Self-aligning Tetris-Like (TILE) modular microfluidic platform for mimicking multi-organ interactions.

Multi-organ perfusion systems offer the unique opportunity to mimic different physiological systemic interactions. However, existing multi-organ culture platforms have limited flexibility in specifying the culture conditions, device architectures, and fluidic connectivity simultaneously. Here, we report a modular microfluidic platform that addresses this limitation by enabling easy conversion of existing microfluidic devices into tissue and fluid control modules with self-aligning magnetic interconnects. This enables a 'stick-n-play' approach to assemble planar perfusion circuits that are amenable to both bioimaging-based and analytical measurements. A myriad of tissue culture and flow control TILE modules were successfully constructed with backward compatibility. Finally, we demonstrate applications in constructing recirculating multi-organ systems to emulate liver-mediated bioactivation of nutraceuticals and prodrugs to modulate their therapeutic efficacies in the context of atherosclerosis and cancer. This platform greatly facilitates the integration of existing organs-on-chip models to provide an intuitive and flexible way for users to configure different multi-organ perfusion systems.

A reconfigurable stick-n-play modular microfluidic system using magnetic interconnects.

A reconfigurable "stick-n-play" modular microfluidic system that can be assembled, disassembled, reconfigured and assembled again for building different integrated microfluidic systems is presented. Magnetic interconnects, comprising ring magnets and sealing gaskets, are integrated into each microfluidic module's inlet(s) and outlet(s) for both module-to-module and world-to-chip fluidic interconnects. The magnetic interconnects reversibly "stick" each individual microfluidic module together and provide leak-free fluidic communication between connected microfluidic modules in order to form a larger integrated microfluidic system. Because of the magnetic interconnects, connected microfluidic modules can be easily disconnected, reconfigured and connected again to form a different integrated microfluidic system. Using a fused deposition modeling (FDM)/fused filament fabrication (FFF)-based 3D printer, a reconfigurable stick-n-play modular microfluidic system, comprising a serpentine channel base platform and various microfluidic modules as well as inlet/outlet modules for world-to-chip fluidic interconnects, was first 3D printed. Magnetic interconnects were then integrated into each 3D printed module. Finally, the stick-n-play modular microfluidic system was used to demonstrate its reconfigurability to build various integrated microfluidic systems by simply and reversibly sticking various modules together. Based on the magnetic interconnects, customized multi-dimensional stick-n-play modular microfluidic systems can be easily designed and built providing a convenient platform for designing large scale microfluidic systems.

Embedding objects during 3D printing to add new functionalities.

A novel method for integrating and embedding objects to add new functionalities during 3D printing based on fused deposition modeling (FDM) (also known as fused filament fabrication or molten polymer deposition) is presented. Unlike typical 3D printing, FDM-based 3D printing could allow objects to be integrated and embedded during 3D printing and the FDM-based 3D printed devices do not typically require any post-processing and finishing. Thus, various fluidic devices with integrated glass cover slips or polystyrene films with and without an embedded porous membrane, and optical devices with embedded Corning(®) Fibrance™ Light-Diffusing Fiber were 3D printed to demonstrate the versatility of the FDM-based 3D printing and embedding method. Fluid perfusion flow experiments with a blue colored food dye solution were used to visually confirm fluid flow and/or fluid perfusion through the embedded porous membrane in the 3D printed fluidic devices. Similar to typical 3D printed devices, FDM-based 3D printed devices are translucent at best unless post-polishing is performed and optical transparency is highly desirable in any fluidic devices; integrated glass cover slips or polystyrene films would provide a perfect optical transparent window for observation and visualization. In addition, they also provide a compatible flat smooth surface for biological or biomolecular applications. The 3D printed fluidic devices with an embedded porous membrane are applicable to biological or chemical applications such as continuous perfusion cell culture or biocatalytic synthesis but without the need for any post-device assembly and finishing. The 3D printed devices with embedded Corning(®) Fibrance™ Light-Diffusing Fiber would have applications in display, illumination, or optical applications. Furthermore, the FDM-based 3D printing and embedding method could also be utilized to print casting molds with an integrated glass bottom for polydimethylsiloxane (PDMS) device replication. These 3D printed glass bottom casting molds would result in PDMS replicas with a flat smooth bottom surface for better bonding and adhesion.

A pump-free membrane-controlled perfusion microfluidic platform.

In this article, we present a microfluidic platform for passive fluid pumping for pump-free perfusion cell culture, cell-based assay, and chemical applications. By adapting the passive membrane-controlled pumping principle from the previously developed perfusion microplate, which utilizes a combination of hydrostatic pressure generated by different liquid levels in the wells and fluid wicking through narrow strips of a porous membrane connecting the wells to generate fluid flow, a series of pump-free membrane-controlled perfusion microfluidic devices was developed and their use for pump-free perfusion cell culture and cell-based assays was demonstrated. Each pump-free membrane-controlled perfusion microfluidic device comprises at least three basic components: an open well for generating fluid flow, a micron-sized deep chamber/channel for cell culture or for fluid connection, and a wettable porous membrane for controlling the fluid flow. Each component is fluidically connected either by the porous membrane or by the micron-sized deep chamber/channel. By adapting and incorporating the passive membrane-controlled pumping principle into microfluidic devices, all the benefits of microfluidic technologies, such as small sample volumes, fast and efficient fluid exchanges, and fluid properties at the micro-scale, can be fully taken advantage of with this pump-free membrane-controlled perfusion microfluidic platform.

Methods for advanced hepatocyte cell culture in microwells utilizing air bubbles.

Flat, two-dimensional (2D) cell culture substrates are simple to use but offer little control over cell morphologies and behavior. In this article, we present a number of novel and unique methods for advanced cell culture in microwells utilizing air bubbles as a way to seed cells in order to provide substantial control over cellular microenvironments and organization to achieve specific cell-based applications. These cell culture methods enable controlled formation of stable air bubbles in the microwells that spontaneously formed when polar solvents such as cell culture media are loaded. The presence of air bubbles (air bubble masking) enables highly controllable cell patterning and organization of seeded cells as well as cell co-culture in microwells. In addition, these cell culture methods are simple to use and implement, yet versatile, and have the potential to provide a wide range of microenvironments to improve in vivo-like behavior for a number of cell types and applications. The air bubble masking technique can also be used to produce a micron thick layer of collagen film suspended on top of the microwells. These collagen film enclosed microwells could provide an easy way for high throughput drug screening and cytotoxicity assays as different drug compounds could be pre-loaded and dried in selected microwells and then released during cell culture.

Microstructured multi-well plate for three-dimensional packed cell seeding and hepatocyte cell culture.

In this article, we present a microstructured multi-well plate for enabling three-dimensional (3D) high density seeding and culture of cells through the use of a standard laboratory centrifuge to promote and maintain 3D tissue-like cellular morphology and cell-specific functionality in vitro without the addition of animal derived or synthetic matrices or coagulants. Each well has microfeatures on the bottom that are comprised of a series of ditches/open microchannels. The dimensions of the microchannels promote and maintain 3D tissue-like cellular morphology and cell-specific functionality in vitro. After cell seeding with a standard pipette, the microstructured multi-well plates were centrifuged to tightly pack cells inside the ditches in order to enhance cell-cell interactions and induce formation of 3D cellular structures during cell culture. Cell-cell interactions were optimized based on cell packing by considering dimensions of the ditches/open microchannels, orientation of the microstructured multi-well plate during centrifugation, cell seeding density, and the centrifugal force and time. With the optimized cell packing conditions, we demonstrated that after 7 days of cell culture, primary human hepatocytes adhered tightly together to form cord-like structures that resembled 3D tissue-like cellular architecture. Importantly, cell membrane polarity was restored without the addition of animal derived or synthetic matrices or coagulants.

A polystyrene-based microfluidic device with three-dimensional interconnected microporous walls for perfusion cell culture.

In this article, we present a simple, rapid prototyped polystyrene-based microfluidic device with three-dimensional (3D) interconnected microporous walls for long term perfusion cell culture. Patterned 3D interconnected microporous structures were created by a chemical treatment together with a protective mask and the native hydrophobic nature of the microporous structures were selectively made hydrophilic using oxygen plasma treatment together with a protective mask. Using this polystyrene-based cell culture microfluidic device, we successfully demonstrated the support of four days perfusion cell culture of hepatocytes (C3A cells).

Accelerating drug discovery via organs-on-chips.

Considerable advances have been made in the development of micro-physiological systems that seek to faithfully replicate the complexity and functionality of animal and human physiology in research laboratories. Sometimes referred to as "organs-on-chips", these systems provide key insights into physiological or pathological processes associated with health maintenance and disease control, and serve as powerful platforms for new drug development and toxicity screening. In this Focus article, we review the state-of-the-art designs and examples for developing multiple "organs-on-chips", and discuss the potential of this emerging technology to enhance our understanding of human physiology, and to transform and accelerate the drug discovery and preclinical testing process. This Focus article highlights some of the recent technological advances in this field, along with the challenges that must be addressed for these technologies to fully realize their potential.

Fluid control in microfluidic devices using a fluid conveyance extension and an absorbent microfluidic flow modulator.

This article presents a simple method for controlling fluid in microfluidic devices without the need for valves or pumps. A fluid conveyance extension is fluidly coupled to the enclosed outlet chamber of a microfluidic device. After a fluid is introduced into the microfluidic device and saturates the fluid conveyance extension, a fluid flow in the microfluidic device is generated by contacting an absorbent microfluidic flow modulator with the fluid conveyance extension to absorb the fluid from the fluid conveyance extension through capillary action. Since the fluid in the microfluidic device is fluidly coupled with the fluid conveyance extension and the fluid conveyance extension is fluidly coupled with the absorbent microfluidic flow modulator, the absorption rate of the absorbent microfluidic flow modulator, which is the rate at which the absorbent microfluidic flow modulator absorbs fluid, matches the fluid flow rate in the microfluidic device. Thus, the fluid flow rate in the microfluidic device is set by the absorption rate of the absorbent microfluidic flow modulator. Sheath flow and fluid switching applications are demonstrated using this simple fluid control method without the need for valves or pumps. Also, the ability to control the fluid flow rate in the microfluidic device is demonstrated using absorbent microfluidic flow modulators with various absorbent characteristics and dimensions.

A continuous perfusion microplate for cell culture.

We describe a 96-well microplate with fluidically connected wells that enables the continuous fluid perfusion between wells without the need for external pumping. A single unit in such a perfusion microplate consists of three wells: a source well, a sample (cell culture) well in the middle and a waste well. Fluid perfusion is achieved using a combination of the hydrostatic pressure generated by different liquid levels in the wells and the fluid wicking through narrow strips of a cellulose membrane connecting the wells. There is an excellent correspondence between the observed perfusion flow dynamics and the flow simulations based on Darcy's Law. Hepatocytes (C3A cells) cultured for 4 days in the perfusion microplate with no media exchange in the cell culture well had the same viability as hepatocytes exposed to a daily exchange of media. EOC 20 cells that require media conditioned by LADMAC cells were shown to be equally viable in the adjacent cell culture well of the perfusion microplate with LADMAC cells cultured in the source well. Tegafur, a prodrug, when added to primary human hepatocytes in the source well, was metabolized into a cytotoxic metabolite that kills colon cancer cells (HCT 116) cultured in the adjacent cell culture well; no toxicity was observed when only medium was in the source well. These results suggest that the perfusion microplate is a useful tool for a variety of cell culture applications with benefits ranging from labor savings to enabling in vivo-like toxicity studies.

Microfluidic platforms for hepatocyte cell culture: new technologies and applications.

In this article, we summarize the key elements of microfluidic platforms for mimicking in vivo hepatocyte cell culture and the major recent advances in this area. Specifically, we will give brief background and rationale for key design requirements for mimicking in vivo hepatocyte cell culture, and then summarize findings, applications, and limitations from microfluidic platforms that addressed these design requirements. Although no ideal microfluidic platform has so far been developed for fully mimicking in vivo hepatocyte cell culture, some approaches and designs have demonstrated great potential in this area.

Flexible microfluidic devices with three-dimensional interconnected microporous walls for gas and liquid applications.

This article presents a simple, low-cost method of fabrication and the applications of flexible polystyrene microfluidic devices with three-dimensional (3D) interconnected microporous walls based on treatment using a solvent/non-solvent mixture at room temperature. The complete fabrication process from device design concept to working device can be completed in less than an hour in a regular laboratory setting, without the need for expensive equipment. Microfluidic devices were used to demonstrate gas generation and absorption reactions by acidifying water with carbon dioxide (CO(2)) gas. By selectively treating the microporous structures with oxygen plasma, acidification of water by acetic acid (distilled white vinegar) perfusion was also demonstrated with the same device design.

Three-dimensional interconnected microporous poly(dimethylsiloxane) microfluidic devices.

This technical note presents a fabrication method and applications of three-dimensional (3D) interconnected microporous poly(dimethylsiloxane) (PDMS) microfluidic devices. Based on soft lithography, the microporous PDMS microfluidic devices were fabricated by molding a mixture of PDMS pre-polymer and sugar particles in a microstructured mold. After curing and demolding, the sugar particles were dissolved and washed away from the microstructured PDMS replica revealing 3D interconnected microporous structures. Other than introducing microporous structures into the PDMS replica, different sizes of sugar particles can be used to alter the surface wettability of the microporous PDMS replica. Oxygen plasma assisted bonding was used to enclose the microstructured microporous PDMS replica using a non-porous PDMS with inlet and outlet holes. A gas absorption reaction using carbon dioxide (CO(2)) gas acidified water was used to demonstrate the advantages and potential applications of the microporous PDMS microfluidic devices. We demonstrated that the acidification rate in the microporous PDMS microfluidic device was approximately 10 times faster than the non-porous PDMS microfluidic device under similar experimental conditions. The microporous PDMS microfluidic devices can also be used in cell culture applications where gas perfusion can improve cell survival and functions.

Perfusion-based microfluidic device for three-dimensional dynamic primary human hepatocyte cell culture in the absence of biological or synthetic matrices or coagulants.

We describe a perfusion-based microfluidic device for three-dimensional (3D) dynamic primary human hepatocyte cell culture. The microfluidic device was used to promote and maintain 3D tissue-like cellular morphology and cell-specific functionality of primary human hepatocytes by restoring membrane polarity and hepatocyte transport function in vitro without the addition of biological or synthetic matrices or coagulants. A unique feature of our dynamic cell culture device is the creation of a microenvironment, without the addition of biological or synthetic matrices or coagulants, that promotes the 3D organization of hepatocytes into cord-like structures that exhibit functional membrane polarity as evidenced by the expression of gap junctions and the formation of an extended, functionally active, bile canalicular network.

Low-cost rapid prototyping of flexible microfluidic devices using a desktop digital craft cutter.

Low-cost and straight forward rapid prototyping of flexible microfluidic devices using a desktop digital craft cutter is presented. This rapid prototyping method can consistently achieve microchannels as thin as 200 microm in width and can be used to fabricate three-dimensional (3D) microfluidic devices using only double-sided pressure sensitive adhesive (PSA) tape and laser printer transparency film. Various functional microfluidic devices are demonstrated with this rapid prototyping method. The complete fabrication process from device design concept to working device can be completed in minutes without the need of expensive equipment.

Multidimensional modular microfluidic system.

An extension of our previous work on a genuinely plug-n-play modular microfluidic system is presented for designing and building customized multidimensional (planar, three-dimensional (3D) and their combinations) microfluidic systems as well as for better system integration by allowing direct plug-in of active components such as micropumps.

SmartBuild-a truly plug-n-play modular microfluidic system.

In this Technical Note, for the first time, a truly "plug-n-play" modular microfluidic system (SmartBuild Plug-n-Play Modular Microfluidic System) is presented for designing and building integrated modular microfluidic systems for biological and chemical applications. The modular microfluidic system can be built by connecting multiple microfluidic components together to form a larger integrated system. The SmartBuild System comprises of a motherboard with interconnect channels/grooves, fitting components, microchannel inserts with different configurations and microchips/modules with different functionalities. Also, heaters, micropumps and valving systems can be designed and used in the system. Examples of an integrated mixing system and reaction systems are presented here to demonstrate the versatility of the SmartBuild System.

Characteristics of dynamic mass redistribution of epidermal growth factor receptor signaling in living cells measured with label-free optical biosensors.

This paper reported the identification of a novel optical signature for epidermal growth factor (EGF) receptor signaling in human epidermoid carcinoma A431 cells mediated by EGF. The optical signature was based on dynamic mass redistribution (DMR) in living cells triggered by EGFR activation, as monitored in real time with resonant waveguide grating biosensors. Analysis of the modulation of the EGF-induced DMR signals by a variety of known modulators provided links of various targets to distinct steps in the cellular responses. Results showed that the dynamic mass redistribution in quiescent A431 cells mediated by EGF required EGFR tyrosine kinase activity, actin polymerization, and dynamin and mainly proceeded through MEK. The DMR signals obtained serve as integrated signatures for interaction networks in the EGFR signaling.

Self-referencing a single waveguide grating sensor in a micron-sized deep flow chamber for label-free biomolecular binding assays.

In order to allow the design of increasingly sensitive label-free biosensors, compensation of environmental fluctuations is emerging as the dominant hurdle. The system and technique presented here utilize a unique combination of microfluidics, optical instrumentation, and image processing to provide a reference signal for each label-free biomolecular binding assay. Moreover, this reference signal is generated from the same sensor used to detect the biomolecular binding events. In this manner, the reference signal and the binding signal share nearly all common-mode noise sources (temperature, pressure, vibration, etc.) and their subtraction leaves the purest binding signal possible. Computational fluid dynamic simulations have been used to validate the flow behavior and thermal characteristics of the fluids inside the sensing region. This system has been demonstrated in simple bulk refractive index tests, as well as small molecule (biotin/streptavidin) binding experiments. The ability to perform not only simple binding but also control experiments has been discussed, indicating the wide applicability of the technique.

Rare earth-doped glass microbarcodes.

The development of ultraminiaturized identification tags has applications in fields ranging from advanced biotechnology to security. This paper describes micrometer-sized glass barcodes containing a pattern of different fluorescent materials that are easily identified by using a UV lamp and an optical microscope. A model DNA hybridization assay using these "microbarcodes" is described. Rare earth-doped glasses were chosen because of their narrow emission bands, high quantum efficiencies, noninterference with common fluorescent labels, and inertness to most organic and aqueous solvents. These properties and the large number (>1 million) of possible combinations of these microbarcodes make them attractive for use in multiplexed bioassays and general encoding.