Mechanochemical actuators of embryonic epithelial contractility.

Spatiotemporal regulation of cell contractility coordinates cell shape change to construct tissue architecture and ultimately directs the morphology and function of the organism. Here we show that contractility responses to spatially and temporally controlled chemical stimuli depend much more strongly on intercellular mechanical connections than on biochemical cues in both stimulated tissues and adjacent cells. We investigate how the cell contractility is triggered within an embryonic epithelial sheet by local ligand stimulation and coordinates a long-range contraction response. Our custom microfluidic control system allows spatiotemporally controlled stimulation with extracellular ATP, which results in locally distinct contractility followed by mechanical strain pattern formation. The stimulation-response circuit exposed here provides a better understanding of how morphogenetic processes integrate responses to stimulation and how intercellular responses are transmitted across multiple cells. These findings may enable one to create a biological actuator that actively drives morphogenesis.

How do control-based approaches enter into biology?

Control is intrinsic to biological organisms, whose cells are in a constant state of sensing and response to numerous external and self-generated stimuli. Diverse means are used to study the complexity through control-based approaches in these cellular systems, including through chemical and genetic manipulations, input-output methodologies, feedback approaches, and feed-forward approaches. We first discuss what happens in control-based approaches when we are not actively examining or manipulating cells. We then present potential methods to determine what the cell is doing during these times and to reverse-engineer the cellular system. Finally, we discuss how we can control the cell's extracellular and intracellular environments, both to probe the response of the cells using defined experimental engineering-based technologies and to anticipate what might be achieved by applying control-based approaches to affect cellular processes. Much work remains to apply simplified control models and develop new technologies to aid researchers in studying and utilizing cellular and molecular processes.

Three-dimensional chemical profile manipulation using two-dimensional autonomous microfluidic control.

The ability to specify or control spatiotemporal chemical environments is critical for controlling diverse processes from chemical synthesis to cellular responses. When established by microfluidics methods, this chemical control has largely been limited to two dimensions and by the need for using complex approaches. The ability to create three-dimensional (3D) chemical patterns is becoming more critical as microfluidics is beginning to have novel applications at larger millifluidic scales, including model organism behavior, embryonic development, and optofluidics. Here, we present a simple approach to create 3D chemical patterns that can be controlled in space and time via two-dimensional (2D), single-layer fluidic modules. Not only can we employ autonomous flow in a 2D fluidic configuration to produce a 3D pattern, but with very simple changes in the 2D configuration, the chemical pattern can be "focused and defocused" within the 3D cross section. We also show that these chemical patterns can be predicted by computational fluid dynamics simulations with high experimental correlation. These simulations allow analyses of the characteristics of interface behaviors with respect to three basic yet critical parameters that need to be thoroughly considered in scaling-up from microfluidic to millifluidic research: Reynolds number (Re), inlet geometry, and channel height. The findings not only indicate proof of concept for 3D pattern creation but also reveal that a number of fluidic experiments may have inherent limitations resulting from unrecognized 3D profiles that depend on these parameter choices. These results will be useful for research areas including embryonic development, cellular stimulation, and chemical fabrication approaches.

Modulation of fluidic resistance and capacitance for long-term, high-speed feedback control of a microfluidic interface.

Existing microfluidic systems can control local chemical environments by directing the interface between laminar flowing streams for applications ranging from subcellular stimulation to fuel cells. However, conventional flow modulation methods have not yet provided a robust and reliable way to dynamically control laminar flow interfaces for very long time periods. Such control is important in biological investigations, since response times for living cells and tissues can be as long as several days. Here, we describe a novel long-term, high-speed approach that employs modulation of fluidic resistance and fluidic capacitance between a fluid reservoir and a microfluidic network with feedback control to enable long-term dynamic control of a microfluidic interface in time and space. Our method involves constricting a narrow tube through a pinching approach to modulate fluidic resistance while also controlling a small variable reservoir in the fluidic network through a squeezing approach to modulate fluidic capacitance. We designed a well-tuned proportional-integral-derivative (PID) controller for the closed-loop control system that resulted in control of pressure for short-term (2 s) and long-term (15 h) experiments. Further, we integrated a pressure-based feedback control approach into this method, which enables both long-term spatiotemporal control of our microfluidic interface at frequencies greater than 1 Hz and a reservoir capacity to enable experiments for longer than 60 days. This long-term and high-speed control is not possible with standard microfluidic laboratory practices. Our system has a diversity of potential applications including long-term cellular studies in cancer metastasis or embryonic development.

Pressure-driven spatiotemporal control of the laminar flow interface in a microfluidic network.

We describe a system for dynamically adjusting the position of the laminar interface between two fluids flowing inside a microfluidic channel, with a time response of less than 0.1 s, through feedback control of the channel inlet pressure.

Sudden motility reversal indicates sensing of magnetic field gradients in Magnetospirillum magneticum AMB-1 strain.

Many motile unicellular organisms have evolved specialized behaviors for detecting and responding to environmental cues such as chemical gradients (chemotaxis) and oxygen gradients (aerotaxis). Magnetotaxis is found in magnetotactic bacteria and it is defined as the passive alignment of these cells to the geomagnetic field along with active swimming. Herein we show that Magnetospirillum magneticum (AMB-1) show a unique set of responses that indicates they sense and respond not only to the direction of magnetic fields by aligning and swimming, but also to changes in the magnetic field or magnetic field gradients. We present data showing that AMB-1 cells exhibit sudden motility reversals when we impose them to local magnetic field gradients. Our system employs permalloy (Ni(80)Fe(20)) islands to curve and diverge the magnetic field lines emanating from our custom-designed Helmholtz coils in the vicinity of the islands (creating a drop in the field across the islands). The three distinct movements we have observed as they approach the permalloy islands are: unidirectional, single reverse and double reverse. Our findings indicate that these reverse movements occur in response to magnetic field gradients. In addition, using a permanent magnet we found further evidence that supports this claim. Motile AMB-1 cells swim away from the north and south poles of a permanent magnet when the magnet is positioned less than ∼30 mm from the droplet of cells. All together, these results indicate previously unknown response capabilities arising from the magnetic sensing systems of AMB-1 cells. These responses could enable them to cope with magnetic disturbances that could in turn potentially inhibit their efficient search for nutrients.

3D bio-etching of a complex composite-like embryonic tissue.

Morphogenesis involves a complex series of cell signaling, migration and differentiation events that are coordinated as tissues self-assemble during embryonic development. Collective cell movements such as those that occur during morphogenesis have typically been studied in 2D with single layers of cultured cells adhering to rigid substrates such as glass or plastic. In vivo, the intricacies of the 3D microenvironment and complex 3D responses are pivotal in the formation of functional tissues. To study such processes as collective cell movements within 3D multilayered tissues, we developed a microfluidic technique capable of producing complex 3D laminar multicellular structures. We call this technique "3D tissue-etching" because it is analogous to techniques used in the microelectromechanics (MEMS) field where complex 3D structures are built by successively removing material from a monolithic solid through subtractive manufacturing. We use a custom-designed microfluidic control system to deliver a range of tissue etching reagents (detergents, chelators, proteases, etc.) to specific regions of multilayered tissues. These tissues were previously isolated by microsurgical excision from embryos of the African claw-toed frog, Xenopus laevis. The ability to shape the 3D form of multicellular tissues and to control 3D stimulation will have a high impact on tissue engineering and regeneration applications in bioengineering and medicine as well as provide significant improvements in the synthesis of highly complex 3D integrated multicellular biosystems.

Controlling magnetotactic bacteria through an integrated nanofabricated metallic island and optical microscope approach.

Herein, we demonstrate the control of magnetotactic bacteria through the application of magnetic field gradients with real-time visualization. We accomplish this control by integrating a pair of macroscale Helmholtz coils and lithographically fabricated nanoscale islands composed of permalloy (Ni80Fe20). This system enabled us to guide and steer amphitrichous Magnetospirillum magneticum strain AMB-1 to specific location via magnetic islands. The geometries of the islands allowed us to have control over the specific magnetic field gradients on the bacteria. We estimate that magnetotactic bacteria located less than 1 µm from the edge of a diamond shaped island experience a maximum force of approximately 34 pN, which engages the bacteria without trapping them. Our system could be useful for a variety of applications including magnetic fabrication, self-assembly, and probing the sensing apparatus of magnetotactic bacteria.

Detection of dynamic spatiotemporal response to periodic chemical stimulation in a Xenopus embryonic tissue.

Embryonic development is guided by a complex and integrated set of stimuli that results in collective system-wide organization that is both time and space regulated. These regulatory interactions result in the emergence of highly functional units, which are correlated to frequency-modulated stimulation profiles. We have determined the dynamic response of vertebrate embryonic tissues to highly controlled, time-varying localized chemical stimulation using a microfluidic system with feedback control. Our approach has enabled localized spatiotemporal manipulation of the steroid hormone dexamethasone (DEX) in Animal Cap (AC) tissues isolated from gastrulating Xenopus embryos. Using this approach we investigated cell-scale responses to precisely controlled stimulation by tracking the redistribution of a GFP-tagged DEX-reporter constructed from the human glucocorticoid receptor (GR). We exposed defined regions of a single AC explant to different stimulation conditions--continuous stimulation, periodic stimulation, and no stimulation. We observed collective behavior of the GR transport into the nucleus was first-order. Furthermore, the dynamic response was well-modeled by a first-order differential equation with a single time derivative. The model predicted that responses to periodic stimulations closely matched the results of the frequency-based experiments. We find that stimulation with localized bursts versus continuous stimulation can result in highly distinct responses. This finding is critical as controlled space and time exposure to growth factors is a hallmark of complex processes in embryonic development. These complex responses to cellular signaling and transport machinery were similar to emergent behaviors in other complex systems, suggesting that even within a complex embryonic tissue, the overall system can converge toward a predictive first-order response.

Dynamic control of 3D chemical profiles with a single 2D microfluidic platform.

Dynamic control of three-dimensional (3D) chemical patterns with both high precision and high speed is important in a range of applications from chemical synthesis, flow cytometry, and multi-scale biological manipulation approaches. A central challenge in controlling 3D chemical patterns is the inability to create rapidly tunable 3D profiles with simple and direct approaches that avoid complicated microfabrication. Here, we present the ability to rapidly and precisely create 3D chemical patterns using a single two-dimensional (2D) microfluidic platform. We are not only able to create these 3D patterns, but can rapidly switch from one mode to another (e.g. from a focused to a defocused pattern in less than 1 second) via simple changes in inlet pressures. A feedback control scheme with a pressure modulation mechanism controls the pressure changes. In addition to experiments, we conducted computational simulations for guiding the optimum design of the channels as well as revealing the sensitivity of the patterns to the channel dimensions; these simulations have high experimental correlations. We also show that microvortices play an important role in creating these tunable 3D patterns in this microfluidic platform. We quantitatively determine the degrees of the focused patterns in 2D cross-sections using a focus index with a 2D Gaussian function. Our integrated approach combining feedback control with simple microfluidics will be useful for researchers in diverse disciplines including chemistry, engineering, physics, and biology.

Probing cellular dynamics with a chemical signal generator.

Observations of material and cellular systems in response to time-varying chemical stimuli can aid the analysis of dynamic processes. We describe a microfluidic "chemical signal generator," a technique to apply continuously varying chemical concentration waveforms to arbitrary locations in a microfluidic channel through feedback control of the interface between parallel laminar (co-flowing) streams. As the flow rates of the streams are adjusted, the channel walls are exposed to a chemical environment that shifts between the individual streams. This approach can be used to probe the dynamic behavior of objects or substances adherent to the interior of the channel. To demonstrate the technique, we exposed live fibroblast cells to ionomycin, a membrane-permeable calcium ionophore, while assaying cytosolic calcium concentration. Through the manipulation of the laminar flow interface, we exposed the cells' endogenous calcium handling machinery to spatially-contained discrete and oscillatory intracellular disturbances, which were observed to elicit a regulatory response. The spatiotemporal precision of the generated signals opens avenues to previously unapproachable areas for potential investigation of cell signaling and material behavior.