Available online at www.sciencedirect.com
ScienceDirect Fluidic and microfluidic tools for quantitative systems biology Burak Okumus1,3, Sadik Yildiz2 and Erdal Toprak2,3 Abstract Understanding genes and their functions is a daunting task due to the level of complexity in biological organisms. For discovering how genotype and phenotype are linked to each other, it is essential to carry out systematic studies with maximum sensitivity and high-throughput. Recent developments in fluid-handling technologies, both at the macro and micro scale, are now allowing us to apply engineering approaches to achieve this goal. With these newly developed tools, it is now possible to identify genetic factors that are responsible for particular phenotypes, perturb and monitor cells at the single-cell level, evaluate cell-to-cell variability, detect very rare phenotypes, and construct faithful in vitro disease models. Addresses 1 Department of Systems Biology, Harvard Medical School, Boston, MA, USA 2 Faculty of Engineering and Natural Sciences, Sabanci University, Istanbul, Turkey 3 These authors contributed equally to this work. Corresponding authors: Okumus, Burak ([email protected]), Toprak, Erdal ([email protected])
Current Opinion in Biotechnology 2014, 25:30–38 This review comes from a themed issue on Analytical biotechnology Edited by Frank L Jaksch and Savas¸ Tay
0958-1669/$ – see front matter, # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.copbio.2013.08.016
Introduction Deciphering how genetic networks function inside cells is essential to understand life. Classical approaches used molecular biology tools to discover functions of genes, one at a time, where, genes of interest are either deleted, overexpressed, fluorescently labeled, or systematically mutated [1–5]. The resulting phenotypic changes are quantified using available physical tools [6]. Although these studies revealed a great deal of information about cell physiology in general, linking genotype and phenotype unequivocally has been virtually impossible since for a given phenotype, several thousand genes are regulated at a very complex level (Figure 1a) as a result of interactions between them and their products [7,8]. In this review article, we will summarize two experimental platforms that can potentially address this complexity especially when combined with next-generation sequencing Current Opinion in Biotechnology 2014, 25:30–38
technologies and fluorescence microscopy [9,10,11]. These methods are: (A) long-term adaptation experiments in large volumes for acquiring evolved populations that are specialized in certain environments and identifying genetic changes responsible for the emerging phenotypes, (B) microfluidic devices to perturb and/or monitor cell populations in small volumes to understand cellular responses using sensitive imaging or detection, and sequencing tools. Long-term adaptation experiments for understanding gene function
Long-term adaptation experiments have been useful in many fields of biology. Typically in these experiments, isogenic populations of reference wild type (WT) strains are exposed to stress environments for several hundred generations until a diminishing return in adaptation is observed [10,12,13,14,15]. Evolved populations are then further characterized using available phenotyping and genotyping tools (Figure 1a). These experiments can be as simple as serially diluting cells in test tubes and petri dishes. The seminal example is Richard Lenski’s epic experiment on Escherichia coli (E. coli) that has been running continuously since 1988 [16,17]. Lenski and his colleagues started these experiments by growing 12 isogenic WT E. coli strains in growth media containing glucose and citrate. Each day, they transferred 1% of the overnight grown populations to sterile flasks containing fresh media. This process, which has been continuing for more than 50,000 generations, revealed several interesting phenotypic changes such as increased cell size, enhanced growth rate, and utilization of citrate as a carbon source in aerobic conditions. Furthermore, next-generation sequencing technologies allowed determination of the genetic changes and epistatic interactions responsible for the emergence of these novel phenotypes [16–18]. Although Lenski’s long-term evolution experiment is a milestone in experimental evolution field, this system has an intrinsic limitation. Once evolved populations are diluted by hundred-fold and transferred to new culture tubes, cells start duplicating and cell density quickly reaches to saturation. Since cell populations remain in the stationary phase for most of the day and experience non-optimal conditions for cell growth, it becomes difficult to understand how selection takes place. To this end, there are several continuous-culture devices that are designed to run long-term evolution experiments in well-controlled environments to minimize the interference of other environmental factors during the emergence of the novel phenotypes. These tools often rely on www.sciencedirect.com
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Biological organisms have several thousand genes that form complex interaction networks. (a) Left. A hypothetical genetic network where each gene cluster is represented with a circle and labeled with a capital letter. Solid lines depict interactions. Right. Same network after the adaptation. Evolved gene clusters and interactions are shown in red. (b) Typical design schematics of continuous-culture devices with milliliter volumes. OD and environmental variables such as temperature, pH, and dissolved oxygen are measured by detectors and recorded for further analysis. These measurements are processed by digital or analog feedback control circuits and growing cell cultures inside vials are accordingly diluted or chemically perturbed by pumps injecting growth media or growth media containing chemicals (e.g. drugs). Volumes of cell cultures are kept constant with the help of waste pumps. (c) Simulated representative growth curves. Growth rate is 1 h 1 for all simulations. (i) Unperturbed growth displaying a log-logistic curve. (ii) Chemostat. Cells are diluted by 10% every 15 min. OD of cells reaches to a steady-state around 0.6 where the growth rate is equal to the dilution rate of the system. (iii) Turbidostat. Cells are diluted by 20% whenever OD reaches 0.2. This allows continuous growth at the exponential phase. (iv) Morbidostat. Cells are diluted by either fresh media or drug solution by 8% every 15 min. Cell growth is inhibited by adding antibiotic solution whenever OD exceeds a threshold value of 0.2 (marked by red arrows). As drug concentration fluctuates due to media and drug injections, OD of the cell population also fluctuates and average growth rate of the cells remains close to the dilution rate of the system.
feedback circuits (Figure 1b) that clamp experimental variables such as acidity, turbidity, dissolved oxygen, growth rate, and chemically induced growth inhibition. Below, we will elaborate on three such continuous-culture devices – the chemostat, turbidostat and morbidostat – and note relatively recent studies performed using these systems. www.sciencedirect.com
Chemostat
Chemostats are commonly used bioreactors, especially in microbiology, where cell populations are grown in culture vials that are fed with fresh growth media at a constant flow rate. Chemostats have been very powerful in revealing several biological phenomena such as carbon Current Opinion in Biotechnology 2014, 25:30–38
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metabolism, antibiotic resistance, population dynamics, and survival of populations [12,19,20]. The operational algorithm of chemostats is quite simple. The only parameter that has to be controlled is the flow rate of the inflowing fresh media (Figure 1b). When cells are grown in a culture vial without any external perturbation, they grow obeying a log-logistic function (Figure 1c-(i)) in which they start growing exponentially after a lag phase. Following the exponential regime, the growth rate of the population starts decreasing due to the limiting available nutrients (and space) as well as the accumulation of metabolic waste. Finally, cells enter into a stationary phase where the net growth rate of the population becomes zero. However, in a chemostat, cell populations grow and reach to a steady-state where the average growth rate of cells and the influx rate of the chemostat are equal (Figure 1c-(ii)). After this equilibrium is reached, optical density (OD) of the cell population stays constant with minor fluctuations. As mentioned before, it is possible (and also important) to monitor several experimental parameters (i.e. pH, dissolved oxygen) at the equilibrium. In fact, this information revealed invaluable clues about temporal oscillations in the cell cycle [19]. Using a chemostat, it is possible to run experiments on a time scale suitable for studying evolution and cell physiology. One limitation of chemostats is that it is not possible to clamp the OD of the cells when cells grow exponentially since any small difference between the population growth rate and the dilution rate of the system causes an experimental catastrophe: either the cells are completely washed out or a new, undesired equilibrium is reached where cells grow at a rate slower than that of the exponential growth. In order to overcome this problem, another continuous-culture device, turbidostat, is used. Turbidostat
Turbidostats are continuous-culture devices in which cells are grown within a narrow turbidity range (practically constant OD). In a turbidostat, using a feedback circuit, cells are diluted whenever OD exceeds a preset threshold (Figure 1c-(iii)). In this way, it becomes possible to grow cells in the exponential phase for several hundred generations in relatively short time periods. For example, for E. coli cells that double in 20 min when grown in rich conditions, one can grow 500 generations within only one week. This feature of the turbidostat imposes a considerable selective pressure on cell populations and therefore any mutation that increases the growth rate has a chance to get fixed. Since turbidostats are great tools for understanding principles of evolutionary dynamics and expedited evolution in the lab, researchers recently used this system to study stochastic switching in fluctuating environments, evolve biomolecules, and eliminate Current Opinion in Biotechnology 2014, 25:30–38
certain amino acids and nucleotides from the genetic code of bacterial cells [15,21,22,23]. Morbidostat
Morbidostat is a relatively recent continuous-culture device that was developed for studying the evolution of antibiotic resistance in a well-controlled laboratory environment [10,11]. Similar continuous-culture devices were designed and used in the past [12,15,24]. The control algorithm of the morbidostat is quite similar to the chemostat in the sense that evolving cell populations are periodically diluted and the growth rates of the cell populations are precisely measured within every growth period (i.e. every 10 min). As opposed to dilution by media in the case of a turbidostat, whenever OD exceeds a threshold, cell growth is inhibited by adding antibiotics to the culture vial. By taking the advantage of continuous-growth rate measurements, it becomes possible to keep the cell density and growth rate within a regime where cells do not experience any other stress besides drug induced growth inhibition. Cells then evolve high levels of drug resistance allowing easy identification of mutations that confer resistby using next-generation sequencing ance technologies [10]. The morbidostat has been proven to be useful for studying bacterial drug resistance and has the potential for studying evolution of resistance against cancer drugs as well. Advantages and limitations of fluidic devices
Unlike microfluidic devices, there is no need for specialized laboratory tools or fabrication facilities for constructing and maintaining fluidic devices. Owing to their macroscopic sizes, it is also possible to grow large volumes of cells in these devices, and evolution takes place in an environment where clonal interference is allowed [10]. However, using large volumes sometimes poses a drawback for maintaining the experiments for prolonged periods, especially if high-cost growth media or chemicals are used. Besides, fluidic devices have two other major limitations. First, most of these devices rely on OD measurements, which can be misleading especially for some cell types where it is not possible to distinguish between dead vs. live cells. Second, in almost all bioreactors, biofilm formation is a very significant problem. Although this problem can partially be addressed by self-cleaning bioreactors, no silver bullet yet exists to fully solve the issue [15]. Microfluidics
Microfluidic approaches are garnering attention due to their enabling capabilities that open up new experimental avenues, which used to be otherwise completely inaccessible, prohibitively expensive or tedious. We will www.sciencedirect.com
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elaborate on such significantly distinctive aspects of microfluidics through specific examples below. Culturing of non-adherent cell types in micro-chemostats and ultra-small volumes
Cell culturing is an art in its own right, but non-adherent cells (especially small ones such as bacteria and yeast) pose a particular challenge for imaging and manipulation. By providing cell immobilization within well-defined geometries, and precise spatio-temporal control over media conditions, microfluidics comes to the rescue at this point, offering solutions for researchers to study nonadherent cells with unprecedented capabilities. As an elegant example, Wang et al. recently devised a design so-called mother machine (Figure 2a), constraining cells within linear tracks, which facilitates cell tracking, and assignment of lineages [25]. The mother machine allows long-term monitoring of steady-state growth and division patterns of E. coli at the single-cell level for over 200-generations, which is more than an order of magnitude improvement over the total observation periods attainable with commonplace agar pads. Another mother machine enabled studying of aging for the budding yeast, Saccharomyces cerevisiae (S. cerevisiae) by squeezing the mother cells between a polydimethylsiloxane (PDMS) micropad and coverglass (Figure 2b). A constant flow of fresh media washed away the smaller daughter cells also providing a chemostatic growth condition [26]. As opposed to the conventional manual dissection method used in other studies, this microfluidic dissection platform required minimal human labor and offered high-resolution microscopic imaging and continuous monitoring of cells. Using a microfluidic device also with linear trenches, Leibler and co-workers exposed E. coli to antibiotics and discovered that the rare survivors were cells that differed from the rest of the population only phenotypically (i.e. without acquiring any mutations): these antibiotic persisters simply had reduced growth rate before antibiotic treatment [9]. They could then spontaneously switch to normal growth to generate a genetically identical population following the removal of the drug. On a similar platform, Makamoto et al. recently discovered an alternative mechanism of persistence (so-called dynamic persistence) where the cells that happen to express KatG (an enzyme that converts a pro-drug into its active antibiotic form) in low levels by the time the drug is around, evade succumbing to the antibiotic [27]. By patterning linear tracks within agar (instead of PDMS) facilitated the growth of several different strains of E. coli auxotrophs that complemented one another for amino-acids since agar allows small molecule exchange between bacteria, making it possible to study bacterial communication in general [28]. Aside from maintaining cells in isolation or within linear tracks, researchers employed microfluidic designs that www.sciencedirect.com
allow them to grow cells in small-volume chambers in 2D or in 3D. Elf and co-workers report on utilizing a geometry that restricts E. coli cells to grow within a single layer micro-colony to extract excessive statistics on the expression and localization of a low-copy number transcription factor throughout a total of 3000 bacterial lifespans [29]. Note that, unlike the mother machine, this number for the observed lifespans does not come from tracking one particular cell. Another micro-chemostat optimized for preventing biofilm formation was used to grow bacterial populations in 16-nanoliter chambers to study the dynamics of synthetic biological circuits [30]. Using this miniaturized chip compared to a macroscopic tube reduces the population size by 105 (100 to 10,000 bacteria per chamber) yielding a proportionate decrease in the total mutation rate, which ensures a stable, prolonged monitoring of a genetically homogenous population. For instance, while a macro-scaled study only hinted at expected oscillations between the two distinct E. coli populations due to accumulated mutations that broke the stability of the circuit, this micro-chemostat revealed clear oscillations (Figure 2c). The observations recapitulated the theoretical expectations from the circuit behavior providing an ideal quantitative platform to test the designed biological circuits both analytically and experimentally. The same platform was used earlier to observe the dynamics of a quorum-sensing based synthetic control-circuit that regulates cell density [31]. In addition, Hasty and co-workers grew E. coli in 2D or 3D features of varying dimensions to test the behavior of a coupled intracellular synthetic oscillator [32]. The system produced colony-wide rhythmic oscillations with temporal and spatial patterns depending on the communication between the cells, which can be modeled taking the geometry of the growth chambers into account. Mammalian cells are mostly adherent and hence easier to culture but for the non-adherent mammalian cells types, similar challenges still apply. To this end, Lecault et al. developed a PDMS chip for monitoring single hematopoetic stem cells. The chip, (only as big as a matchbox that contains 1600 of 4-nanoliter chambers) enabled precise control of conditions for growth of clones over multiple days, immunostaining and recovery of viable cells [33]. Such small volumes inside the microfluidic compartments not only provide scalability but also allow for low-reagent consumption, faster reaction rates, rapid diffusive mixing, and higher detection sensitivity. It is also possible to isolate non-adherent cells in plugs or emulsion droplets enclosing volumes in the range of pico-liters to nano-liters. Boedicker et al. used a plugbased microfluidic chip to detect the presence of Current Opinion in Biotechnology 2014, 25:30–38
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(a) E. coli ‘mother machine’ allows tracking a single mother cell over 200 generations, hence the name. The device provides a well-controlled environment for growth with the constant feeding of fresh media, resembling a microfluidic version of a chemostat. (b) S. cerevisiae mother machine relies on the size difference between mother and daughter cells to achieve ‘microfluidic dissection’. (c) Observation of oscillatory dynamics between predator (blue) and prey (red) population made possible by the micro-chemostat developed by Balagadde et al. (d) Instead of growing and tracking cells, the E. coli mother machine (a) was used for lysing single bacterium in linear tracks (so-called the ‘piston channel’) and manipulating chromosomes (stained in red). The chromosomes liberated after cell lysis were perturbed: chemically (by means of cycling between buffers with and without crowding agents through the main trench, top panel) and mechanically (using a ‘micro-piston’ that was achieved via moving a bead using optical tweezers, bottom panel). Adapted from references [25,26,30,40].
methicillin-resistant Staphylococcus aureus (MRSA), determine its sensitivity to different antibiotics, measure the minimal inhibitory concentration (MIC) of a drug (cefoxitin), and distinguish between sensitive and resistant S. aureus strains in human blood plasma samples, all in a 1–4 h experiment, while the impediments with the current diagnosis and characterization techniques would require days to carry out detection of bacteria alone [34]. An extensive review further summarizes the applications of droplet microfluidics for studying cell growth assays, cell-cell interactions, and bacterial persistence [35]. Current Opinion in Biotechnology 2014, 25:30–38
Single-cell lysis and manipulation
Biological inquiries at the level of single-cells can reveal information otherwise hidden in bulk measurements and the capabilities offered by microfluidics is well suited to meet such a promise (see excellent review by Lecault et al. for a more detailed discourse [36]). Aside from observing single-cell behavior in the platforms described above, such as the mother machine, researchers carry out post-mortem analysis to further extract clues within intracellular scenes using microfluidic systems. Lysis of [single] cells is akin to biochemical fractionation, only at www.sciencedirect.com
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(a) Left. Nanofabricated channels forcing E. coli to grow into a 0.25-micron constriction, which is much narrower than typical width (0.8 mm) of E. coli. Right. Fluorescence images of squeezed bacteria growing into aberrant shapes in the channel. Despite this anomalous morphology, E. coli cells are mostly still able to divide into two equally sized daughters with accuracy similar to that of normal conditions. Scale bar: 5 mm. (b) The microchannels where bacteria confer antibiotic resistance to the antibiotic ciprofloxacin far more rapidly compared to the situation in flasks. Since the environments bacteria reside in real-life scenarios is far-removed from well-shaken cultures, the observed behavior might be a better representation of the reality. (c) Left. The opposite sides of the porous flexible membrane were coated with cultured human lung (top) and capillary blood vessel cells (bottom) to mimic the alveolar-capillary interface. Cyclic suction from the side chambers makes the membrane stretch and relax rhythmically to imitate breathing. Right. Air flows from the top, over the lung cells, and the liquid medium containing human white blood cells flow from the bottom, over the capillary (endothelial) cells. To mimic an infection, (airborne) bacteria (green) are introduced in the top layer (0 min). Rapidly responding to the invaders, white blood cells (red) from the bottom stream readily go through the porous membrane to engulf the bacteria (4 min). Scale bar: 50 mm. Figures adapted from references [13,42,46].
the micro scale, allowing one to spread cell contents for minimizing overlap and cross-talk between entities to be measured, achieving purity and handling of the only desired components, and in a particular example circumvent the issues with background fluorescence and optical diffraction limit for precise enumeration of low-abundance proteins in insect and bacterial cells [37]. This was achieved via cell lysis, fluorescent labeling of proteins of interest, and subsequent separation (of the labeled proteins from unbound dyes) by capillary electrophoresis, and single-molecule imaging – all achieved on a single chip. Another device allowed for all steps of single-cell www.sciencedirect.com
processing (from cell capture to quantitive PCR) from 3300 single-cells to measure miRNA expression levels in immortalized human leukemia (K562) cells, co-regulation of a miRNA and its target transcript in embryonic stem cells, and to obtain single nucleotide variant detection in breast cancer cells [38]. A microfluidic system with highly parallel sample processing was used for sequencing the entire genome of 100 individual sperms from a 40-yearold human male to generate a recombination map and mutation rates to reveal the extent of genetic variation at the level of single-cells [39]. Furthermore, on-chip singlecell lysis strategies were used to mechanically manipulate Current Opinion in Biotechnology 2014, 25:30–38
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chromosomes: Pelletier et al. used the E. coli mother machine to study the mechanical properties of bacterial chromosomes (Figure 2d) [40].
An artistic rendering of the E. coli mother machine. Image credit: Dr. Raul Fernandez-Lopez.
Microfluidics allows for mechanical and chemical perturbations, and mimicking of biologically relevant niches
Owing to the mechanical flexibility of PDMS and the fine spatio-temporal control over changing buffers within flow chambers, microfluidic devices are used for the well-controlled mechanical and chemical perturbation of cells. Moreover, fabrication of structures with precise geometries and dimensions can be used to create environments that mimic the biological niches better than the macro scale assays. In this vein, Boulant et al. mechanically stretched mammalian cells, using elastic properties of PDMS substrates, to study the actin dependence of endocytotic coat formation in the presence of membrane tension [41]. Stretching, which cells of a developing tissue regularly experience, promotes arrest of endocytosis at the adherent surface of the plasma membrane, unless actin dynamics operate to rescue the process. In another study, E. coli cells were squeezed into nanofabricated channels (Figure 3a) towards understanding how bacteria move in natural habitats such as soil, tissues and biofilms with constrictions that are comparable to their sizes [42]. Controlled chemical perturbations on microfluidic platforms are useful to study the response properties of biological circuits. A device that allows for rapid periodic changes in media was used to measure signaling pathway response in S. cerevisae over various input frequencies [43]. Applying temporal stimulations of the tumor necrosis factor (TNF-a), Tay et al. studied the dynamics of NFkB response in single mammalian cells on a microfluidic cell culture, which better mimics physiological conditions in terms of volume, flow and concentration of ligands since in conventional cultures, the secreted signaling molecules are quickly diluted into large volumes of media [44]. In addition to the active control of media exchange, the chemical gradients that passively form within small volumes of microstructures also prove useful. Zhang et al. studied the effects of gradients of chemicals and Current Opinion in Biotechnology 2014, 25:30–38
nutrients on growth of bacterial populations within an array of connected microchambers (Figure 3b), which may potentially better represent the heterogeneous environments in soil or within an animal’s body as opposed to wellmixed liquid cultures [13]. Finally, researchers take advantage of microfluidics to construct in vitro disease models. Mimicking occlusion in capillaries by red blood cells in sickle-cell disease, Higgins et al. were able to trigger, control, and reverse the collective jamming event in PDMS channels [45]. In a recent, very exciting report, Ingber and colleagues described a bio-inspired micro-device (so-called lung-ona-chip, Figure 3c) that can recapitulate the essential phenomena at the alveolar-capillary interface of the human lung allowing studies of complex organ-level responses to bacterial infections, cancer drugs and airborne nanoparticles at the alveolar space [46]. Along similar lines, the same group reported on [human] gut-on-a-chip that can imitiate physical and functional features of human intestine [47]. Along with the cultured epithelium, they were able to co-culture a native intestinal microbe in these chips for extended periods that would allow for studies on human gut flora. In general, such organ-on-a-chip models holds the potential to set the stage for the next surge of studies that may serve as an alternative for animal testing to provide more tractable disease models and eventually bring therapies to patients, faster and cheaper.
More than just cool gadgets Although Agent 007, James Bond might be going a long way with his wit, charisma, and fighting skills (not to forget his unmatched luck), he sometimes achieves narrow escapes thanks to his ever-expanding repertoire of gizmos. Similarly, for researchers, the advent of fluidic and microfluidic systems offer more than just cool gadgets. As documented in this review, augmented by next-generation sequencing technologies, highly sensitive microscopy, and elaborate engineering, as well as the involvement of interdisciplinary research teams, fluidic systems are taking an exciting twist in applications to biological problems. Being the new kid on the block, microfluidics is finally starting to make a difference for biological and biomedical research after going through an initial technology development phase. With fluidic and microfluidic approaches finding traction for systems-level understanding of biological problems, we anticipate to see more of the broad adoption and long-term impact of these technologies in the near future.
Acknowledgements We thank Rishi Jajoo and Andreas Hilfinger for their careful reading of the manuscript, and Raul Fernandez-Lopez for his depiction of the E. coli mother machine. Burak Okumus is supported by the NIH GM081563 grant and Novartis Fellowship in Systems Biology. Erdal Toprak is supported by Marie Curie Career Integration Grant (303786) and EMBO Installation Grant (2552). www.sciencedirect.com
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17. Khan AI, Dinh DM, Schneider D, Lenski RE, Cooper TF: Negative epistasis between beneficial mutations in an evolving bacterial population. Science 2011, 332:1193-1196. 18. Blount ZD, Borland CZ, Lenski RE: Historical contingency and the evolution of a key innovation in an experimental population of Escherichia coli. Proc Natl Acad Sci U S A 2008, 105: 7899-7906. 19. Tu BP, Kudlicki A, Rowicka M, McKnight SL: Logic of the yeast metabolic cycle: temporal compartmentalization of cellular processes. Science 2005, 310:1152-1158. 20. Dai L, Vorselen D, Korolev KS, Gore J: Generic indicators for loss of resilience before a tipping point leading to population collapse. Science 2012, 336:1175-1177. 21. Pezo V, Louis D, Gue´rineau V, Le Caer J-P, Gaillon L, Mutzel R, Marlie`re P: A metabolic prototype for eliminating tryptophan from the genetic code. Sci Rep 2013, 3:1359. 22. Esvelt KM, Carlson JC, Liu DR: A system for the continuous directed evolution of biomolecules. Nature 2012, 472:499-503. Authors developed a system that can continuously evolve biological molecules in E. coli. 23. Acar M, Mettetal JT, van Oudenaarden A: Stochastic switching as a survival strategy in fluctuating environments. Nat Genet 2008, 40:471-475. 24. Bryson V, Szybalski W: Microbial selection. Science 1952, 116:45-51. 25. Wang P, Robert L, Pelletier J, Dang WL, Taddei F, Wright A, Jun S: Robust growth of Escherichia coli. Current Biology 2010 http:// dx.doi.org/10.1016/j.cub.2010.04.045. Using the mother machine, authors concluded that growth rate of E. coli remains constant over generations, and all cells within the steady-state growing population are essentially indistinguishable from each other regardless of their replicative age. With such advantages that enable long-term monitoring of many cells, the assay can be utilized to address a multitude of problems such as the extent of cell-to-cell variation due to stochastic gene expression and the longevity of such variations, as well as heterogeneity of cells in responding to different media conditions or perturbations with familial relationships. 26. Lee SS, Avalos Vizcarra I, Huberts DHEW, Lee LP, Heinemann M: Whole lifespan microscopic observation of budding yeast aging through a microfluidic dissection platform. Proc Natl Acad Sci U S A 2012, 109:4916-4920. 27. Wakamoto Y, Dhar N, Chait R, Schneider K, Signorino-Gelo F, Leibler S, McKinney JD: Dynamic persistence of antibioticstressed mycobacteria. Science 2013, 339:91-95. 28. Moffitt JR, Lee JB, Cluzel P: The single-cell chemostat: an agarose-based, microfluidic device for high-throughput, single-cell studies of bacteria and bacterial communities. Lab Chip 2012, 12:1487. 29. Ullman G, Wallden M, Marklund EG, Mahmutovic A, Razinkov I, Elf J: High-throughput gene expression analysis at the level of single proteins using a microfluidic turbidostat and automated cell tracking. Philos Trans R Soc B: Biol Sci 2012, 368 20120025. 30. Balagadde´ FK, Song H, Ozaki J, Collins CH, Barnet M, Arnold FH, Quake SR, You L: A synthetic Escherichia coli predator-prey ecosystem. Mol Syst Biol 2008:4. 31. Balagadde FK: Long-term monitoring of bacteria undergoing programmed population control in a microchemostat. Science 2005, 309:137-140. 32. Danino T, Mondrago´n-Palomino O, Tsimring L, Hasty J: A synchronized quorum of genetic clocks. Nature 2010, 463:326-330. 33. Lecault V, VanInsberghe M, Sekulovic S, Knapp DJHF, Wohrer S, Bowden W, Viel F, McLaughlin T, Jarandehei A, Miller M et al.: High-throughput analysis of single hematopoietic stem cell proliferation in microfluidic cell culture arrays. Nat Methods 2011, 8:581-586. 34. Boedicker JQ, Li L, Kline TR, Ismagilov RF: Detecting bacteria and determining their susceptibility to antibiotics by stochastic confinement in nanoliter droplets using plug-based microfluidics. Lab Chip 2008, 8:1265. Current Opinion in Biotechnology 2014, 25:30–38
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35. Guo MT, Rotem A, Heyman JA, Weitz DA: Droplet microfluidics for high-throughput biological assays. Lab Chip 2012, 12:2146.
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38. White AK, VanInsberghe M, Petriv OI, Hamidi M, Sikorski D, Marra MA, Piret J, Aparicio S, Hansen CL: High-throughput microfluidic single-cell RT-qPCR. Proc Natl Acad Sci U S A 2011, 108:13999-14004. 39. Quake JWHNFBBS, Fan HC, Behr B, Quake SR: Genome-wide single-cell analysis of recombination activity and de novo mutation rates in human sperm. Cell 2012, 150:402-412. 40. Pelletier J, Halvorsen K, Ha B-Y, Paparcone R, Sandler SJ, Woldringh CL, Wong WP, Jun S: Physical manipulation of the Escherichia coli chromosome reveals its soft nature. Proc Natl Acad Sci U S A 2012, 109:E2649-E2656. Authors do extensive characterization of mechanical properties of the E. coli chromosome. Based on their findings, they speculate that the physical properties of the chromosome might solely be the major driving force for chromosome segregation in E. coli, which lacks any active segregation machinery. 41. Boulant S, Kural C, Zeeh J-C, Ubelmann F, Kirchhausen T: Actin dynamics counteract membrane tension during clathrinmediated endocytosis. Nat Cell Biol 2011, 13:1124-1131. 42. Ma¨nnik J, Wu F, Hol FJH, Bisicchia P, Sherratt DJ, Keymer JE, Dekker C: Robustness and accuracy of cell division in
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45. Higgins JM, Eddington DT, Bhatia SN, Mahadevan L: Sickle cell vasoocclusion and rescue in a microfluidic device. Proc Natl Acad Sci U S A 2007, 104:20496-20500. In this sickle cell disease model, by manipulating the channel sizes as well as oxygen levels, authors reported on the processes that may be governing the occlusion (by red-blood cells) and its rescue within capillaries of different sizes under a range of oxygenation levels. 46. Huh D, Matthews BD, Mammoto A, Montoya-Zavala M, Hsin HY, Ingber DE: Reconstituting organ-level lung functions on a chip. Science 2010, 328:1662-1668. Authors used the lung-on-a-chip platform to mimic the bacterial infection in lungs, and study the side effects of a cancer drug that causes leaking of blood into alveoli. They also studied the toxic effects of airborne nanoparticles on the lung, where they found that the mechanical strain during breathing increased particles’ absorption into the bloodstream. 47. Kim HJ, Huh D, Hamilton G, Ingber DE: Human gut-on-achip inhabited by microbial flora that experiences intestinal peristalsis-like motions and flow. Lab Chip 2012, 12:2165.
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ScienceDirect Fluidic and microfluidic tools for quantitative systems biology Burak Okumus1,3, Sadik Yildiz2 and Erdal Toprak2,3 Abstract Understanding genes and their functions is a daunting task due to the level of complexity in biological organisms. For discovering how genotype and phenotype are linked to each other, it is essential to carry out systematic studies with maximum sensitivity and high-throughput. Recent developments in fluid-handling technologies, both at the macro and micro scale, are now allowing us to apply engineering approaches to achieve this goal. With these newly developed tools, it is now possible to identify genetic factors that are responsible for particular phenotypes, perturb and monitor cells at the single-cell level, evaluate cell-to-cell variability, detect very rare phenotypes, and construct faithful in vitro disease models. Addresses 1 Department of Systems Biology, Harvard Medical School, Boston, MA, USA 2 Faculty of Engineering and Natural Sciences, Sabanci University, Istanbul, Turkey 3 These authors contributed equally to this work. Corresponding authors: Okumus, Burak ([email protected]), Toprak, Erdal ([email protected])
Current Opinion in Biotechnology 2014, 25:30–38 This review comes from a themed issue on Analytical biotechnology Edited by Frank L Jaksch and Savas¸ Tay
0958-1669/$ – see front matter, # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.copbio.2013.08.016
Introduction Deciphering how genetic networks function inside cells is essential to understand life. Classical approaches used molecular biology tools to discover functions of genes, one at a time, where, genes of interest are either deleted, overexpressed, fluorescently labeled, or systematically mutated [1–5]. The resulting phenotypic changes are quantified using available physical tools [6]. Although these studies revealed a great deal of information about cell physiology in general, linking genotype and phenotype unequivocally has been virtually impossible since for a given phenotype, several thousand genes are regulated at a very complex level (Figure 1a) as a result of interactions between them and their products [7,8]. In this review article, we will summarize two experimental platforms that can potentially address this complexity especially when combined with next-generation sequencing Current Opinion in Biotechnology 2014, 25:30–38
technologies and fluorescence microscopy [9,10,11]. These methods are: (A) long-term adaptation experiments in large volumes for acquiring evolved populations that are specialized in certain environments and identifying genetic changes responsible for the emerging phenotypes, (B) microfluidic devices to perturb and/or monitor cell populations in small volumes to understand cellular responses using sensitive imaging or detection, and sequencing tools. Long-term adaptation experiments for understanding gene function
Long-term adaptation experiments have been useful in many fields of biology. Typically in these experiments, isogenic populations of reference wild type (WT) strains are exposed to stress environments for several hundred generations until a diminishing return in adaptation is observed [10,12,13,14,15]. Evolved populations are then further characterized using available phenotyping and genotyping tools (Figure 1a). These experiments can be as simple as serially diluting cells in test tubes and petri dishes. The seminal example is Richard Lenski’s epic experiment on Escherichia coli (E. coli) that has been running continuously since 1988 [16,17]. Lenski and his colleagues started these experiments by growing 12 isogenic WT E. coli strains in growth media containing glucose and citrate. Each day, they transferred 1% of the overnight grown populations to sterile flasks containing fresh media. This process, which has been continuing for more than 50,000 generations, revealed several interesting phenotypic changes such as increased cell size, enhanced growth rate, and utilization of citrate as a carbon source in aerobic conditions. Furthermore, next-generation sequencing technologies allowed determination of the genetic changes and epistatic interactions responsible for the emergence of these novel phenotypes [16–18]. Although Lenski’s long-term evolution experiment is a milestone in experimental evolution field, this system has an intrinsic limitation. Once evolved populations are diluted by hundred-fold and transferred to new culture tubes, cells start duplicating and cell density quickly reaches to saturation. Since cell populations remain in the stationary phase for most of the day and experience non-optimal conditions for cell growth, it becomes difficult to understand how selection takes place. To this end, there are several continuous-culture devices that are designed to run long-term evolution experiments in well-controlled environments to minimize the interference of other environmental factors during the emergence of the novel phenotypes. These tools often rely on www.sciencedirect.com
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Biological organisms have several thousand genes that form complex interaction networks. (a) Left. A hypothetical genetic network where each gene cluster is represented with a circle and labeled with a capital letter. Solid lines depict interactions. Right. Same network after the adaptation. Evolved gene clusters and interactions are shown in red. (b) Typical design schematics of continuous-culture devices with milliliter volumes. OD and environmental variables such as temperature, pH, and dissolved oxygen are measured by detectors and recorded for further analysis. These measurements are processed by digital or analog feedback control circuits and growing cell cultures inside vials are accordingly diluted or chemically perturbed by pumps injecting growth media or growth media containing chemicals (e.g. drugs). Volumes of cell cultures are kept constant with the help of waste pumps. (c) Simulated representative growth curves. Growth rate is 1 h 1 for all simulations. (i) Unperturbed growth displaying a log-logistic curve. (ii) Chemostat. Cells are diluted by 10% every 15 min. OD of cells reaches to a steady-state around 0.6 where the growth rate is equal to the dilution rate of the system. (iii) Turbidostat. Cells are diluted by 20% whenever OD reaches 0.2. This allows continuous growth at the exponential phase. (iv) Morbidostat. Cells are diluted by either fresh media or drug solution by 8% every 15 min. Cell growth is inhibited by adding antibiotic solution whenever OD exceeds a threshold value of 0.2 (marked by red arrows). As drug concentration fluctuates due to media and drug injections, OD of the cell population also fluctuates and average growth rate of the cells remains close to the dilution rate of the system.
feedback circuits (Figure 1b) that clamp experimental variables such as acidity, turbidity, dissolved oxygen, growth rate, and chemically induced growth inhibition. Below, we will elaborate on three such continuous-culture devices – the chemostat, turbidostat and morbidostat – and note relatively recent studies performed using these systems. www.sciencedirect.com
Chemostat
Chemostats are commonly used bioreactors, especially in microbiology, where cell populations are grown in culture vials that are fed with fresh growth media at a constant flow rate. Chemostats have been very powerful in revealing several biological phenomena such as carbon Current Opinion in Biotechnology 2014, 25:30–38
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metabolism, antibiotic resistance, population dynamics, and survival of populations [12,19,20]. The operational algorithm of chemostats is quite simple. The only parameter that has to be controlled is the flow rate of the inflowing fresh media (Figure 1b). When cells are grown in a culture vial without any external perturbation, they grow obeying a log-logistic function (Figure 1c-(i)) in which they start growing exponentially after a lag phase. Following the exponential regime, the growth rate of the population starts decreasing due to the limiting available nutrients (and space) as well as the accumulation of metabolic waste. Finally, cells enter into a stationary phase where the net growth rate of the population becomes zero. However, in a chemostat, cell populations grow and reach to a steady-state where the average growth rate of cells and the influx rate of the chemostat are equal (Figure 1c-(ii)). After this equilibrium is reached, optical density (OD) of the cell population stays constant with minor fluctuations. As mentioned before, it is possible (and also important) to monitor several experimental parameters (i.e. pH, dissolved oxygen) at the equilibrium. In fact, this information revealed invaluable clues about temporal oscillations in the cell cycle [19]. Using a chemostat, it is possible to run experiments on a time scale suitable for studying evolution and cell physiology. One limitation of chemostats is that it is not possible to clamp the OD of the cells when cells grow exponentially since any small difference between the population growth rate and the dilution rate of the system causes an experimental catastrophe: either the cells are completely washed out or a new, undesired equilibrium is reached where cells grow at a rate slower than that of the exponential growth. In order to overcome this problem, another continuous-culture device, turbidostat, is used. Turbidostat
Turbidostats are continuous-culture devices in which cells are grown within a narrow turbidity range (practically constant OD). In a turbidostat, using a feedback circuit, cells are diluted whenever OD exceeds a preset threshold (Figure 1c-(iii)). In this way, it becomes possible to grow cells in the exponential phase for several hundred generations in relatively short time periods. For example, for E. coli cells that double in 20 min when grown in rich conditions, one can grow 500 generations within only one week. This feature of the turbidostat imposes a considerable selective pressure on cell populations and therefore any mutation that increases the growth rate has a chance to get fixed. Since turbidostats are great tools for understanding principles of evolutionary dynamics and expedited evolution in the lab, researchers recently used this system to study stochastic switching in fluctuating environments, evolve biomolecules, and eliminate Current Opinion in Biotechnology 2014, 25:30–38
certain amino acids and nucleotides from the genetic code of bacterial cells [15,21,22,23]. Morbidostat
Morbidostat is a relatively recent continuous-culture device that was developed for studying the evolution of antibiotic resistance in a well-controlled laboratory environment [10,11]. Similar continuous-culture devices were designed and used in the past [12,15,24]. The control algorithm of the morbidostat is quite similar to the chemostat in the sense that evolving cell populations are periodically diluted and the growth rates of the cell populations are precisely measured within every growth period (i.e. every 10 min). As opposed to dilution by media in the case of a turbidostat, whenever OD exceeds a threshold, cell growth is inhibited by adding antibiotics to the culture vial. By taking the advantage of continuous-growth rate measurements, it becomes possible to keep the cell density and growth rate within a regime where cells do not experience any other stress besides drug induced growth inhibition. Cells then evolve high levels of drug resistance allowing easy identification of mutations that confer resistby using next-generation sequencing ance technologies [10]. The morbidostat has been proven to be useful for studying bacterial drug resistance and has the potential for studying evolution of resistance against cancer drugs as well. Advantages and limitations of fluidic devices
Unlike microfluidic devices, there is no need for specialized laboratory tools or fabrication facilities for constructing and maintaining fluidic devices. Owing to their macroscopic sizes, it is also possible to grow large volumes of cells in these devices, and evolution takes place in an environment where clonal interference is allowed [10]. However, using large volumes sometimes poses a drawback for maintaining the experiments for prolonged periods, especially if high-cost growth media or chemicals are used. Besides, fluidic devices have two other major limitations. First, most of these devices rely on OD measurements, which can be misleading especially for some cell types where it is not possible to distinguish between dead vs. live cells. Second, in almost all bioreactors, biofilm formation is a very significant problem. Although this problem can partially be addressed by self-cleaning bioreactors, no silver bullet yet exists to fully solve the issue [15]. Microfluidics
Microfluidic approaches are garnering attention due to their enabling capabilities that open up new experimental avenues, which used to be otherwise completely inaccessible, prohibitively expensive or tedious. We will www.sciencedirect.com
Fluidic and microfluidic tools for systems biology Okumus, Yildiz and Toprak 33
elaborate on such significantly distinctive aspects of microfluidics through specific examples below. Culturing of non-adherent cell types in micro-chemostats and ultra-small volumes
Cell culturing is an art in its own right, but non-adherent cells (especially small ones such as bacteria and yeast) pose a particular challenge for imaging and manipulation. By providing cell immobilization within well-defined geometries, and precise spatio-temporal control over media conditions, microfluidics comes to the rescue at this point, offering solutions for researchers to study nonadherent cells with unprecedented capabilities. As an elegant example, Wang et al. recently devised a design so-called mother machine (Figure 2a), constraining cells within linear tracks, which facilitates cell tracking, and assignment of lineages [25]. The mother machine allows long-term monitoring of steady-state growth and division patterns of E. coli at the single-cell level for over 200-generations, which is more than an order of magnitude improvement over the total observation periods attainable with commonplace agar pads. Another mother machine enabled studying of aging for the budding yeast, Saccharomyces cerevisiae (S. cerevisiae) by squeezing the mother cells between a polydimethylsiloxane (PDMS) micropad and coverglass (Figure 2b). A constant flow of fresh media washed away the smaller daughter cells also providing a chemostatic growth condition [26]. As opposed to the conventional manual dissection method used in other studies, this microfluidic dissection platform required minimal human labor and offered high-resolution microscopic imaging and continuous monitoring of cells. Using a microfluidic device also with linear trenches, Leibler and co-workers exposed E. coli to antibiotics and discovered that the rare survivors were cells that differed from the rest of the population only phenotypically (i.e. without acquiring any mutations): these antibiotic persisters simply had reduced growth rate before antibiotic treatment [9]. They could then spontaneously switch to normal growth to generate a genetically identical population following the removal of the drug. On a similar platform, Makamoto et al. recently discovered an alternative mechanism of persistence (so-called dynamic persistence) where the cells that happen to express KatG (an enzyme that converts a pro-drug into its active antibiotic form) in low levels by the time the drug is around, evade succumbing to the antibiotic [27]. By patterning linear tracks within agar (instead of PDMS) facilitated the growth of several different strains of E. coli auxotrophs that complemented one another for amino-acids since agar allows small molecule exchange between bacteria, making it possible to study bacterial communication in general [28]. Aside from maintaining cells in isolation or within linear tracks, researchers employed microfluidic designs that www.sciencedirect.com
allow them to grow cells in small-volume chambers in 2D or in 3D. Elf and co-workers report on utilizing a geometry that restricts E. coli cells to grow within a single layer micro-colony to extract excessive statistics on the expression and localization of a low-copy number transcription factor throughout a total of 3000 bacterial lifespans [29]. Note that, unlike the mother machine, this number for the observed lifespans does not come from tracking one particular cell. Another micro-chemostat optimized for preventing biofilm formation was used to grow bacterial populations in 16-nanoliter chambers to study the dynamics of synthetic biological circuits [30]. Using this miniaturized chip compared to a macroscopic tube reduces the population size by 105 (100 to 10,000 bacteria per chamber) yielding a proportionate decrease in the total mutation rate, which ensures a stable, prolonged monitoring of a genetically homogenous population. For instance, while a macro-scaled study only hinted at expected oscillations between the two distinct E. coli populations due to accumulated mutations that broke the stability of the circuit, this micro-chemostat revealed clear oscillations (Figure 2c). The observations recapitulated the theoretical expectations from the circuit behavior providing an ideal quantitative platform to test the designed biological circuits both analytically and experimentally. The same platform was used earlier to observe the dynamics of a quorum-sensing based synthetic control-circuit that regulates cell density [31]. In addition, Hasty and co-workers grew E. coli in 2D or 3D features of varying dimensions to test the behavior of a coupled intracellular synthetic oscillator [32]. The system produced colony-wide rhythmic oscillations with temporal and spatial patterns depending on the communication between the cells, which can be modeled taking the geometry of the growth chambers into account. Mammalian cells are mostly adherent and hence easier to culture but for the non-adherent mammalian cells types, similar challenges still apply. To this end, Lecault et al. developed a PDMS chip for monitoring single hematopoetic stem cells. The chip, (only as big as a matchbox that contains 1600 of 4-nanoliter chambers) enabled precise control of conditions for growth of clones over multiple days, immunostaining and recovery of viable cells [33]. Such small volumes inside the microfluidic compartments not only provide scalability but also allow for low-reagent consumption, faster reaction rates, rapid diffusive mixing, and higher detection sensitivity. It is also possible to isolate non-adherent cells in plugs or emulsion droplets enclosing volumes in the range of pico-liters to nano-liters. Boedicker et al. used a plugbased microfluidic chip to detect the presence of Current Opinion in Biotechnology 2014, 25:30–38
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(a) E. coli ‘mother machine’ allows tracking a single mother cell over 200 generations, hence the name. The device provides a well-controlled environment for growth with the constant feeding of fresh media, resembling a microfluidic version of a chemostat. (b) S. cerevisiae mother machine relies on the size difference between mother and daughter cells to achieve ‘microfluidic dissection’. (c) Observation of oscillatory dynamics between predator (blue) and prey (red) population made possible by the micro-chemostat developed by Balagadde et al. (d) Instead of growing and tracking cells, the E. coli mother machine (a) was used for lysing single bacterium in linear tracks (so-called the ‘piston channel’) and manipulating chromosomes (stained in red). The chromosomes liberated after cell lysis were perturbed: chemically (by means of cycling between buffers with and without crowding agents through the main trench, top panel) and mechanically (using a ‘micro-piston’ that was achieved via moving a bead using optical tweezers, bottom panel). Adapted from references [25,26,30,40].
methicillin-resistant Staphylococcus aureus (MRSA), determine its sensitivity to different antibiotics, measure the minimal inhibitory concentration (MIC) of a drug (cefoxitin), and distinguish between sensitive and resistant S. aureus strains in human blood plasma samples, all in a 1–4 h experiment, while the impediments with the current diagnosis and characterization techniques would require days to carry out detection of bacteria alone [34]. An extensive review further summarizes the applications of droplet microfluidics for studying cell growth assays, cell-cell interactions, and bacterial persistence [35]. Current Opinion in Biotechnology 2014, 25:30–38
Single-cell lysis and manipulation
Biological inquiries at the level of single-cells can reveal information otherwise hidden in bulk measurements and the capabilities offered by microfluidics is well suited to meet such a promise (see excellent review by Lecault et al. for a more detailed discourse [36]). Aside from observing single-cell behavior in the platforms described above, such as the mother machine, researchers carry out post-mortem analysis to further extract clues within intracellular scenes using microfluidic systems. Lysis of [single] cells is akin to biochemical fractionation, only at www.sciencedirect.com
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(a) Left. Nanofabricated channels forcing E. coli to grow into a 0.25-micron constriction, which is much narrower than typical width (0.8 mm) of E. coli. Right. Fluorescence images of squeezed bacteria growing into aberrant shapes in the channel. Despite this anomalous morphology, E. coli cells are mostly still able to divide into two equally sized daughters with accuracy similar to that of normal conditions. Scale bar: 5 mm. (b) The microchannels where bacteria confer antibiotic resistance to the antibiotic ciprofloxacin far more rapidly compared to the situation in flasks. Since the environments bacteria reside in real-life scenarios is far-removed from well-shaken cultures, the observed behavior might be a better representation of the reality. (c) Left. The opposite sides of the porous flexible membrane were coated with cultured human lung (top) and capillary blood vessel cells (bottom) to mimic the alveolar-capillary interface. Cyclic suction from the side chambers makes the membrane stretch and relax rhythmically to imitate breathing. Right. Air flows from the top, over the lung cells, and the liquid medium containing human white blood cells flow from the bottom, over the capillary (endothelial) cells. To mimic an infection, (airborne) bacteria (green) are introduced in the top layer (0 min). Rapidly responding to the invaders, white blood cells (red) from the bottom stream readily go through the porous membrane to engulf the bacteria (4 min). Scale bar: 50 mm. Figures adapted from references [13,42,46].
the micro scale, allowing one to spread cell contents for minimizing overlap and cross-talk between entities to be measured, achieving purity and handling of the only desired components, and in a particular example circumvent the issues with background fluorescence and optical diffraction limit for precise enumeration of low-abundance proteins in insect and bacterial cells [37]. This was achieved via cell lysis, fluorescent labeling of proteins of interest, and subsequent separation (of the labeled proteins from unbound dyes) by capillary electrophoresis, and single-molecule imaging – all achieved on a single chip. Another device allowed for all steps of single-cell www.sciencedirect.com
processing (from cell capture to quantitive PCR) from 3300 single-cells to measure miRNA expression levels in immortalized human leukemia (K562) cells, co-regulation of a miRNA and its target transcript in embryonic stem cells, and to obtain single nucleotide variant detection in breast cancer cells [38]. A microfluidic system with highly parallel sample processing was used for sequencing the entire genome of 100 individual sperms from a 40-yearold human male to generate a recombination map and mutation rates to reveal the extent of genetic variation at the level of single-cells [39]. Furthermore, on-chip singlecell lysis strategies were used to mechanically manipulate Current Opinion in Biotechnology 2014, 25:30–38
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chromosomes: Pelletier et al. used the E. coli mother machine to study the mechanical properties of bacterial chromosomes (Figure 2d) [40].
An artistic rendering of the E. coli mother machine. Image credit: Dr. Raul Fernandez-Lopez.
Microfluidics allows for mechanical and chemical perturbations, and mimicking of biologically relevant niches
Owing to the mechanical flexibility of PDMS and the fine spatio-temporal control over changing buffers within flow chambers, microfluidic devices are used for the well-controlled mechanical and chemical perturbation of cells. Moreover, fabrication of structures with precise geometries and dimensions can be used to create environments that mimic the biological niches better than the macro scale assays. In this vein, Boulant et al. mechanically stretched mammalian cells, using elastic properties of PDMS substrates, to study the actin dependence of endocytotic coat formation in the presence of membrane tension [41]. Stretching, which cells of a developing tissue regularly experience, promotes arrest of endocytosis at the adherent surface of the plasma membrane, unless actin dynamics operate to rescue the process. In another study, E. coli cells were squeezed into nanofabricated channels (Figure 3a) towards understanding how bacteria move in natural habitats such as soil, tissues and biofilms with constrictions that are comparable to their sizes [42]. Controlled chemical perturbations on microfluidic platforms are useful to study the response properties of biological circuits. A device that allows for rapid periodic changes in media was used to measure signaling pathway response in S. cerevisae over various input frequencies [43]. Applying temporal stimulations of the tumor necrosis factor (TNF-a), Tay et al. studied the dynamics of NFkB response in single mammalian cells on a microfluidic cell culture, which better mimics physiological conditions in terms of volume, flow and concentration of ligands since in conventional cultures, the secreted signaling molecules are quickly diluted into large volumes of media [44]. In addition to the active control of media exchange, the chemical gradients that passively form within small volumes of microstructures also prove useful. Zhang et al. studied the effects of gradients of chemicals and Current Opinion in Biotechnology 2014, 25:30–38
nutrients on growth of bacterial populations within an array of connected microchambers (Figure 3b), which may potentially better represent the heterogeneous environments in soil or within an animal’s body as opposed to wellmixed liquid cultures [13]. Finally, researchers take advantage of microfluidics to construct in vitro disease models. Mimicking occlusion in capillaries by red blood cells in sickle-cell disease, Higgins et al. were able to trigger, control, and reverse the collective jamming event in PDMS channels [45]. In a recent, very exciting report, Ingber and colleagues described a bio-inspired micro-device (so-called lung-ona-chip, Figure 3c) that can recapitulate the essential phenomena at the alveolar-capillary interface of the human lung allowing studies of complex organ-level responses to bacterial infections, cancer drugs and airborne nanoparticles at the alveolar space [46]. Along similar lines, the same group reported on [human] gut-on-a-chip that can imitiate physical and functional features of human intestine [47]. Along with the cultured epithelium, they were able to co-culture a native intestinal microbe in these chips for extended periods that would allow for studies on human gut flora. In general, such organ-on-a-chip models holds the potential to set the stage for the next surge of studies that may serve as an alternative for animal testing to provide more tractable disease models and eventually bring therapies to patients, faster and cheaper.
More than just cool gadgets Although Agent 007, James Bond might be going a long way with his wit, charisma, and fighting skills (not to forget his unmatched luck), he sometimes achieves narrow escapes thanks to his ever-expanding repertoire of gizmos. Similarly, for researchers, the advent of fluidic and microfluidic systems offer more than just cool gadgets. As documented in this review, augmented by next-generation sequencing technologies, highly sensitive microscopy, and elaborate engineering, as well as the involvement of interdisciplinary research teams, fluidic systems are taking an exciting twist in applications to biological problems. Being the new kid on the block, microfluidics is finally starting to make a difference for biological and biomedical research after going through an initial technology development phase. With fluidic and microfluidic approaches finding traction for systems-level understanding of biological problems, we anticipate to see more of the broad adoption and long-term impact of these technologies in the near future.
Acknowledgements We thank Rishi Jajoo and Andreas Hilfinger for their careful reading of the manuscript, and Raul Fernandez-Lopez for his depiction of the E. coli mother machine. Burak Okumus is supported by the NIH GM081563 grant and Novartis Fellowship in Systems Biology. Erdal Toprak is supported by Marie Curie Career Integration Grant (303786) and EMBO Installation Grant (2552). www.sciencedirect.com
Fluidic and microfluidic tools for systems biology Okumus, Yildiz and Toprak 37
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