Systematic prevention of bubble formation and accumulation for long-term culture of pancreatic islet cells in microfluidic device

Reliable long-term cell culture in microfluidic system is limited by air bubble formation and accumulation. In this study, we developed a bubble removal system capable of both trapping and discharging air bubbles in a consistent and reliable manner. Combined with PDMS (Polydimethylsiloxane) hydrophilic surface treatment and vacuum filling, a microfluidic perifusion system equipped with the bubble trap was successfully applied for long-term culture of mouse pancreatic islets with no bubble formation and no flow interruption. In addition to demonstrating normal cell viability and islet morphology, post-cultured islets exhibited normal insulin secretion kinetics, intracellular calcium signaling, and changes in mitochondrial potentials in response to glucose challenge. This design could be easily adapted by other microfluidic systems due to its simple design, ease of fabrication, and portability.     

Bubble removal with the use of a vacuum pressure generated by a converging-diverging nozzle

Bubbles are an intrinsic problem in microfluidic devices and they can appear during the initial filling of the device or during operation. This report presents a generalizable technique to extract bubbles from microfluidic networks using an adjacent microfluidic negative pressure network over the entire microfluidic channel network design. We implement this technique by superimposing a network of parallel microchannels with a vacuum microfluidic channel and characterize the bubble extraction rates as a function of negative pressure applied. In addition, we generate negative pressure via a converging-diverging (CD) nozzle, which only requires inlet gas pressure to operate. Air bubbles generated during the initial liquid filling of the microfluidic network are removed within seconds and their volume extraction rate is calculated. This miniaturized vacuum source can achieve a vacuum pressure of 7.23 psi which corresponds to a bubble extraction rate of 9.84 pL/s, in the microfluidic channels we characterized. Finally, as proof of concept it is shown that the bubble removal system enables bubble removal on difficult to fill microfluidic channels such as circular or triangular shaped channels. This method can be easily integrated into many microfluidic experimental protocols.     

Hemodialysis dialyzers contribute to contamination of air microemboli that bypass the alarm system in the air trap

BACKGROUND: Previous studies have shown that micrometer-sized air bubbles are introduced into the patient during hemodialysis. The aim of this study was to investigate, in vitro, the influence of dialysis filters on the generation of air bubbles. METHODS: Three different kind of dialyzers were tested: one high-flux FX80 dry filter (Fresenius Medical Care AG&Co. KGaA, Bad Homburg, Germany), one low-flux F8HPS dry filter (Fresenius Medical Care AG&Co. KGaA, Bad Homburg, Germany) and a wet-stored APS-18u filter (Asahi Kasei Medical, Tokyo, Japan). The F8HPS was tested with pump flow ranging between 100 to 400 ml/min. The three filters were compared using a constant pump flow of 300 ml/min. Measurements were performed using an ultrasound Doppler instrument. RESULTS: In 90% of the series, bubbles were measured after the outlet line of the air trap without triggering an alarm. There were significantly more bubbles downstream than upstream of the filters F8HPS and FX80, while there was a significant reduction using the APS-18u. There was no reduction in the number of bubbles after passage through the air trap versus before the air trap (after the dialyzer). Increased priming volume reduced the extent of bubbles in the system. CONCLUSIONS: Data indicate that the air trap does not prevent air microemboli from entering the venous outlet part of the dialysis tubing (entry to the patient). More extended priming of the dialysis circuit may reduce the extent of microemboli that originate from dialysis filters. A wet filter may be favorable instead of dry-steam sterilized filters.     

A 3-D microfluidic combinatorial cell array

We present the development of a three-dimensional (3-D) combinatorial cell culture array device featured with integrated three-input, eight-output combinatorial mixer and cell culture chambers. The device is designed for cell-based screening of multiple compounds simultaneously on a microfluidic platform. The final assembled device is composed of a porous membrane integrated in between a Parylene 3-D microfluidic chip and a PDMS microfluidic chip. The membrane turned the cell culture chambers into two-level configuration to facilitate cell loading and to maintain cells in a diffusion dominated space during device operation. Experimentally, we first characterized the combined compound concentration profile at each chamber using a fluorescence method. We then successfully demonstrated the functionality of the quantitative cell-based assay by culturing B35 rat neuronal cells on this device and screening the ability of three compounds (1,5-dihydroxyisoquinoline, deferoxamine, and 3-aminobenzoic acid) to attenuate cell death caused by cytotoxic hydrogen peroxide. In another experiment, we assayed for the combinatorial effects of three chemotherapeutic compound exposures (vinorelbine, paclitaxel, and γ-linolenic acid) on human breast cancer cells, MDA-MB-231. The same technology will enable the construction of inexpensive lab-on-a-chip devices with high-input combinatorial mixer for performing high-throughput cell-based assay and highly parallel and combinatorial chemical or biochemical reactions.     

Air filtering capacity of an integrated cardiopulmonary bypass unit

To limit the morbidity of cardiopulmonary bypass (CPB), a new concept of integrating pumping, oxygenation, and air removal into a single unit has been developed (CardioVention Inc., Santa Clara, CA). The air filtration capacity of this system was tested. Three calves (73.2 +/- 2 kg) were connected to the integrated system by jugular and carotid cannulation. The integrated unit was challenged with injections of boluses of air of 5, 10, and 20 ml, three times each, and for a blood flow of 3 L/min and 5 L/min, respectively. The bubble count and size were recorded downstream of the unit with a Doppler ultrasound. At 3 L/min, bubbles were detected after injections of 20 ml only (n = 7 for the nine boluses). At 5 L/min, 1 bubble was detected with the nine injections of 5 ml, 14 bubbles were detected with nine injections of 10 ml, and 25 bubbles were detected with nine injections of 20 ml. No bubble exceeded 40 microm in diameter as determined by the Doppler ultrasound. The air filtering capacity of the CardioVention system is excellent both in terms of bubble count and of size after injection of large boluses of air. Its integrated concept offers a simplification of the circuit with fewer devices and connections, which further reduces the risk of accidental air introduction.     

Membrane-activated microfluidic rotary devices for pumping and mixing

Microfluidic devices are operated at a low-Reynolds-number flow regime such that the transportation and mixing of fluids are naturally challenging. There is still a great need to integrate fluid control systems such as pumps, valves and mixers with other functional microfluidic devices to form a micro-total-analysis-system. This study presents a new pneumatic microfluidic rotary device capable of transporting and mixing two different kinds of samples in an annular microchannel by using MEMS (Micro-electro-mechanical-systems) technology. Pumping and mixing can be achieved using a single device with different operation modes. The micropump has four membranes with an annular layout and is compact in size. The new device has a maximum pumping rate of 165.7 μL/min at a driving frequency of 17 Hz and an air pressure of 30 psi. Experimental data show that the pumping rate increases as higher air pressure and driving frequency are applied. In addition, not only can the microfluidic rotary device work as a peristaltic pumping device, but it also is an effective mixing device. The performance of the micromixer is extensively characterized. Experimental data indicate that a mixing index as high as 96.3% can be achieved. The developed microfluidic rotary device can be easily integrated with other microfluidic devices due to its simple and reliable PDMS fabrication process. The development of the microfluidic rotary device can be promising for micro-total-analysis-systems.     

Development of a novel microfluidic device for long-term in situ monitoring of live cells in 3-dimensional matrices

Using the latest innovations in microfabrication technology, 3-dimensional microfluidic cell culture systems have been developed as an attractive alternative to traditional 2-dimensional culturing systems as a model for long-term microscale cell-based research. Most microfluidic systems are based on the embedding of cells in hydrogels. However, physiologically realistic conditions based on hydrogels are difficult to obtain and the systems are often too complicated. We have developed a microfluidic cell culture device that incorporates a biodegradable rigid 3D polymer scaffold using standard soft lithography methods. The device permits repeated high-resolution fluorescent imaging of live cell populations within the matrix over a 4?week period. It was also possible to track cell development at the same spatial location throughout this time. In addition, human primary periodontal ligament cells were induced to produce quantifiable calcium deposits within the system. This simple and versatile device should be readily applicable for cell-based studies that require long-term culture and high-resolution bioimaging.     

Automatic microfluidic platform for cell separation and nucleus collection

This study reports a new biochip capable of cell separation and nucleus collection utilizing dielectrophoresis (DEP) forces in a microfluidic system comprising of micropumps and microvalves, operating in an automatic format. DEP forces operated at a low voltage (15 V_p–p) and at a specific frequency (16 MHz) can be used to separate cells in a continuous flow, which can be subsequently collected. In order to transport the cell samples continuously, a serpentine-shape (S-shape) pneumatic micropump device was constructed onto the chip device to drive the samples flow through the microchannel, which was activated by the pressurized air injection. The mixed cell samples were first injected into an inlet reservoir and driven through the DEP electrodes to separate specific samples. Finally, separated cell samples were collected individually in two outlet reservoirs controlled by microvalves. With the same operation principle, the nucleus of the specific cells can be collected after the cell lysis procedure. The pumping rate of the micropump was measured to be 39.8 μl/min at a pressure of 25 psi and a driving frequency of 28 Hz. For the cell separation process, the initial flow rate was 3 μl/min provided by the micropump. A throughput of 240 cells/min can be obtained by using the developed device. The DEP electrode array, microchannels, micropumps and microvalves are integrated on a microfluidic chip using micro-electro-mechanical-systems (MEMS) technology to perform several crucial procedures including cell transportation, separation and collection. The dimensions of the integrated chip device were measured to be 6 × 7 cm. By integrating an S-shape pump and pneumatic microvalves, different cells are automatically transported in the microchannel, separated by the DEP forces, and finally sorted to specific chambers. Experimental data show that viable and non-viable cells (human lung cancer cell, A549-luc-C8) can be successfully separated and collected using the developed microfluidic platform. The separation accuracy, depending on the DEP operating mode used, of the viable and non-viable cells are measured to be 84 and 81%, respectively. In addition, after cell lysis, the nucleus can be also collected using a similar scheme. The developed automatic microfluidic platform is useful for extracting nuclear proteins from living cells. The extracted nuclear proteins are ready for nuclear binding assays or the study of nuclear proteins.     

Elimination of microbubbles from the extracorporeal circuit: dynamic bubble trap versus arterial filter

BACKGROUND: Open heart surgery is associated with important risk of cerebral and peripheral organ dysfunction, attributed in part to microbubbles generated in or not eliminated from the ECC. For elimination of microbubbles, a dynamic bubble trap (DBT) was developed for the arterial line of ECCs. METHODS: Bubble eliminating properties of an arterial filter were evaluated in four CABG patients and compared to the performance of the DBT in four patients. One patient received both devices. RESULTS: Elimination of bubbles between 40-120 microm was significantly higher with the DBT (88% vs. 57% with arterial filter, p=0.034). Reduction of bubbles below 40 microm was equivalent in both groups. The combination of both devices was most effective (94% for bubbles >40 microm). CONCLUSION: Arterial filter and DBT are equally effective in elimination of smaller gas bubbles. However, bigger bubbles possibly causing cerebral and peripheral organ damage are eliminated to a greater degree by the DBT.     

Microfluidics/CMOS orthogonal capabilities for cell biology

The study of individual cells and cellular networks can greatly benefit from the capabilities of microfabricated devices for the stimulation and the recording of electrical cellular events. In this contribution, we describe the development of a device, which combines capabilities for both electrical and pharmacological cell stimulation, and the subsequent recording of electrical cellular activity. The device combines the unique advantages of integrated circuitry (CMOS technology) for signal processing and microfluidics for drug delivery. Both techniques are ideally suited to study electrogenic mammalian cells, because feature sizes are of the same order as the cell diameter, ∼ 50 μm. Despite these attractive features, we observe a size mismatch between microfluidic devices, with bulky fluidic connections to the outside world, and highly miniaturized CMOS chips. To overcome this problem, we developed a microfluidic flow cell that accommodates a small CMOS chip. We simulated the performances of a flow cell based on a 3-D microfluidic system, and then fabricated the device to experimentally verify the nutrient delivery and localized drug delivery performance. The flow-cell has a constant nutrient flow, and six drug inlets that can individually deliver a drug to the cells. The experimental analysis of the nutrient and drug flow mass transfer properties in the flowcell are in good agreement with our simulations. For an experimental proof-of-principle, we successfully delivered, in a spatially resolved manner, a ‘drug’ to a culture of HL-1 cardiac myocytes.     

Embryonic body culturing in an all-glass microfluidic device with laser-processed 4 μm thick ultra-thin glass sheet filter

In this paper, we report the development and demonstration of a method to fabricate an all-glass microfluidic cell culturing device without circulation flow. On-chip microfluidic cell culturing is an indispensable technique for cellular replacement therapies and experimental cell biology. Polydimethylsiloxane (PDMS) have become a popular material for fabricating microfluidic cell culture devices because it is a transparent, biocompatible, deformable, easy-to-mold, and gas-permeable. However, PDMS is also a chemically and physically unstable material. For example, PDMS undergoes aging easily even in room temperature conditions. Therefore, it is difficult to control long term experimental culturing conditions. On the other hand, glass is expected to be stable not only in physically but also chemically even in the presence of organic solvents. However, cell culturing still requires substance exchanges such as gases and nutrients, and so on, which cannot be done in a closed space of a glass device without circulation flow that may influence cell behavior. Thus, we introduce a filter structure with micropores onto a glass device to improve permeability to the cell culture space. Normally, it is extremely difficult to fabricate a filter structure on a normal glass plate by using a conventional fabrication method. Here, we demonstrated a method for fabricating an all-glass microfluidic cell culturing device having filters structure. The function of this all-glass culturing device was confirmed by culturing HeLa, fibroblast and ES cells. Compared with the closed glass devices without a filter structure, the numbers of cells in our device increased and embryonic bodies (EBs) were formed. This method offers a new tool in microfluidic cell culture technology for biological analysis and it expands the field of microfluidic cell culture.     

Multiparameter evaluation of the longevity in C. elegans under stress using an integrated microfluidic device

Caenorhabditis elegans (C. elegans) is an excellent model organism for the study of aging and longevity. In this work, we presented a microfluidic approach for the evaluation of longevity in C. elegans under stress. The microfluidic device integrated multiple microvalves with parallel channels, which enabled the long-term culture and flexible manipulation of C. elegans in real-time. The utility of the device was demonstrated by characterizing the lifespan, mobility behavior and fluorescence expression of oxidative stress in mutant strain CL2166 simultaneously at single animal resolution. A certain dose of polydatin was found to enable the extension of mean lifespan of CL2166 for the first time, and the prolonged longevity activity was possibly mediated by the protective response to oxidative stress, indicating the promising role of polydatin involved in aging process. The device is simple to operate, easy for real-time imaging and multiparatemer evaluations in parallel, providing the powerful platform for drug evaluation/screening in highthroughput format.     

An analysis of contact stiffness between a finger and an object when wearing an air-cushioned glove: the effects of the air pressure

Air-cushioned gloves have the advantages of lighter weight, lower cost, and unique mechanical performance, compared to gloves made of conventional engineering materials. The goal of this study is to analyze the contact interaction between fingers and object when wearing an air-cushioned glove. The contact interactions between the the fingertip and air bubbles, which is considered as a cell of a typical air-cushioned glove, has been analyzed theoretically. Two-dimensional finite element models were developed for the analysis. The fingertip model was assumed to be composed of skin layers, subcutaneous tissue, bone, and nail. The air bubbles were modeled as air sealed in the container of nonelastic membrane. We simulated two common scenarios: a fingertip in contact with one single air bubble and with two air cushion bubbles simultaneously. Our simulation results indicated that the internal air pressure can modulate the fingertip-object contact characteristics. The contact stiffness reaches a minimum when the initial air pressure is equal to 1.3 and 1.05 times of the atmosphere pressure for the single air bubble and the double air bubble contact, respectively. Furthermore, the simulation results indicate that the double air bubble contact will result in smaller volumetric tissue strain than the single air bubble contact for the same force.     

A self-contained microfluidic cell culture system

Conventional in vitro cell culture that utilizes culture dishes or microtiter plates is labor-intensive and time-consuming, and requires technical expertise and specific facilities to handle cell harvesting, media exchange and cell subculturing procedures. A microfluidic array platform with eight microsieves in each cell culture chamber is presented for continuous cell culture. With the help of the microsieves, uniform cell loading and distribution can be obtained. Within the arrays, cells grown to the point of confluency can be trypsinized and recovered from the device. Cells trapped in the microsieves after trypsinization function to seed the chambers for subsequent on-chip culturing, creating a sustainable platform for multiple cycles. The capability of the microfluidic array platform was demonstrated with a BALB/3T3 (murine embryonic fibroblast) cell line. The present microfluidic cell culture platform has potential to develop into a fully automated cell culture system integrated with temperature control, fluidic control, and micropumps, maximizing cell culture health with minimal intervention.     

Particle manipulation in a microfluidic channel using acoustic trap

A high frequency sound beam was employed to explore an experimental method that could control particle motions in a microfluidic device. A 24 MHz single element lead zirconate titanate (PZT) transducer was built to transmit a focused ultrasound of variable duty factors (pulse duration/pulse repetition time), and its 1–3 piezocomposite structure established a tight focusing with f-number (focal depth/aperture size) of one. The transducer was excited by the Chebyshev windowed chirp signal sweeping from 18 MHz to 30 MHz with a 50% of duty factor, in order to ensure that enough sound beams were penetrated into the microfluidic device. The device was fabricated from a polydimethylsiloxane (PDMS) mold, and had a main channel composed of three subchannels among which particles flowed in the middle. A 60~70 μm diameter single droplet in the flow could be trapped near the channel bifurcation, and subsequently diverted into the sheath flow by releasing or shifting the acoustic trap. Hence, the results showed the potential use of a focused sound beam in microfluidic devices, and further suggested that this method could be exploited in the development of ultrasound-based flow cytometry and cell sorting devices.     

Microbubble transport through a bifurcating vessel network with pulsatile flow

Motivated by two-phase microfluidics and by the clinical applications of air embolism and a developmental gas embolotherapy technique, experimental and theoretical models of microbubble transport in pulsatile flow are presented. The one-dimensional time-dependent theoretical model is developed from an unsteady Bernoulli equation that has been modified to include viscous and unsteady effects. Results of both experiments and theory show that roll angle (the angle the plane of the bifurcating network makes with the horizontal) is an important contributor to bubble splitting ratio at each bifurcation within the bifurcating network. When compared to corresponding constant flow, pulsatile flow was shown to produce insignificant changes to the overall splitting ratio of the bubble despite the order one Womersley numbers, suggesting that bubble splitting through the vasculature could be modeled adequately with a more modest constant flow model. However, bubble lodging was affected by the flow pulsatility, and the effects of pulsatile flow were evident in the dependence of splitting ratio of bubble length. The ability of bubbles to remain lodged after reaching a steady state in the bifurcations is promising for the effectiveness of gas embolotherapy to occlude blood flow to tumors, and indicates the importance of understanding where lodging will occur in air embolism. The ability to accurately predict the bubble dynamics in unsteady flow within a bifurcating network is demonstrated and suggests the potential for bubbles in microfluidics devices to encode information in both steady and unsteady aspects of their dynamics.     

Surface passivation of a microfluidic device to glial cell adhesion: a comparison of hydrophobic and hydrophilic SAM coatings.

Cell adhesion in a microfluidic structure can lead to catastrophic flow problems due to the comparable size of the cell with the microfabricated device. Such issues are important in the growing research area involving the merging of biological materials and MEMS devices. We have examined the surface compatibility of uncoated and coated microfabricated glass and semiconductor surfaces under static solution (cell culture) and flow experiments (microfluidic device) using glial (astrocyte and glioblastoma) cells. Bare semiconductor and glass surfaces were most attractive to cell adhesion, promoting biofouling under both static and flow conditions. Passivation of the surfaces was performed with silane coupling agents octadecyltrimethoxysilane (OTMS) or N-(triethoxysilylpropyl)-O-polyethylene oxide urethane (TESP) on SiO2 surfaces via self-assembled monolayer (SAM) deposition. The hydrophilic TESP coating was effective at inhibiting biofouling of the microfluidic structure, allowing greater than several minutes of fluid flow. The hydrophobic OTMS coating, on the other hand, promoted cell adhesion leading to restricted flow within a few minutes. Interestingly, under cell culture conditions the TESP surface exhibited biocompatible properties for glial cell adhesion and proliferation, in contrast to the OTMS surface which resisted cell growth. These studies suggest that cell adhesion is dependent upon the time domain of the cell-surface interaction.     

Development of disposable PDMS micro cell culture analog devices with photopolymerizable hydrogel encapsulating living cells

Microscale cell culture devices with two or more cell types, such as the micro cell culture analog (microCCA), are promising devices to predict mammalian response to toxic drug and chemical exposure. A polydimethylsiloxane (PDMS) version of such microfluidic devices has been challenging to construct due to the difficulty of patterning multi cell types directly into designated individual cell culture chambers in an oxygen plasma bonded PDMS device. Approaches with micro-valves for flow control are complex, expensive and inconvenient to use. In this study, an alternative approach using polyethylene glycol diacrylate (PEG-DA) for spatially controlled multi-cell type patterning inside a bonded microCCA device is described. We constructed a three-cell type PDMS microCCA following a human physiologically based pharmacokinetic (PBPK) modeling, and applied continuous cell culture medium recirculation within the device as a blood surrogate. A fluorescence microscope based direct pattern writing method was used to form cell/hydrogel microstructures with higher cell viability than the traditional UV lamp based method. The positive effect of mixed molecular weight PDG-DA on hydrogel-encapsulated cell membrane integrity was also studied. This prototype PDMS microCCA device was then tested with Triton X-100 as a model toxicant. The combination of hydrogel photo-patterning and the microfluidic cell culture platform enables the fabrication of simple and low cost multi-cell type biosensors for drug development, toxicity study and clinical diagnosis.     

Immunological Analyses of Whole Blood via “Microfluidic Drifting” Based Flow Cytometric Chip

Cost-effective, high-performance diagnostic instruments are vital to providing the society with accessible, affordable, and high-quality healthcare. Here we present an integrated, “microfluidic drifting” based flow cytometry chip as a potential inexpensive, fast, and reliable diagnostic tool. It is capable of analyzing human blood for cell counting and diagnosis of diseases. Our device achieves a throughput of ~3754 events/s. Calibration with Flow-Check calibration beads indicated good congruency with a commercially available benchtop flow cytometer. Moreover, subjection to a stringent 8-peak rainbow calibration particle test demonstrated its ability to perform high-resolution immunological studies with separation resolution of 4.28 between the two dimmest fluorescent populations. Counting accuracy at different polystyrene bead concentrations showed strong correlation (r = 0.9991) with hemocytometer results. Finally, reliable quantification of CD4+ cells in healthy human blood via staining with monoclonal antibodies was demonstrated. These results demonstrate the potential of our microfluidic flow cytometry chip as an inexpensive yet high-performance point-of-care device for mobile medicine.     

Poly(dimethylsiloxane) thin films as biocompatible coatings for microfluidic devices: cell culture and flow studies with glial cells

Oxygen plasma treatment of poly(dimethylsiloxane) (PDMS) thin films produced a hydrophilic surface that was biocompatible and resistant to biofouling in microfluidic studies. Thin film coatings of PDMS were previously developed to provide protection for semiconductor-based microoptical devices from rapid degradation by biofluids. However, the hydrophobic surface of native PDMS induced rapid clogging of microfluidic channels with glial cells. To evaluate the various issues of surface hydrophobicity and chemistry on material biocompatibility, we tested both native and oxidized PDMS (ox-PDMS) coatings as well as bare silicon and hydrophobic alkane and hydrophilic oligoethylene glycol silane monolayer coated under both cell culture and microfluidic studies. For the culture studies, the observed trend was that the hydrophilic surfaces supported cell adhesion and growth, whereas the hydrophobic ones were inhibitive. However, for the fluidic studies, a glass-silicon microfluidic device coated with the hydrophilic ox-PDMS had an unperturbed flow rate over 14 min of operation, whereas the uncoated device suffered a loss in rate of 12%, and the native PDMS coating showed a loss of nearly 40%. Possible protein modification of the surfaces from the culture medium also were examined with adsorbed films of albumin, collagen, and fibrinogen to evaluate their effect on cell adhesion.