Name: a multilayer microfluidic platform for the conduction of prolonged cell-free gene expression
Uploaded: 2019-10-06T12:30:00.000+00:00
Duration: 11 min 23 s
Description: Watch this Scientific Journal Video about A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression at JoVE.com

This work was supported by the European Research Council, ERC (project n. 677313 BioCircuit) an NWO-VIDI grant from the Netherlands Organization for Scientific Research (NWO, 723.016.003), funding from the Ministry of Education, Culture and Science (Gravity programs, 024.001.035 &#38; 024.003.013), the Human Frontier Science Program Grant RGP0032/2015, the European Research Council under the European Union&#8217;s Horizon 2020 research and innovation program Grant 723106, and a Swiss National Science Foundation Grant 200021_182019.

The authors declare that they have no competing financial interests.

A PDMS-based multilayer microfluidic device has been presented, and its capability to sustain IVTT reactions for prolonged periods of time has been demonstrated. Although well-suited for this specific example, this technology can conceivably be used for numerous other applications. The additional control over fluid flow - paired with the ability to continuously replenish reaction reagents whilst removing (by)products - is ideal for continuous synthesis reactions, the investigation of various dynamic behaviors, and the simultaneous conduction of multiple variations of a single reaction.
Despite the relatively straightforward fabrication process of PDMS based devices, the use thereof requires an extensive hardware setup. Comprising valve arrays, pressure regulators, pressure pumps, incubators, and cooling units, the transition from fabrication to use is not elementary, and requires a significant initial investment. In addition, the ability to consistently set-up and perform successful experiments with these devices requires a significant time-investment; a point which this manuscript aims to address. However, once in place, the entire setup can be modified for a range of purposes. Furthermore, the hardware setup comprises numerous modular elements, each of which can be expanded to allow more complex microfluidic device designs to be employed. Additionally, the modular design enables the replacement of hardware components by similarly functioning alternatives, such that users are not limited to the specific setup described here48,49.
Variability between individual devices, and in the external conditions (such as pressure fluctuations) can result in inaccuracies when performing experiments using these devices. To address this issue, a calibration of the system should be performed prior to each experiment, providing a unique Refresh Ratio for each of the reactors. Whilst the calibration addresses the device-to-device and experiment-to-experiment variations, it is a time consuming process and not flawless. Fluids with differing viscosities will not flow with the same rate when exposed to identical pressure, and as such performing the calibration with multiple reagents may not yield identical Refresh Ratios. This effect is attenuated by utilising three control channels to peristaltically pump the reagents into the microfluidic device, as opposed to regulating the flow by varying the supplied pressure only. As a last resort in cases where the disparity in viscosity is very large, a unique Refresh Ratio can be implemented for each individual reagent by performing multiple calibration experiments.
The use of a peristaltic pump to inject reagents into the microfluidic device attenuates the effects of using solutions with varying viscosities, however it also creates a secondary problem. Using discrete steps to pump fluids into the microfluidic device, means that the resolution of injections into a single reactor, is limited by the volume injected when performing a single pump cycle. Within our research this value - determined during the calibration - is approximately equal to 1%, indicating that a single pump cycle displaces approximately 1% of the reactor volume (about 0.1 nL). As such, displacing 30% of the reactor volume requires the execution of 30 pump cycles, with 23 pump cycles of the IVTT reaction solution being added, and only 7 pump cycles of DNA or ultrapure water being added. Although sufficient for our research, alternative experimental protocols may encounter problems when attempting to add larger numbers of unique reagents, use a lower Refresh Fraction, or add smaller volumes of a single reagent to a reactor. In such cases, the microfluidic device design can be adapted to provide reactors with a larger volume. An example of such is reported in Niederholtmeyer et al.36.
Crucially, the device outlined within this manuscript allows reactions to be sustained for prolonged durations resulting in steady-state transcription and translation rates. By periodically injecting new reagents into the reactors - and removing reaction (by)products - the reactions are sustained and complex dynamic behaviors can be monitored. In this way, a platform has been created that - to some extent - mimics the cellular environment. Furthermore, this platform enables the exploration of the system dynamics, by adapting the period between injections and the specific composition of the injections. As a result, these multilayer microfluidic devices are a powerful tool for the characterisation and optimisation of novel synthetic networks which display complex dynamic behavior.

Cells are able to sense and respond to their environment using complex dynamic regulatory networks1,2. The field of synthetic biology utilizes our knowledge of the naturally occurring components comprising these networks to engineer biological systems that can expand the functionality of cells3,4. Conversely, it is also possible to further our understanding of the natural networks governing life by designing simplified, synthetic analogues of existing circuits or by forward-engineering biological systems which exhibit naturally occurring behaviors. The de novo engineering of such biological systems is performed in a bottom-up fashion where novel genetic circuits or signalling pathways are engineered in a rational manner, using well-defined parts5,6. Combining the rational design of networks with the design of biologically relevant systems enables the in-depth characterization and study of biological regulatory systems with various levels of abstraction7.
The pioneering works of Elowitz and Leibler8 and Gardner et al.9 were the first to demonstrate the successful introduction of synthetic genetic networks into cellular hosts. In the following decade, numerous researchers have continued to build on these initial successes despite the emergence of several limitations regarding the introduction of synthetic circuits into cells7,10,11,12. Ideally, the introduction of synthetic circuits into cellular hosts should occur in a modular fashion. Unfortunately, the complexity of the cellular environment makes this particularly challenging, with the function of many parts and networks being highly context dependent12,13,14. As a result, networks often experience undesired interactions with native host componentry which can affect the function of the synthetic circuit. Similarly, components of the exogenous network can inhibit host processes, compete for shared resources within the host, and influence growth kinetics15,16,17. Consequently, in order to rationally design and predict the behavior of synthetic networks in an in vivo environment, a comprehensive model of all host and circuit-specific dynamics is required18.
A viable alternative to the use of cellular hosts for the characterization of synthetic networks is the application of in vitro transcription and translation (IVTT) technologies. Acting as a testbed for synthetic networks, reactions are performed in solutions comprising all the components required to enable gene expression19,20,21. In this manner, a biologically relevant, albeit artificial, environment is created within which synthetic networks can be tested22,23,24,25,26,27,28. A major advantage of using IVTT solutions is the ability to perform reactions under user-specified conditions, with researchers able to tune the precise composition of each reaction2. Furthermore, the cell-free approach enables high-throughput testing of synthetic networks, since it removes the need to perform time-consuming cellular cloning steps. As a result, the duration of successive design - build - test cycles is significantly reduced29,30,31,32. The design cycle can be further accelerated by utilizing cell-free cloning techniques such as the Gibson assembly to rapidly engineer novel networks, and by constructing networks from linear DNA templates which - unlike the plasmids required for in vivo testing - can be amplified via polymerase chain reactions (PCR)33,34.
Batch reactions are the simplest method by which IVTT reactions can be performed, requiring a single reaction vessel wherein all of the reaction components are combined35. Such reactions are sufficient for protein expression and basic circuit testing yet prove insufficient when attempting to study the long-term dynamic behavior of a network. Over the course of a batch reaction, reagents are either depleted or undergo degradation resulting in a continuous decrease of the transcription and translation rates. Furthermore, as reactions progress by-products accumulate that can interfere with - or completely inhibit - the correct functioning of the network. Ultimately, the use of batch reactors limits the dynamic behavior which can be observed, with negative regulation being particularly challenging to implement5,36.
The versatility of IVTT systems enables multiple alternative methods by which prolonged IVTT reactions can be performed, ranging from continuous flow to droplet based methods as well as simpler dialysis approaches2,30,37,38,39,40. The application of microfluidic devices offers users increased control over their reactions whilst increasing the throughput and minimizing costs35,41,42, with each specific approach having its own advantages. The use of continuous flow can be easily optimized for increasing expression yields however, the inability to effectively remove specific reaction products makes the study of dynamic behavior non-trivial39. Whilst employing droplet based microfluidic systems allows high-throughput screening of novel networks, the difficulty of supplying fresh reagents to the reaction results in the droplets resembling small volume batch reactions43. Dialysis based reactors allow the introduction of fresh reagents as well as the removal of some reaction products however, RNA molecules and larger proteins accumulate within the reactor, being too large to diffuse through the membrane pores. Furthermore, large volumes of reagents are required to sustain these reactions for prolonged periods30,44. In 2013, Maerkl et al. presented a multi-layered microfluidic device designed specifically for conducting prolonged IVTT reactions36,45. The use of multi-layered microfluidic devices permits direct control over fluid flow, allowing for the redirection of flow as well as the isolation of fluid in specific regions of the device46,47. These isolated regions can function as independent nanoliter-scale reaction chambers wherein IVTT reactions can be performed. Over the course of a single IVTT reaction, periodic injections of fresh reagents into the reactor are used to replenish IVTT components and DNA templates. Simultaneously, an equal volume of the old reaction solution is displaced, removing reaction products. In this manner, an out-of-equilibrium environment is maintained where the basal transcription and translation rates remain in steady-state, prolonging the lifetime of IVTT reactions and allowing rich dynamic behaviors to occur. By applying this approach, researchers are able to investigate the kinetic rates of the individual processes occurring within a specific circuit, aiding in the forward-engineering of novel genetic networks. For instance, Niederholtmeyer et al. implemented this approach to characterize various elements of a genetic ring oscillator, determining the kinetic rates thereof36. In further studies, Yelleswarapu et al. showed that the kinetic rates of sigma factor 28 (&#963;28) determined under batch conditions were insufficient to describe the behavior of a &#963;28-based oscillator, and that the addition of flow-based data improved model predictions of the network behavior22.
The goal of this manuscript is to present a complete protocol for the fabrication of multilayer microfluidic devices capable of performing long-term IVTT reactions. In addition, this manuscript will describe all of the hardware and software required to perform prolonged IVTT reactions. The actuation of the microfluidic device - necessary to control the flow of fluids therein - is achieved using a series of pneumatic valves which connect directly to the microfluidic devices via lengths of tubing. In turn, the pneumatic valves are controlled via a custom-built virtual control interface. Fluid flow within the microfluidic devices is achieved using continuous pressure which is provided by a commercially available pressure regulation system. IVTT reactions are typically performed between 29 &#176;C and 37 &#176;C and a microscope incubator is used to regulate the temperature during reactions. However, the functionality of the IVTT mixture gradually degrades when stored above 4 &#176;C. As such, this manuscript will expand on the off-chip cooling system used to cool the IVTT mixture prior to injection into the microfluidic device. In conclusion, this manuscript provides a comprehensive overview of the procedures required to successfully perform prolonged IVTT reactions using a microfluidic flow reactor such that other researchers will be able to replicate this technology with relative ease.

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Acetone</td><td>VWR</td><td>20063.365</td><td></td></tr><tr><td>AZ 40 XT</td><td>Merck KGaA (Darmstadt, Germany)</td><td>-</td><td>Positive Photoresist</td></tr><tr><td>AZ 726 MIF Developer</td><td>Merck KGaA (Darmstadt, Germany)</td><td>-</td><td>Developer Positive Photoresist</td></tr><tr><td>Isopropanol</td><td>Merck KGaA (Darmstadt, Germany)</td><td>109634</td><td></td></tr><tr><td>Microscope slides</td><td>VWR</td><td>ECN 631-1550</td><td></td></tr><tr><td>mr Dev 600</td><td>Microresist Technology GmbH (Berlin, Germany)</td><td>-</td><td>Developer Negative Photoresist</td></tr><tr><td>Silicon Free Heat Sink Grease</td><td>Circuit Works</td><td>CW7270</td><td>Thermal Compound</td></tr><tr><td>Silicon wafers</td><td>Silicon Materials</td><td>-</td><td>&amp;lt;1-0-0&gt; orientation, 100 mm diameter, 525 &amp;micro;m thickness</td></tr><tr><td>SU-8 3050</td><td>Microchem Corp. (Newton, MA)</td><td>-</td><td>Negative Photoresist</td></tr><tr><td>Sylgard 184 Elastomer Kit (PDMS)</td><td>The Dow Chemical Company</td><td>01317318</td><td></td></tr><tr><td>trichloro(1H,1H,2H,2H-perfluorooctyl)silane</td><td>Sigma-Aldrich</td><td>448931-10G</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>4 channel digital input/output module</td><td>WAGO Kontakttechnik GmbH</td><td>750-504</td><td>8x</td></tr><tr><td>Camera lens</td><td>The Imaging Source</td><td>-</td><td></td></tr><tr><td>Compression fitting</td><td>Koolance, Inc.</td><td>FIT-V06X10</td><td>Fitting for tubing with 6mm ID and 10mm OD. 4x</td></tr><tr><td>Controller end module</td><td>WAGO Kontakttechnik GmbH</td><td>750-600</td><td></td></tr><tr><td>Device connecting tubing</td><td>Saint-Gobain Performance Plastics</td><td>AAD04103</td><td>0.02&quot; ID, 0.06&quot; OD, Tygon Tubing (ND-100-80)</td></tr><tr><td>Device connector pins</td><td>Unimed SA (Lausanne, Switserland)</td><td>200.010-A</td><td>AISI 304 tubing, 0.35mm ID, 0.65mm OD, 8mm L</td></tr><tr><td>Ethernet Controller</td><td>WAGO Kontakttechnik GmbH</td><td>750-881</td><td></td></tr><tr><td>Female bus connector</td><td>Encitech</td><td>DTCK15-DBS-K</td><td>15 pole female bus connector</td></tr><tr><td>Fluid reservoirs</td><td>Fluigent</td><td>Fluiwell-4C</td><td></td></tr><tr><td>Fluigent pressure system</td><td>Fluigent</td><td>MFCS-EZ</td><td>0 - 345 mbar</td></tr><tr><td>Hg short arc lamp</td><td>Advanced Radiation Corporation</td><td>-</td><td>350W</td></tr><tr><td>Hot plate</td><td>Torrey Pines Scientific</td><td>HP61</td><td></td></tr><tr><td>Inverted microscope</td><td>Nikon Instruments</td><td>Eclipse Ti-E</td><td></td></tr><tr><td>LabVIEW Software</td><td>de Greef Lab, Eindhoven University of Technology</td><td>https://github.com/tfadgreef/Microfluidic-Device-Control-Software</td><td></td></tr><tr><td>Liquid coolant</td><td>Koolance, Inc.</td><td>LIQ-705CL-B</td><td></td></tr><tr><td>Luer stubs</td><td>Instech Laboratories, Inc.</td><td>LS23</td><td></td></tr><tr><td>Male Luer to barb connectors</td><td>Cole Parmer</td><td>45505-32</td><td>3/32&quot; ID</td></tr><tr><td>Matlab Software</td><td>de Greef Lab, Eindhoven University of Technology</td><td>https://github.com/tfadgreef/Microfluidic-Device-Control-Software</td><td></td></tr><tr><td>Microcamera</td><td>The Imaging Source</td><td>DMK 42AUC03</td><td></td></tr><tr><td>Microscope camera</td><td>Hamamatsu Photonics</td><td>OrcaFlash4.0 V2 (C11440-22CU)</td><td></td></tr><tr><td>Orbital shaker</td><td>Cole Parmer</td><td>EW-513000-05</td><td></td></tr><tr><td>Oven</td><td>Thermo Scientific</td><td>Heraeus T6P 50045757</td><td></td></tr><tr><td>Oxygen plasma asher</td><td>Quorum Technologies</td><td>K1050X</td><td></td></tr><tr><td>PDMS puncher</td><td>SYNEO</td><td>Accu-Pucnh MP10</td><td></td></tr><tr><td>PEEK tubing</td><td>Trajan</td><td>1301005001-5F</td><td>0.005&quot; ID, 1/32&quot; OD, Red</td></tr><tr><td>Peltier element</td><td>European Thermodynamics</td><td>APH-127-10-25-S</td><td></td></tr><tr><td>Peltier temperature controller</td><td>Warner Instruments</td><td>CL-100</td><td></td></tr><tr><td>Photomask</td><td>CAD/Art Services, Inc.</td><td>-</td><td></td></tr><tr><td>Photomask Design</td><td>Maerkl Lab, EPFL</td><td>https://zenodo.org/record/886937#.XBzpA8-2nOQ</td><td></td></tr><tr><td>Pneumatic valve array</td><td>FESTO</td><td>-</td><td>1x 22 valve array and 1x 8 valve array, Normally closed valves.</td></tr><tr><td>Power adapter</td><td>Koolance, Inc.</td><td>ADT-EX004S</td><td>110/220V AC Power Adapter</td></tr><tr><td>PTFE tubing</td><td>Cole Parmer</td><td>06417-21</td><td>#24 AWG Thin Wall PTFE</td></tr><tr><td>Punching pin</td><td>SYNEO</td><td>CR0320245N21R4</td><td>OD: 0.032&quot; (0.8128 mm), ID: 0.024&quot; (0.6090 mm)</td></tr><tr><td>PVC Tubing</td><td>Koolance, Inc.</td><td>HOS-06CL</td><td>6 mm ID, 10 mm OD</td></tr><tr><td>Single edge blades</td><td>GEM Scientific</td><td>-</td><td></td></tr><tr><td>Soft tubing</td><td>Fluigent</td><td>-</td><td>Supplied with fluid reservoirs. (1 mm ID, 3mm OD)</td></tr><tr><td>Spin coater</td><td>Laurell Technologies Corporation</td><td>WS-650MZ-23NPPB</td><td></td></tr><tr><td>Stereo microscope</td><td>Olympus Corporation</td><td>SZ61</td><td></td></tr><tr><td>Thermistor cable</td><td>Warner Instruments</td><td>TA-29</td><td>Cable with bead thermistor</td></tr><tr><td>UV exposure system</td><td>ABM, USA</td><td>-</td><td>Near UV Exposure System, 350W</td></tr><tr><td>Vacuum pump</td><td>Vacuumbrand GmbH</td><td>MD1C</td><td></td></tr><tr><td>Water cooled cold plate block</td><td>Koolance, Inc.</td><td>PLT-UN40F</td><td></td></tr><tr><td>Water cooler</td><td>Koolance, Inc.</td><td>EX2-755</td><td></td></tr><tr><td>Weighing scales</td><td>Sartorius</td><td>M-prove</td><td></td></tr></tbody></table>

a multilayer microfluidic platform for the conduction of prolonged cell-free gene expression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

Watch this Scientific Journal Video about A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression at JoVE.com

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...

a-multilayer-microfluidic-platform-for-conduction-prolonged-cell-free

Research

JoVE Journal

Bioengineering

10.1K Views.  Eindhoven University of Technology. Prototyping synthetische gennetwerken wordt sterk verbeterd door het gebruik van celvrije genexpressiesystemen. Ons protocol beschrijft het fabricageproces voor meerlaagse microfluïde stroomreactor en laat zien dat een dergelijk apparaat kan worden gebruikt voor langdurige celvrije expressie van GFP. Celvrije eiwitexpressies in combinatie met microfluïde stroomreactoren vormen het rapid prototyping platform voor het ontwerp van synthetische biologische apparaten.Deze hele setup is ze...

Video: A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (&ge;105) in milliliter-scale volumes (&ge;0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 &mu;l). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

We have developed an automated cell culture and interrogation platform for micro-scale cell stimulation experiments. The platform offers simple, versatile, and precise control in cultivating and stimulating small populations of cells, and recovering lysates for molecular analyses. The platform is well suited to studies that use precious cells and/or reagents.

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments,&#160;as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier,&#160;more quantifiable extracellular relay of signals to the cells. 
For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media circulation.

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

Sealable Femtoliter Chamber Arrays for Cell-free Biology

Cell-free systems provide a flexible platform for probing specific networks of biological reactions isolated from the complex resource sharing (e.g., global gene expression, cell division) encountered within living cells. However, such systems, used in conventional macro-scale bulk reactors, often fail to exhibit the dynamic behaviors and efficiencies characteristic of their living micro-scale counterparts. Understanding the impact of internal cell structure and scale on reaction dynamics is crucial to understanding complex gene networks. Here we report a microfabricated device that confines cell-free reactions in cellular scale volumes while allowing flexible characterization of the enclosed molecular system. This multilayered poly(dimethylsiloxane) (PDMS) device contains femtoliter-scale reaction chambers on an elastomeric membrane which can be actuated (open and closed). When actuated, the chambers confine Cell-Free Protein Synthesis (CFPS) reactions expressing a fluorescent protein, allowing for the visualization of the reaction kinetics over time using time-lapse fluorescent microscopy. Here we demonstrate how this device may be used to measure the noise structure of CFPS reactions in a manner that is directly analogous to those used to characterize cellular systems, thereby enabling the use of noise biology techniques used in cellular systems to characterize CFPS gene circuits and their interactions with the cell-free environment.

A microfabricated device with sealable femtoliter-volume reaction chambers is described. This report includes a protocol for sealing cell-free protein synthesis reactants inside these chambers for the purpose of understanding the role of crowding and confinement in gene expression.

Cell-free systems provide a flexible platform for probing specific networks of biological reactions isolated from the complex resource sharing (e.g., ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...

Functional Surface-immobilization of Genes Using Multistep Strand Displacement Lithography

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic bioreactor system. In contrast to other approaches towards artificial cellular systems, such a setup allows for a continuous supply with gene expression reagents and simultaneous draining of waste products. This facilitates the implementation of cell-free gene expression processes over extended periods of time, which is important for the realization of dynamic gene regulatory feedback systems. Here we provide a detailed protocol for the fabrication of genetic biochips using a simple-to-use lithographic technique based on DNA strand displacement reactions, which exclusively uses commercially available components. We also provide a protocol on the integration of compartmentalized genes with a polydimethylsiloxane (PDMS)-based microfluidic system. Furthermore, we show that the system is compatible with total internal reflection fluorescence (TIRF) microscopy, which can be used for the direct observation of molecular interactions between DNA and molecules contained in the expression mix.

We describe a simple lithographic procedure for the immobilization of gene-length DNA molecules on a surface, which can be used to perform cell-free gene expression experiments on biochips.

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic ...

Generation of Heterogeneous Drug Gradients Across Cancer Populations on a Microfluidic Evolution Accelerator for Real-Time Observation

Conventional cell culture remains the most frequently used preclinical model, despite its proven limited ability to predict clinical results in cancer. Microfluidic cancer-on-chip models have been proposed to bridge the gap between the oversimplified conventional 2D cultures and more complicated animal models, which have limited ability to produce reliable and reproducible quantitative results. Here, we present a microfluidic cancer-on-chip model that reproduces key components of a complex tumor microenvironment in a comprehensive manner, yet is simple enough to provide robust quantitative descriptions of cancer dynamics. This microfluidic cancer-on-chip model, the &#34;Evolution Accelerator,&#34; breaks down a large population of cancer cells into an interconnected array of tumor microenvironments while generating a heterogeneous chemotherapeutic stress landscape. The progression and the evolutionary dynamics of cancer in response to drug gradient can be monitored for weeks in real time, and numerous downstream experiments can be performed complementary to the time-lapse images taken through the course of the experiments.

We present a microfluidic cancer-on-chip model, the &#34;Evolution Accelerator&#34; technology, which provides a controllable platform for long-term real-time quantitative studies of cancer dynamics within well-defined environmental conditions at the single-cell level. This technology is expected to work as an in vitro model for fundamental research or pre-clinical drug development.

Conventional cell culture remains the most frequently used preclinical model, despite its proven limited ability to predict clinical results in cancer. ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) 1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells 3, and cell-free in vitro translation systems 2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins 5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.
Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein 5,10.
In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

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