitive and -resistant O. dentatum isolates in response to different doses of the anthelmintic drug levamisole. Rezai et al.135 showed that electric fields in the range of 1−12 V cm−1 could be used as a stimulus to control movement of worms in a microfluidic environment. This response, coined electrotaxis, was directional and was mediated by neuronal activity that varied with the age and size of animals. Although the speed of swimming was unaffected by changes in the electric field strength and direction, each worm developmental stage responded to a specific range of electric field with a specific speed. Synchronized animals of different stages (from L1 to young adult) were introduced into the microchannel. L1and L2-stage animals displayed no obvious response to the applied electric field, because they continued to swim randomly regardless of the direction and presence of the field. At later stages (L3 onward), animals responded robustly to the electric field within a certain range that was different for each stage and exhibited directed movement toward the negative pole. The swimming pattern was typical of unexposed animals in a liquid environment, except that the response was directional, suggesting that the electric field did not distort body bends, but rather induced the swimming behavior. The finding that only older worms responded to the electric field suggested that this behavior is developmentally regulated and is likely to be mediated by certain differentiated cell types that may be absent (or immature) at earlier L1 and L2 stages. It should be noted that this type of result is enabled by the microfluidic approach and only achievable when well-defined channel geometries can be defined, in which electric fields can be applied uniformly and in a very controlled way. Also, it was checked that the exposure P


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to the applied electric fields had no discernible effect on the ability of animals to survive and reproduce. When instead of a static field, ac electrical fields in the 1 Hz to 3 kHz range were applied, worms could be momentarily immobilized in the channel (see Figure 11).134 Also, a unidirectional electrotactic-

device, worms from a mixed culture were sorted in a semicontinuous flow manner resulting in synchronized populations of animals. Higher electric fields (25−30 V/cm) in the trap region inhibited most young adult worms, to a lesser extent L4 stage worms, and had virtually no effect on the L3 stage animals. The sorter operated with a selectivity of ∼90% for worms of different ages in a single sample pass through the device. The method was also used to sort electrotaxis-defective worms (neuronal or muscular mutants) from healthy animals. Another passive microfluidic strategy to synchronize worm populations based on their age and size took advantage of adult and juvenile phenotypic differences and the associated animal behavior in different microfluidic architectures.148 In a so-called “smart” maze, adults moved in larger main channels to a desired output port of the maze, while smaller larvae were deviated into smaller interconnecting channels and left the maze at another output port. While electrotaxis is mediated by the worm’s sensory nervous system and it affects only relatively mature worms, the influence of larger electric fields (up to 740 V cm−1) and relatively high frequency (up to MHz) on worm behavior was also investigated.133 In contrast to electrotaxis, under these conditions, the worms were trapped at the location of the maximum electric field intensity and were insensitive to the field’s polarity. The worms were polarized by the electric field and subjected to a net dielectrophoretic (DEP) force when the field was nonuniform in space, like a passive dielectric object. The magnitude of this force could be tuned by adjusting the electric potentials, and worms could be trapped and released on demand. A range of electric field intensities and frequencies were identified for which worms were trapped without apparent adverse effect on their viability. When the frequency was very low (24 h) sustenance of the former’s metabolic functions and protein expression levels,184 when compared to conventional culture methods (where sustenance is only a few hours). Thanks to the continuous flow of perfusate, not only the culture media was replenished, but also the metabolic waste produced by the tissue slice was removed. Even after 24 h of continuous perifusion culture, the tissue slices exposed to the perfusate flow from top and bottom showed a slightly higher level of protein marker expression, when compared to slices exposed to the same flow from only a single side. Alternatively, the same group also reported a stainless steel microneedle array-based device for intratissue perfusion of thick rat liver slices (∼300 μm to 1 mm) (see Figure 16e).180 The microneedle array poking inside the tissue slice was able to interface with the angio-architecture of the latter and allowed the perfusion of medium and reagents to the interior of the tissue slice, thus overcoming the mass transport limitations associated with thick liver slices and noninvasive static culture methods. The tissue slice could be kept viable and functional for up to 3 days. While the perfusion device of Yu’s group was realized using conventional machining techniques, the microneedle array was a microfluidic element providing the flow patterns at the interior of the tissue slice. The Haswell group developed a microfluidic device in glass, for investigating rat liver tissues under continuous perifusion (see Figure 16f).181 The device consisted of two glass layers, which were irreversibly bonded together. The top glass layer (3 mm thick) of the device had a cylindrical chamber (3 mm diameter) for accommodating the tissue, while the bottom layer contained a microfluidic channel network. To enable the sealing of the tissue chamber, the device was accommodated with an internally threaded microport fitting, which could be closed using a threaded adapter. The viability of sectioned liver slices (∼1 mm3) was investigated by monitoring the albumin and urea levels produced by the liver tissue within the microfluidic device. These studies showed that the sectioned liver slices were viable and functional for at least 70 h. They also demonstrated the on-chip enzymatic disaggregation, from the tissue slices, of primary cells of which ∼80% were viable. Also, the viability of the cells within the tissue slice, when exposed to a time-dependent cell lysis buffer, was investigated. Cell viability diminished with repeated exposure to the lysis buffer, due to the reduction in the tissue mass during the removal of peripheral cell layers. An inconvenience of this device is that only one side of the tissue could be exposed to the perfusate, thus diminishing the exposure of the tissue slice. The same type of device was used for studying the viability and functionality of explant liver tissues over 4 days in the present of varying concentrations of ethanol.187 Concentrations of ethanol as low as 20 mM produced already a decrease of metabolism and mitochondrial activity, as well as an increase in lactate dehydrogenase (LDH) release, a sign of cell death. At the same time, a decrease in albumin and urea synthesis was observed. This type of device was also used for culture of human colorectal tissue biopsies, both normal and neoplastic, for periods in excess of 3 days.182 The response of these biopsies to hypoxic conditions (obtained by replacing O2 in the perfusing

controlled and simultaneous release and suction of the medium using, for example, syringe pumps. The authors used a MFP having 40 × 70 μm2 inlet and outlet apertures (spaced 55 μm apart from each other) and an injection and suction flow rate of 5 and 25 nL s−1, respectively. Their experimental setup was comprised of two parts: (i) an open perfusion chamber for studying organotypic brain slices, which could be perifused with culture media continuously for several hours while at the same time providing the flexibility/possibility to perform highresolution imaging using an inverted microscope, and (ii) the MFP that had direct access to the surface of the tissue slice from the top for localized “microperfusion” of the area under investigation, which can be a surface comprising only a few cells, when a distance of 20 μm between the MFP tip and the tissue slice was maintained (see Figure 16d).175 It was possible to simultaneously visualize the tissue slice using confocal microscopy and microperfuse it via the MFP with fluorescent dextran. Confocal microscopy results showed that the dextran was able to diffuse 32 μm inside a 72 μm thick tissue slice after ∼12 min of microperfusion (see Figure 17i). These results clearly confirmed that the MFP can be a useful tool for highly localized perifusion. Additionally, as the tissue slice can be translated underneath the MFP, this provides an extra degree of freedom for microperfusion-based probing of highly specific locations across the entire tissue area. The idea of local perfusion was also used in a microfluidic chamber design that incorporated fluid ports with active suction to achieve localized chemical stimulation of brain slices.177,178 A two-level softlithography process was used to fabricate fluid ports with integrated injection and suction holes that were connected to underlying microchannels. This planar microfluidic platform was placed underneath a brain slice and facilitated optical inspection of the brain slice, while at the same time permitting spectroscopic recording and electrode placement from above the slice, avoiding modification of most commonly used slice physiology setups. The local perfusion capability of the device was illustrated by providing locally a cortical spreading depression, which is a self-propagating wave of cellular depolarization. It was chemically induced by locally injecting a 1 M solution of KCl through a fluidic port. The elevated extracellular [K+] triggered a concentric depolarization of neurons and glial cells originating from that locus. The spreading depolarization causes cells to swell, resulting in observable changes in their optical properties. 3D perfusion microneedles in SU-8 photoresist were proposed to provide convective mass transport to the interior of 0.4 mm thick rat brain tissue slices.179 Such intruding fluidic channels are beneficial for brain slice culturing, because there naturally is limited flow in the slice and therefore nutrients applied exogenously may not reach the interior cells in adequate quantities. Microneedle arrays offer the ability to replace this functionality; furthermore, they were designed to have a sharp tapered shape to minimize the rupture of the tissue upon insertion. The device was tested for its ability to improve viability in slices of harvested brain tissue. Improved viability was visible in the short term, as probed via live-dead cells discriminating fluorescent staining and confocal microscopy techniques. Perfusion systems for studying ex vivo liver tissue slices have also been demonstrated. The liver is a vital organ with a wide range of functions, including detoxification and metabolism control. Experimental microfluidics-based in vitro systems to study liver metabolism and/or toxicity have been discussed in a Z


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analysis system are its speed and the elimination of the necessity to store samples, enabling the analysis of unstable metabolites. The quantification of such unstable compounds in conventional well-plate incubations may not be possible, as detectable concentrations may not be achieved due to the relatively large incubation volume and decomposition during the incubation. In addition, tissue physiology and stability of metabolic enzymes can be monitored directly over time in such integrated analysis system. Cheah et al.189 showed the applicability of a microfluidic device for perifusing heart tissues. Their device was realized by bonding a replicated PDMS layer, comprising a microfluidic channel with a central chamber, with a Petri dish surface for accommodating a rat/human heart tissue slice (Figure 16h). Two sets of wire electrodes were attached to the device, one inserted inside the tissue chamber for electrically stimulating the heart tissue slices and the other placed close to the channel outlet for in situ electrochemical monitoring, using cyclic voltammetry, of the reactive oxygen species (ROS) released from the tissue slice. As the ROS level correlates directly to the proportion of cell damage, continuous assessment of the former can act as a vital information source regarding the tissue viability and health. Using an optimal perfusate medium (2.5 mM CaCl2) and electrical stimulation parameters (3−4 V cm−1, 1.5 Hz), the authors of this study were able to maintain heart tissue slices viable with regular contractions for up to ∼5 and 3.5 h for rat right ventricular and human right atrial tissue, respectively. Damage to the cells was induced by subjecting the tissue slice to a flow of perfusate medium having 2% v/v of Triton X100 or saturated with 95% N2−5% CO2. The study showed that the total ROS levels measured either prior to or after inducing damage were in good agreement with the results of conventional off-chip biochemical assays, which measure the release of LDH and H2O2 after cell damage. The same type of device was also used to evaluate heart tissue viability under redox-magnetohydrodynamic-induced flow conditions.190 This principle uses the ionic current resulting from oxidation and reduction processes produced at electrodes in the presence of a magnetic field for fine-tuning of liquid flow in proximity of the tissue. Figure 16i shows the system used by Berdichevsky et al.167,168 to investigate the communication between different brain regions, and between local circuits in the same brain region. They designed an in vitro platform that captured some of the complexity of mammalian brain pathways but permitted easy experimental manipulation of their constituent parts. Organotypic cultures of brain slices were carried out on multielectrode array surfaces or in interconnected compartments, and it was shown that cocultures from cortex and hippocampus formed functional connections by extending axons through microchannels situated between the two culture chambers. Synchronization of neural activity in cocultures was reported, and selective pharmacological manipulation of activity in the constituent slices was demonstrated. In other work, liver and kidney explants harvested from chicken embryos were cultivated inside PDMS microchannels of different shapes with the aim of studying the migration of liver and kidney cells originating from the explants in the microchannels, and this with and without coating the microchannel by fibronectin.191,192 Migration velocities were typically 100−700 μm/ day, higher values corresponding to results obtained in the fibronectin-coated microchannels. In an embryonic development study, a microfluidic chip was used for providing localized

medium by N2) was assayed by monitoring the release of vascular endothelial growth factor into the media, which was measured off-chip. The system was also used for the maintenance and interrogation of head and neck squamous cell carcinoma (HNNSCC) tumor biopsies.183 Primary HNSCC or metastatic lymph samples were treated with 5fluorouracil and cisplatin to investigate viability and apoptopic biomarker release. Thereby, the device acted as a preclinical model for studying tumor treatment regimes. A group at the University of Groningen developed a microfluidic chip for the perifusion of rat liver slices for metabolism and toxicology studies.64 The device was realized by reversibly assembling two stacks of PDMS layers, with each stack comprising five bonded layers of varying thicknesses. The device also comprised two microporous polycarbonate membranes, of which one was attached to the top stack and the other to the bottom stack (see Figure 16g). Both of the polycarbonate membranes thus acted as the top and bottom lids for the tissue chamber (25 μL volume), while at the same time allowing optimal flow around the tissue slice. To monitor the viability, the leakage of the enzyme LDH from liver tissue slices was measured, confirming that the tissue slices were viable for at least 24 h. Protocols to prepare and incubate rat and human liver and intestinal slices have been described in much detail.226 When such prepared liver slice was embedded in a hydrogel inside the microfluidic chip, the viability of the tissue slice could be extended from 24 to 72 h.186 The same group demonstrated in addition the applicability of their device for on-chip perifusion of intestinal tissue slices.185 Their results showed that the intestinal tissues were viable and functional for up to 3 h. However, slight morphological changes of the intestinal villi in some slices were observed. They also adapted their device for sequential perifusion of two tissue slices, either from the same organ or from two different organs, intestine and liver. The outlet of the first device containing a tissue slice was connected to the inlet of the second having another tissue slice (either from the same organ or from a different organ) via an interconnection tube. Both the liver and the intestinal slices remained functional and viable in such a sequential continuousflow device. Metabolism studies were done using three different model substrates and showed that the metabolic rate/activity of a single liver or intestinal slice was well comparable to the activity of sequentially perifused slices. When the intestinal slice was exposed to bile acid, it induced the expression of the fibroblast growth factor 15, which in turn down-regulated the synthesis of bile acid by the liver tissue slice via suppressing the expression of the enzyme cytochrome P4507A1 by the liver tissue slice. A further study focused on the use of an injection loop, in which the medium containing the metabolites produced by a liver tissue slice was accumulated.188 Once filled, the content of this injection loop was automatically injected onto a high-performance liquid chromatography system (HPLC) with ultraviolet-based detection for analysis. As in previous work, the analysis of metabolites was tested by using as a substrate, 7-hydroxycoumarin (7-HC). Rapid switching between substrate and solvent control was possible, and a direct metabolic response of the liver slice to perifusion with the substrate was detected. Very stable phase II metabolism over a period of 24 h was observed. The inhibitory effect of phloxine B on the formation of 7-hydroxycoumarin glucuronide (phase II product of 7-HC) was also investigated. The results showed a concentration-dependent inhibition of 7HC glucuronide formation. Advantages of this integrated AA


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spatiotemporal gradients of the steroid hormone dexamethasone to animal cap tissues isolated from Xenopus laevis (clawed frog) embryos.65 The tissues of area ∼1 mm2 were placed at the bottom of a microchannel and exposed to well-controlled laminar flows of hormone solution. Animal cap cells are cells situated around the pigmented pole of a blastula or very early gastrula-stage embryo. Animal caps are composed of pluripotent cells that can be induced to form endodermal, mesodermal, or ectodermal cell types, and can therefore serve as a useful substrate to assess the activity of various inducing factors. Patterns of a fluorescent hormone reporter translocation into the cell nuclei in response to continuous or periodic hormone stimulation were analyzed.

were noticed. The obtained results were in good concordance with the clinical results obtained from whole tissue section analysis. In follow-up work,194,228 the microfluidic channel structure was extended to allow for simultaneous immunohistochemical staining of 10 different biomarkers on a single tissue sample. Another type of PDMS-based microfluidic device having a serpentine channel design, covering ∼40% of the tissue slice’s surface area, was used in combination with lanthanide-based immunocomplexes. The device allowed the multiplexed timeresolved luminescent detection of biomarkers (ER, PR, Her2/ neu) on breast cancer tissues, revealing an enhanced sensitivity compared to classical organic dyes.66,196 Figure 17iii characterizes the on-chip immunohistochemical detection of both Her2/ neu and ER. The tests proved to be economical (use of reagent volumes in the μL range) and had high-throughput potential (short assay times of only 20 min versus a minimum of 2 h needed for the classical protocol). Moreover, the results were in complete agreement with those obtained in the clinical laboratory using classical immunohistochemistry settings. In an improved integrated microfluidic design, assay times could be further reduced to 4 min, and analysis was achieved over complete area of the tissue slice, which was as large 256 mm2.195 The high sensitivity partly originated from the timeresolved luminescence capability of the lanthanide probes, which eliminated a large fraction of the background autofluorescence. A group at IBM Research used a MFP for local staining sections of breast cancer tissue slices.176 Nanoliters of antibody solutions was confined over micrometer-sized areas of tissue sections using a MFP for their incubation with primary antibodies. Other parts of the immunohistochemical protocol (dewaxing and rehydratation of the tissue, application of secondary antibodies, and postprocessing) were done in the conventional way. This approach preserved tissue samples and reagents, alleviated antibody cross-reactivity issues, and allows in principle a wide range of staining conditions to be applied on a single tissue section. Microfluidic chips in PDMS were also used for performing in situ hybridizations on formalin-fixed paraffin-embedded sections of a mouse brain.197 Expression signatures of microRNA have been correlated with disease progress in cancers, and this type of assay is therefore complementary to an immunohistochemical diagnostic assay. The 4 μm thick sections were dewaxed, rehydrated, fixed, acetylated, demasked, and blocked for endogenous peroxidase activity, after which the sections were placed in a microfluidic chamber, where an in situ microfluidic hybridization assay was demonstrated using direct fluorescent detection of hybridization to 18S rRNA. Use of flow-based incubations enabled a faster microRNA in situ hybridization assay than the conventional technique (3 h instead of 5 h 40 min). Also, sequential hybridization and detection of two microRNA’s present at the same location on the tissue section was demonstrated.

4.2. Immunohistochemical Analysis of Biopsied Tissue Slices

Immunohistochemistry is widely used for studying tissue morphology and assessing therapeutic biomarkers related to various malignancies of surgical pathology.227 Conventional immunohistochemistry is a macroscale operation, in which immunoreaction times in the range of 30 min to hours are required for achieving uniform exposure of surface antigens on the tissue slice to bioreagents and antibodies to ensure the reproducibility of the immunoassay outcome. This originates from long diffusion times, lack in precision of controlling and dosing of reagents, as well as limited fluidic exchange rates. In addition, long assay and antibody exposure times may result in significant adsorption and nonspecific binding of the antibodies, so that the resultant immunohistochemical signal is not a linear function of the target biomarker concentration on the tissue. A few microfluidic devices have been used for immunohistochemical analysis of biopsy samples, where they contributed to a reduction of labor, tissue consumption, and analysis time, while achieving a high detection sensitivity and precise diagnosis. Research groups have used PDMS-based microfluidic devices for multiplexed immunohistochemical detection of different breast cancer biomarkers expressed on clinical breast tissue samples. The microfluidic device realized by Kim et al.193 had four parallel reaction channels (800 μm in width and 5 mm in length), with each reaction channel dedicated for detecting one specific biomarker of interest, estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and ki-67. Figure 17ii shows the configuration of their microfluidics-based multiplexed immunohistochemistry platform. A PDMS block containing four microfluidic trenches was reversibly assembled, like a “microfluidic sticker”, against a tissue-bearing glass slide, in such a way that the tissue area with highest density of cancer cells aligned directly beneath the reaction channel network. To facilitate this alignment, the tissue samples were prestained with hematoxylin and eosin, and, after assembly, a weight was placed on top of the PDMS block to ensure leak-proof sealing between the PDMS channel network and the tissue slice. One side of the reaction channels was appropriately connected to different reagent (buffer, antibodies, etc.) reservoirs via individual gating valves, whereas the other side was connected to a common outlet. For performing the immunohistochemical staining, different reagents were sequentially sucked inside the reaction channels via the common outlet port under appropriate actuation of valves. As compared to conventional methods, for optimized on-chip assay parameters (flow rate 100 μL/h, incubation period of 10 min), a 6× decrease in total assay time (90 min) and a 200-fold decrease in antibody consumption

5. CONCLUSIONS AND OUTLOOK 5.1. General

This Review shows that microfluidics is at the basis of the development of extremely promising tools for investigation of whole living multicellular organisms, organs, and tissues. An obvious upper size for the biological specimen/species that can be studied is posed by the millimeter range, as dictated by the AB


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dimension of a typical microfluidic chip. However, thin and large tissue slices, up to a square centimeter in size, were studied, because these are naturally compatible with planar microfluidic technology. A recurrent theme in research was the use of multicellular organisms and organs as “integrated biosensors” to investigate the effects of drugs and other chemical substances. The easy integration of control and detection modules with microfluidic technology promoted the latter for automated applications. Future high-throughput microfluidic screening platforms with high-resolution imaging and manipulation capabilities should permit large-scale in vivo studies of many biological processes. The latter can include organ development, neurological studies, stem cell proliferation, cardiovascular, immune, endocrine and nervous system functions, pathogenesis, and cancer detection and prognosis.

relevance to pharmaceutical or genetic developments related to the treatment of human diseases and injuries. As demonstrated in this Review, microfluidics has shown already a strong impact on the study of biological processes in invertebrates, enabled by clever microsystem designs and the availability of proper microfabrication technologies. PDMS has been the dominant material in these studies, as PDMS parts and membranes can be relatively easy replicated and assembled into 3D microstructures, resulting in transparent microfluidic circuits with valves and control channels that can be operated using automated protocols. The question is now if these microfluidic circuits will evolve into a standardized ubiquitous commercial product, similar like the microwell plate is today? Experience learns that such microfluidic solutions require microfabrication technologies that are not immediately part of the background of a traditional biologist. Most of the reported breakthroughs were therefore only possible thanks to the collective effort of multidisciplinary research teams. Indeed, at present, no standardized and robust “plug-and-play”-type industrial platform for invertebrate studies exists, which would permit biologists to profit in a straightforward way from microfluidics. Moreover, the widely practiced PDMS technology has an “artisanal” character and is less suited for mass production; especially fabricating the more complex multiple fluidic layer devices requires specialized skills. So for industrialization to occur, considerable further development in engineering of new materials will be required, fluidic connectivity issues need to be resolved, and improving automated animal handling and image acquisition techniques established. Therefore, meanwhile, strong multidisciplinary research teams will continue to be further responsible for advancing this exciting research area of integrated microfluidic systems for multicellular organism studies.

5.2. Multicellular Organisms

Small vertebrate and invertebrate animal models are intensively studied by biologists. This holds in particular for C. elegans, due to its small size, well-mapped neuronal system, good transparency, and a relatively short lifecycle, which makes it an attractive animal for behavioral, toxicological, and genetic screening. Traditionally, microwell plates are used to study populations of animals, and automated positioning of a microscope objective followed by wide-view optical and fluorescent imaging of the content of a whole well is state-ofthe-art. A very attractive advantage of microfluidic methods is that they allow the environment of an organism to be precisely spatiotemporally controlled. Using microfluidic flow streams of different temperature, thermal gradients can be engineered onchip, with direct implication on organism development. The ability to manipulate single animals in microfluidic chips allows novel ways of phenotyping, like revealing physiological responses of individual neurons through high-throughput functional imaging. A future multiple-well-type microfluidic platform could play a key role in tracking behavioral and physiological phenotypes in individual worms on a long time scale, which is of high relevance for development, learning and memory, and aging studies. In addition, such platform would allow the study of variations in a population of genetically identical worms and worm-based genetic screening assays. When liquid chambers are combined with electrodes to deliver electrical stimuli, electrotaxis of C. elegans can be used in movement-based behavioral microfluidic screening assays for drugs/chemicals. Moreover, electric field-based changes in locomotive behavior within a microfluidic channel may facilitate future high-throughput screening of drugs that influence movement, thereby identifying potential candidates to treat human muscular dystrophy disorders. Droplet-based microfluidics can be exploited for confining and transporting vertebrate eggs, embryos, and invertebrate animals inside microfluid segments without significant inhibition to their development, suggesting the potential for using droplet-based devices as an alternative to microwell plates. Finally, laser microsurgery has been used for the sectioning of neurons within a microfluidic environment for subsequent in vivo screening of chemicals affecting neuronal regeneration. While many chemicals have been found to modulate neurite growth in vitro, in vivo validation of these effects and identification of their mechanisms of action represent an important step forward. Experiments on higher organisms will show how much these mechanisms studied in invertebrates may be of

5.3. Organs

Blood vessels and blood vessel generation have been studied under very controlled experimental conditions on microfluidic chips. A local and spatiotemporal stimulation could be established, opening new directions in in vivo myography methods. The ability to keep microarteries in culture also opens perspective for the transfer of gene transfection procedures onto a microfluidic platform, which can be at the basis of the study of complex multistep morphogenetic processes. The technology has potential to be combined with human stem cells and offers new possibilities for studying basic mechanisms of human embryonic development. In plant research, investigation of the survival and culture of live roots in a microfluidic device revealed the advantages of laminar flow patterns for local chemical stimulation and environmental control. In this field, a microfluidic chip has the potential to be used more generally for any chemical component of interest such as nitrogen, phosphate, salts, and hormones influencing plant development. Moreover, microfluidic devices can enable control of microbial communities, an approach that may further elucidate the chemistry and biology of plant root−microbe interactions. 5.4. Tissues

Microfluidic chips have permitted the controlled exposure of living intestinal, liver, heart, and brain tissue slices to biological or chemical solutions. Also, tissue devices were used for metabolism studies of intestinal slices and liver slices in a multiorgan/microchamber incubation setup, enabling the in vitro monitoring of interorgan interactions. Moreover, the microfluidic approach has potential for on-chip analysis of AC


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AUTHOR INFORMATION

chemically unstable, reactive metabolites. Chip-based studies thereby will open new lines of research in toxicology, gene regulation, and drug−drug interactions and also will facilitate future investigations of normal and pathological organ functions ex vivo, while mimicking the in vivo environment. For localized microperfusion of cells within a tissue slice, a microfluidic probe can be used under concurrent microscopic imaging. The method has the potential to deliver multiple chemicals for promoting the local regeneration of cells, or for delivery of viruses for localized transfection. Such device could be used in the future to explore neuron interactions, while offering long-term, steady-state perifusion of drugs, like in the study of synapse remodelling within organotypic slices following injury. While study of ex vivo tissue slices may be close to true in vivo experimentation and will be essential for understanding basic biological processes and their sensitivity to drugs or environmental factors, it is a low-throughput technique, as the quantity of tissues is limited by the number of donor animals and the duration of the explantation protocols. Also, ex vivo tissues may lack robustness, and the survival rate of an explanted slice in an in vitro environment can be of the order of hours only. Therefore, a more likely option for microfluidic high-throughput drug and environmental factors screening applications may be offered by cell (co)culture systems.27,229 Although in vitro cell models are simpler than the biological reality and can lack resemblance with an in vivo organ or tissue, such cultures can be more easily parallelized and cells of controlled origin can be used. What is here offered as a plus by microfluidic technology, beyond the classical culture approach, is the possibility of control of the microenvironmental parameters of such in vitro cultures. Microenvironment engineering could therefore be a tool for generation of simple, robust, and biologically more realistic culture conditions, while avoiding much of the problems associated with the use of a biologically more complex system.29 Another interesting option for high-throughput research is the use of microscale tissues, consisting of no more than a few thousand of cells, for example, ensembles of individual islets isolated from pancreatic tissue.230,231 Such samples are close to having the functions and complexity of a true biological tissue or organ, but are better associable with a high-throughput-like microfluidic approach than the original complete tissue or organ. Microfluidic systems have also been used for analysis of fixed human clinical tissue slice samples and histopathological cancer diagnosis. Several breast cancer biomarkers could be simultaneously investigated on a single tumor section, giving clear advantages over standard immunohistochemical methods. A very important feature of the microfluidic chip-based approach for diagnosis is the reduced reagent volumes and the possibility of rapidly exposing the tissue slice to reagents and antibodies, benefiting from the short diffusion times that are obtainable in a thin microfluidic cell. A proper microfluidic design should also avoid underexposure of certain parts of the tissue located in the microfluidic chamber. A significantly shortened incubation time also limits in time the nonspecific adsorption of molecules during incubation of the tissue slice with the various reagents. Moreover, a rapid detection opens perspectives for rapid insurgery room detection of cancers, decreasing the number of surgical interventions, risks, costs, and anxiety for the patients, as well as saving a significant amount of health resources.

Corresponding Author

*Tel.: (+41)216936734. Fax: (+41)216935950. E-mail: martin. gijs@epfl.ch. Present Address †

EPFL Middle East, Al Hamra − Jazeera, AE-Ras Al Khaimah.

Notes

The authors declare no competing financial interest. Biographies

Venkataragavalu (Venkat) Sivagnanam obtained the B.Eng. degree in mechatronics from Thiagarajar College of Engineering, Madurai Kamaraj University, India, in 2002 and the M.Sc. degree in microelectronics and microsystems, from Hamburg University of Technology (TUHH), Germany, in 2005. From August 2005 to March 2010, he pursued his doctoral research, guided by Prof. Martin Gijs, at the Institute of Microengineering, Ecole Polytechnique Fédérale de Lausanne (EPFL), Switzerland. He has over six years of experience in clean-room-based microfabrication. His doctoral research experiences are in the domains of self-assembly-driven micropatterning of functional micro- and nanoparticles (“beads”), labon-a-chip bioassays, and tissue-based cancer diagnostics using microfluidics. Soon after his Ph.D. graduation, he joined the bacteriology research group of Prof. John McKinney, at the Global Health Institute of EPFL. There he worked for a year as microfluidics research engineer and was involved in developing novel microfluidic tools for single-cell-level microbiology studies, real-time and long-term imaging of mycobacterium (TB) infection, as well as high-throughput drug screening against microbes. He has published over 10 research papers in peer-reviewed journals. In 2010, he was awarded with Dr. N. Chorafas foundation’s research prize in appreciation of his multidisciplinary doctoral research. In July 2011, he joined the Dean’s Office of EPFL Middle East, an international branch of EPFL based in the UAE. Presently, he is a Senior Program Manager in charge of developing and managing research collaborations between EPFL Middle East and its regional industry partners. AD


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Martin A. M. Gijs received his degree in physics in 1981 from the Katholieke Universiteit Leuven, Belgium, and his Ph.D. degree in physics at the same university in 1986. He joined the Philips Research Laboratories in Eindhoven, The Netherlands, in 1987. Subsequently, he has worked there on micro- and nanofabrication processes of high critical temperature superconducting Josephson and tunnel junctions, the microfabrication of microstructures in magnetic multilayers showing the giant magnetoresistance effect, the design and realization of miniaturised motors for hard disk applications, and the design and realization of planar transformers for miniaturized power applications. He joined the Ecole Polytechnique Fédérale de Lausanne (EPFL) in 1997. He presently is a professor in the Institute of Microengineering, where he is responsible for the Microsystems Technology Group. His main interests are in developing technologies for novel magnetic devices, new microfabrication technologies for microsystems fabrication in general, and the development and use of microfluidics for biomedical applications in particular. He is on the editorial board of Microfluidics and Nanofluidics and the Journal of Micromechanics and Microengineering. He has published over 200 papers in peer-reviewed journals and holds over 20 patents.

ACKNOWLEDGMENTS We gratefully acknowledge Hans-Anton Lehr, Jean-Claude Bünzli, Ata Tuna Ciftlik, Bo Song, and Caroline Vandevyver for helpful discussions and collaboration. This work is part of the research programs supported by the Swiss National Science Foundation, and in particular the SystemsX.ch project: “Timeresolved luminescence of cells and tissue in a Lab-on-a-Chip using lanthanide-doped nanoparticle labels for breast cancer detection”. REFERENCES (1) Dittrich, P. S.; Tachikawa, K.; Manz, A. Anal. Chem. 2006, 78, 3887. (2) Reyes, D. R.; Iossifidis, D.; Auroux, P. A.; Manz, A. Anal. Chem. 2002, 74, 2623. (3) Vilkner, T.; Janasek, D.; Manz, A. Anal. Chem. 2004, 76, 3373. (4) El-Ali, J.; Sorger, P. K.; Jensen, K. F. Nature 2006, 442, 403. (5) Andersson, H.; van den Berg, A. Sens. Actuators, B 2003, 92, 315. (6) Di Carlo, D.; Lee, L. P. Anal. Chem. 2006, 78, 7918. (7) Le Gac, S.; van den Berg, A. Trends Biotechnol. 2010, 28, 55. (8) Andersson-Svahn, H.; van den Berg, A. Lab Chip 2007, 7, 544. (9) Kim, S.; Kim, H. J.; Jeon, N. L. Integr. Biol 2010, 2, 584. (10) van der Meer, A. D.; Poot, A. A.; Duits, M. H. G.; Feijen, J.; Vermes, I. J. Biomed. Biotechnol. 2009, 823148. (11) Wang, J. Y.; Ren, L.; Li, L.; Liu, W. M.; Zhou, J.; Yu, W. H.; Tong, D. W.; Chen, S. L. Lab Chip 2009, 9, 644. (12) Taylor, A. M.; Jeon, N. L. Curr. Opin. Neurobiol. 2010, 20, 640. (13) Ziolkowska, K.; Kwapiszewski, R.; Brzozka, Z. New J. Chem. 2011, 35, 979. AE


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