Authors acknowledge the technical support from Zhifeng Cheng of Chansn Instrument (China) LTD. This work was supported by grants (National Natural Science Foundation of China,51927804).

1. Microfluidic chips design
<ol>
	<li>Design the microfluidic multiplexer consisting of 18 inlets, each of which is controlled by an individual valve and a peristaltic pump. To increase the liquid volume driven by per pumping cycle, have the peristaltic pump be composed of 3 control channels, which was purposely widened to 200 &#181;m, and 10 connected flow lines.</li>
	<li>Design the shear-free culture chamber. Replication of the 2-level culture unit is composed by a lower cell culture chamber (400 &#181;m x 400 &#181;m x 150 &#181;m) and a higher buffer layer (400 &#181;m x 400 &#181;m x 75 &#181;m), which prevents unwanted shear stress on cells during medium exchange (Figure 1).</li>
	<li>Design high-throughput features. Duplicate the culture unit to form a 30 x 50 matrix layout, occupying an area of approximately 7 cm by 5 cm in size.</li>
</ol>
2. Chip fabrication and operation
<ol>
	<li>Fabrication of the replica molding using UV lithography 
	NOTE: The replica molding was fabricated on silicon wafer according to the standard photolithography protocol8.
	<ol>
		<li>Fabrication the channel structures
		<ol>
			<li>Spinning photoresist: Spin coat 5 mL of the SU-8 3025 negative photoresist on a silicon wafer at 500 rpm for 10 s and 3000 rpm for 30 s.</li>
			<li>Soft bake: Put the wafer on a hotplate at 65 &#176;C for 2 min and then 95 &#176;C for 10 min, Cool it down to room temperature.</li>
			<li>Alignment and curing: Fix the wafer and mask on the holder of the aligner and turn on the light source for 18 s to cure the exposed photoresist.</li>
			<li>Pre post exposure bake: Ramp up the wafer to 95&#176;C at 110&#176;C/h from room temperature and keep it at least 40 min till removing.</li>
			<li>Develop: Dip the wafer in the developing solution (SU-8 developer) and agitate it for 2.5 min to wash off redundant photoresist and get the 25 &#181;m-high channel structure.</li>
			<li>Hard bake: Cover the wafer with glass Petri dish and bake at 65 &#176;C for 2 min, then ramp up to 160&#176;C at 120 &#176;C/h and keep it for 3 hours.</li>
		</ol>
		</li>
		<li>Fabrication of the cell culture chamber with the buffer layer
		<ol>
			<li>Use the parameters of step 2.1.1.1 to spin coat 7 mL of the SU-8 3075 negative photoresist on the above wafer.</li>
			<li>Soft bake (described in step 2.1.1.2) the wafer and change the mask to align well with markers on the wafer. Then turn on the light source for 24 s to cure the exposed photoresist.</li>
			<li>Dip the wafer to the developing solution (SU-8 developer) and agitate it for 4 minutes and 40 seconds to wash off redundant photoresist and get a 75 &#181;m-high layer structure that around the 25 &#181;m-high channel structure fabricated before. Hard bake (described in step 2.1.1.6) the wafer to make the complex structure stronger.</li>
			<li>Repeat steps 2.1.2.1-2.1.2.3 to fabricate a 75 &#181;m-high chamber structure that stack on the layer structure.</li>
		</ol>
		</li>
		<li>Fabrication of the valve structures
		<ol>
			<li>Using the parameter of step 2.1.1.1 to spin coat 5 mL of the AZ 50x positive photoresist on the above wafer.</li>
			<li>Soft bake (described in step 2.1.1.2) the wafer and make the mask aligned well with markers on the wafer, then keep the light source on for 20 s and off immediately for 30 s. Repeat the light on/ off procedure in 5 circles to cure the exposed photoresist.</li>
			<li>Dip the wafer to matched developer and agitate it for about 8 min to wash off redundant photoresist and get the round-shaped valves that overlap with the control channels to ensure good connection. Hard bake (described in step 2.1.1.6) the patterned wafer to make the whole model stronger.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Microfluidic chip production using soft lithography
	<ol>
		<li>Treat the patterned and blank silicon wafers with rimethylchlorosilane for 15 min.</li>
		<li>Prepare 3 portions of PDMS gel (10:1 of monomer/catalyst ratio) corresponding to 50 g of flow layer, 20 g of control layer 2, and 20 g of membrane, respectively.</li>
		<li>Cast 50 g of PDMS gel on the patterned silicon wafer, and de-gas them for 1-2 hours in the vacuum chamber at -0.85 MPa to copy the flow layer.</li>
		<li>Degas the 2 portions of 20 g of PDMS and spin it on the patterned wafer and a blank silicon wafer at 2000-2800 rpm for 30 s as to prepare a control layer and membrane layer.</li>
		<li>Put the PDMS-covered wafers into ventilating oven for 60 min at 80 &#176;C for incubation.</li>
		<li>Align and bond the different layers together through customized optical device (zoom in 100x) and plasma etching machine. Then keep it in a ventilating oven for 2 hours at 80 &#176;C to enhance the bonding of the chip.</li>
		<li>Punch the inlet holes on the chip, and then bond it onto a PDMS-coated coverslip and cured for at least 12 hours at 80 &#176;C before use.</li>
	</ol>
	</li>
	<li>Chip operation
	<ol>
		<li>Connect miniature pneumatic solenoid valves to the control layer of the chip, and open the customized MATLAB graphical user interface8 to link and control the switch.</li>
		<li>Set the closing pressures of push-up PDMS membrane valves to 25 psi.</li>
		<li>Deliver dynamically changing combinatorial/sequential inputs to designated chambers (Figure 2d) by timely on-off of the valves.</li>
	</ol>
	</li>
</ol>
3. Generation of dynamic inputs in cellular microenvironments
<ol>
	<li>Chip treatment and cell loading
	<ol>
		<li>Maintain the standard culture conditions (37 &#176;C, 5% CO2) on the microscope for at least 5 hours.</li>
		<li>Fill the chip with coating medium (i.e., mentioned in the NOTE) and incubate it at the standard culture conditions (described in step 3.1.1) for at least one hour.</li>
		<li>Flush the chip by phosphate-buffered saline (PBS) or cell culture medium (Dulbecco&#39;s Modified Eagle&#39;s medium, DMEM) to build a healthy culture environment.</li>
		<li>Harvest cells at 80% confluency, and resuspend the cells using culture media (DMEM) at a density of ~ 106/mL. Then load cells into the chip by pressurizing the cell-containing solution. 
		NOTE: Culturing different cell lines on chip requires corresponding coating medium to treat the cell culture chamber. Typically, for experiments on 3T3 fibroblast and adherent culture of hen all valves controlling the culture chambers are open, cells flow into all culture chambers within the same column.</li>
	</ol>
	</li>
	<li>Setup for high throughput live-cell imaging 
	NOTE: For image acquisition, an inverted microscope with an automated translational stage and a digital complementary metal-oxide semiconductor (CMOS) camera were used. The stage and image acquisition were controlled via the customized software.
	<ol>
		<li>Visualize the matrix of culture chambers using 10x objective lens in bright field to affirm and define the location coordinates of each chamber of the 30 by 50 chamber matrix.</li>
		<li>Transform the objective lens to the 20x or 40x, then select the location coordinates of the desired chamber and the translational stage moves to the assigned position after confirmation. Fine tune the x, y, z focal plane to get an optimal image.</li>
		<li>Optimal the light intensity, expose time, and other imaged parameters were determined individually for each channel (i.e., bright-field and fluorescence imaging).</li>
		<li>Set the interval and duration of imaging cycle, save the path and then start imaging.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Various microfluidic devices have been developed to perform multiplexed and complex experiments17,18,19,20. For example, microwells made of an array of topological recesses can trap individual cells without the use of external force, showing advantageous characters including small sample size, parallelization, lower material cost, faster response, high sensitivity21...

High throughput techniques are crucial for biomedical and clinical studies. By parallelly conducting millions of chemical, genetic, or live cell and organoid tests, researchers can rapidly identify genes that modulate a bio-molecular pathway, and customize sequential drug input to one's specific needs. Robotics1 and microfluidic chips in combination with a device control program allow complex experimental procedures to be automated, covering cell/tissue manipulation, liquid handling, imaging, and data processing/control2,3. Therefore, hundreds and thousands of experimental conditions can be maintained on a single chip, according to the desired throughput4,5. 
In this protocol, we described the design and fabrication procedure of a microfluidic device, which consists of 1500 culture units, an array of enhanced peristaltic pumps and on-site mixing modulus. The 2-level cell culture chamber prevents unnecessary shear during medium exchange, which ensures an undisturbed culture environment for long-term live cell imaging. The studies demonstrate that the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery. Moreover, the advanced features of the microfluidic chip allow automated reconstitution of highly complex and dynamic microenvironmental conditions in vivo, like the everchanging cytokines and ligands compositions6,7, the completion of which takes months for conventional platforms like 96-well plate.

<table><tbody><tr><td>2713 Loker Avenue West</td><td>Torrey pines scientific</td><td></td><td></td></tr><tr><td>AZ-50X</td><td>AZ Electronic Materials, Luxembourg</td><td></td><td></td></tr><tr><td>Chlorotrimethylsilane(TMCS) 92360-25mL</td><td>Sigma</td><td></td><td></td></tr><tr><td>CO2 Incubator HP151</td><td>Heal Force</td><td></td><td></td></tr><tr><td>Desktop Hole Puncher for PDMS chips WH-CF-14</td><td>Suzhou Wenhao Microfluidic Technology Co., Ltd.</td><td></td><td></td></tr><tr><td>DMEM(L-glutamine, High Glucose, henol Red)</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>Electronic Balance UTP-313 Max:600g, e:0.1g, d:0.01g</td><td>Shanghai Hochoice Apparatus Manufacturer Co.,LTD.</td><td></td><td></td></tr><tr><td>FBS</td><td>Sigma</td><td></td><td></td></tr><tr><td>Fibronection 0.25 mg/mL</td><td>Millipore, Austria</td><td></td><td></td></tr><tr><td>Glutamax 100x</td><td>Gibco</td><td></td><td></td></tr><tr><td>Heating Incubator BGG-9240A</td><td>Shanghai bluepard instruments Co.,Ltd.</td><td></td><td></td></tr><tr><td>Nikon Model Eclipse Ti2-E</td><td>Nikon</td><td></td><td></td></tr><tr><td>Pen/Strep 10 Units/mL Penicillin 10 ug/mL Streptomycin</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>Plasma cleaner PDC-002</td><td>Harrick Plasma</td><td></td><td></td></tr><tr><td>polydimethylsiloxane(PDMS)</td><td>Momentive</td><td></td><td></td></tr><tr><td>polylysine 0.01%</td><td>Sigma</td><td></td><td></td></tr><tr><td>Spin coater ARE-310</td><td>Awatori Rentaro</td><td></td><td></td></tr><tr><td>Spin coater TDZ5-WS</td><td>Cence</td><td></td><td></td></tr><tr><td>Spin coater WH-SC-01</td><td>Suzhou Wenhao Microfluidic Technology Co., Ltd.</td><td></td><td></td></tr><tr><td>SU-8 3025</td><td>MicroChem, Westborough, MA, USA</td><td></td><td></td></tr><tr><td>SU-8 3075</td><td>MicroChem, Westborough, MA, USA</td><td></td><td></td></tr></tbody></table>

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.

The conventional on-chip peristaltic pump was firstly described by Stephen Quake in 2000, using which the peristalsis was actuated by the pattern 101, 100, 110, 010, 011, 001 8,10. The number 0 and 1 indicate &#34;open&#34; and &#34;close&#34; of the 3 horizontal control lines. Studies using more than 3 valves (e.g., five) have also been reported11. Even though the peristaltic pump composed by 3 control lines and 3 flow lines provides nano...

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

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 ...

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3.9K Views.  Northwest University. 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.

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

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 Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

Capillary-based Centrifugal Microfluidic Device for Size-controllable Formation of Monodisperse Microdroplets

Here, we demonstrate a simple method for the rapid production of size-controllable, monodisperse, W/O microdroplets using a capillary-based centrifugal microfluidic device. W/O microdroplets have recently been used in powerful methods that enable miniaturized chemical experiments. Therefore, developing a versatile method to yield monodisperse W/O microdroplets is needed. We have developed a method for generating monodisperse W/O microdroplets based on a capillary-based centrifugal axisymmetric co-flowing microfluidic device. We succeeded in controlling the size of microdroplets by adjusting the capillary orifice. Our method requires equipment that is easier-to-use than with other microfluidic techniques, requires only a small volume (0.1-1 &#181;l) of sample solution for encapsulation, and enables the production of hundreds of thousands number of W/O microdroplets per second. We expect this method will assist biological studies that require precious biological samples by conserving the volume of the samples for rapid quantitative analysis biochemical and biological studies.

Here, we demonstrate a simple production method for size-controllable, monodisperse, water-in-oil (W/O) microdroplets using a capillary-based centrifugal microfluidic device. This method requires only a small sample volume and enables high-yield production. We expect this method will be useful for rapid biochemical and cellular analyses.

Here, we demonstrate a simple method for the rapid production of size-controllable, monodisperse, W/O microdroplets using a capillary-based centrifugal ...

Engineering

Designing Microfluidic Devices for Studying Cellular Responses Under Single or Coexisting Chemical/Electrical/Shear Stress Stimuli

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other physical or chemical stimuli. They have been commonly used for in vitro cell studies by providing in vivo like surroundings. Especially, how cells response to chemical gradients, electrical fields, and shear stresses has drawn many interests since these phenomena are important in understanding cellular properties and functions. These microfluidic chips can be made of glass substrates, silicon wafers, polydimethylsiloxane (PDMS) polymers, polymethylmethacrylate (PMMA) substrates, or polyethyleneterephthalate (PET) substrates. Out of these materials, PMMA substrates are cheap and can be easily processed using laser ablation and writing. Although a few microfluidic devices have been designed and fabricated for generating multiple, coexisting chemical and electrical stimuli, none of them was considered efficient enough in reducing experimental repeats, particular for screening purposes. In this report, we describe our design and fabrication of two PMMA-based microfluidic chips for investigating cellular responses, in the production of reactive oxygen species and the migration, under single or coexisting chemical/electrical/shear stress stimuli. The first chip generates five relative concentrations of 0, 1/8, 1/2, 7/8, and 1 in the culture regions, together with a shear stress gradient produced inside each of these areas. The second chip generates the same relative concentrations, but with five different electric field strengths created within each culture area. These devices not only provide cells with a precise, controllable micro-environment but also greatly increase the experimental throughput.

Micro-fabricated devices integrated with fluidic components provide an in vitro platform for cell studies mimicking the in vivo micro-environment. We developed polymethylmethacrylate-based microfluidic chips for studying cellular responses under single or coexisting chemical/electrical/shear stress stimuli.

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other ...

Window on a Microworld: Simple Microfluidic Systems for Studying Microbial Transport in Porous Media

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in the environment, as well as directly for the transmission of pathogens to drinking water supplies. Natural porous media is composed of an intricate physical topology, varied surface chemistries, dynamic gradients of nutrients and electron acceptors, and a patchy distribution of microbes. These features vary substantially over a length scale of microns, making the results of macro-scale investigations of microbial transport difficult to interpret, and the validation of mechanistic models challenging. Here we demonstrate how simple microfluidic devices can be used to visualize microbial interactions with micro-structured habitats, to identify key processes influencing the observed phenomena, and to systematically validate predictive models. Simple, easy-to-use flow cells were constructed out of the transparent, biocompatible and oxygen-permeable material poly(dimethyl siloxane). Standard methods of photolithography were used to make micro-structured masters, and replica molding was used to cast micro-structured flow cells from the masters. The physical design of the flow cell chamber is adaptable to the experimental requirements: microchannels can vary from simple linear connections to complex topologies with feature sizes as small as 2 &mu;m. Our modular EcoChip flow cell array features dozens of identical chambers and flow control by a gravity-driven flow module. We demonstrate that through use of EcoChip devices, physical structures and pressure heads can be held constant or varied systematically while the influence of surface chemistry, fluid properties, or the characteristics of the microbial population is investigated. Through transport experiments using a non-pathogenic, green fluorescent protein-expressing Vibrio bacterial strain, we illustrate the importance of habitat structure, flow conditions, and inoculums size on fundamental transport phenomena, and with real-time particle-scale observations, demonstrate that microfluidics offer a compelling view of a hidden world.

Microfluidic devices can be used to visualize complex natural processes in real time and at the appropriate physical scales. We have developed a simple microfluidic device that mimics key features of natural porous media for studying growth and transport of bacteria in the subsurface.

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in ...

Biology

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.

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 ...

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. ...

Generating Controlled, Dynamic Chemical Landscapes to Study Microbial Behavior

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the microscale&#8213;to create micro-environments for microbial chemotaxis experiments. To create chemical pulses, we developed a system to introduce amino acid sources near-instantaneously by photolysis of caged amino acids within a polydimethylsiloxane (PDMS) microfluidic chamber containing a bacterial suspension. We applied this method to the chemotactic bacterium, Vibrio ordalii, which can actively climb these dynamic chemical gradients while being tracked by video microscopy. Amino acids, rendered biologically inert (&#39;caged&#39;) by chemical modification with a photoremovable protecting group, are uniformly present in the suspension but not available for consumption until their sudden release, which occurs at user-defined points in time and space by means of a near-UV-A focused LED beam. The number of molecules released in the pulse can be determined by a calibration relationship between exposure time and uncaging fraction, where the absorption spectrum after photolysis is characterized by using UV-Vis spectroscopy. A nanoporous polycarbonate (PCTE) membrane can be integrated into the microfluidic device to allow the continuous removal by flow of the uncaged compounds and the spent media. A strong, irreversible bond between the PCTE membrane and the PDMS microfluidic structure is achieved by coating the membrane with a solution of 3-aminopropyltriethoxysilane (APTES) followed by plasma activation of the surfaces to be bonded. A computer-controlled system can generate user-defined sequences of pulses at different locations and with different intensities, so as to create resource landscapes with prescribed spatial and temporal variability. In each chemical landscape, the dynamics of bacterial movement at the individual scale and their accumulation at the population level can be obtained, thereby allowing the quantification of chemotactic performance and its effects on bacterial aggregations in ecologically relevant environments.

A protocol for the generation of dynamic chemical landscapes by photolysis within microfluidic and millifluidic setups is presented. This methodology is suitable to study diverse biological processes, including the motile behavior, nutrient uptake, or adaptation to chemicals of microorganisms, both at the single cell and population level.

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the ...

Automated Microbial Cultivation and Adaptive Evolution using Microbial Microdroplet Culture System (MMC)

Conventional microbial cultivation methods usually have cumbersome operations, low throughput, low efficiency, and large consumption of labor and reagents. Moreover, microplate-based high-throughput cultivation methods developed in recent years have poor microbial growth status and experiment parallelization because of their low dissolved oxygen, poor mixture, and severe evaporation and thermal effect. Due to many advantages of micro-droplets, such as small volume, high throughput, and strong controllability, the droplet-based microfluidic technology can overcome these problems, which has been used in many kinds of research of high-throughput microbial cultivation, screening, and evolution. However, most prior studies remain&#160;at the stage of laboratory construction and application. Some key issues, such as high operational requirements, high construction difficulty, and lack of automated integration technology, restrict the wide application of droplet microfluidic technology in microbial research. Here, an automated Microbial Microdroplet Culture system (MMC) was successfully developed based on droplet microfluidic technology, achieving the integration of functions such as inoculation, cultivation, online monitoring, sub-cultivation, sorting, and sampling required by the process of microbial droplet cultivation. In this protocol, wild-type Escherichia coli (E. coli) MG1655 and a methanol-essential E. coli strain (MeSV2.2) were taken as examples to introduce how to use the MMC to conduct automated and relatively high-throughput microbial cultivation and adaptive evolution in detail. This method is easy to operate, consumes less labor and reagents, and has high experimental throughput and good data parallelity, which has great advantages compared with conventional cultivation methods. It provides a low-cost, operation-friendly, and result-reliable experimental platform for scientific researchers to conduct related microbial research.

Automated Microbial Cultivation and Adaptive Evolution using Microbial Microdroplet Culture System MMC

This protocol describes how to use the Microbial Microdroplet Culture system (MMC) to conduct automated microbial cultivation and adaptive evolution. MMC can cultivate and sub-cultivate microorganisms automatically and continuously and monitor online their growth with relatively high throughput and good parallelization, reducing labor and reagent consumption.

Conventional microbial cultivation methods usually have cumbersome operations, low throughput, low efficiency, and large consumption of labor and ...

Mechanostimulation of Multicellular Organisms Through a High-Throughput Microfluidic Compression System

During embryogenesis, coordinated cell movement generates mechanical forces that regulate gene expression and activity. To study this process, tools such as aspiration or coverslip compression have been used to mechanically stimulate whole embryos. These approaches limit experimental design as they are imprecise, require manual handling, and can process only a couple of embryos simultaneously. Microfluidic systems have great potential for automating such experimental tasks while increasing throughput and precision. This article describes a microfluidic system developed to precisely compress whole Drosophila melanogaster (fruit fly) embryos. This system features microchannels with pneumatically actuated deformable sidewalls and enables embryo alignment, immobilization, compression, and post-stimulation collection. By parallelizing these microchannels into seven lanes, steady or dynamic compression patterns can be applied to hundreds of Drosophila embryos simultaneously. Fabricating this system on a glass coverslip facilitates the simultaneous mechanical stimulation and imaging of samples with high-resolution microscopes. Moreover, the utilization of biocompatible materials, like PDMS, and the ability to flow fluid through the system make this device capable of long-term experiments with media-dependent samples. This approach also eliminates the requirement for manual mounting which mechanically stresses samples. Furthermore, the ability to quickly collect samples from the microchannels enables post-stimulation analyses, including -omics assays which require large sample numbers unattainable using traditional mechanical stimulation approaches. The geometry of this system is readily scalable to different biological systems, enabling numerous fields to benefit from the functional features described herein including high sample throughput, mechanical stimulation or immobilization, and automated alignment.

The present protocol describes the design, fabrication, and characterization of a microfluidic system capable of aligning, immobilizing, and precisely compressing hundreds of Drosophila melanogaster embryos with minimal user intervention. This system enables high-resolution imaging and recovery of samples for post-stimulation analysis and can be scaled to accommodate other multicellular biological systems.

During embryogenesis, coordinated cell movement generates mechanical forces that regulate gene expression and activity. To study this process, tools such ...