Hydrogel-based sealed microchamber arrays for rapid medium exchange and drug testing of cell spheroids.

Culturing cell spheroids in microchamber arrays is a widely used method in regenerative medicine and drug discovery while it requires laborious procedures during medium exchange and drug administration. Here, we report a simple method for the medium exchange and drug testing using a hydrogel-based sealed microchamber arrays. Owing to the high molecular permeability of poly(vinyl alcohol) hydrogel, the sealed microchamber allows nutrients and drugs in outer medium to pass through. Thus, automatic medium exchange and drug testing for all the cell spheroids inside the microchamber arrays are achieved by simply transferring the microchamber from old medium to fresh medium. Cell spheroids of human induced pluripotent stem cell-derived cardiomyocytes were cultured inside the sealed microchambers, and it was confirmed that the spheroids were stably positioned inside the microchamber even after transferring 10 times. The cell spheroids showed high viability after culturing for 7 days in the sealed microchamber with the transfer-based medium exchange, which allowed cardiac maturation by simultaneous electrical stimulation. Isoproterenol, a model cardiac drug, was administrated from outside the sealed microchamber to demonstrate the feasibility of drug testing by the rapid transfer method.

Fabrication of unconventional inertial microfluidic channels using wax 3D printing.

Inertial microfluidics has emerged over the past decade as a powerful tool to accurately control cells and microparticles for diverse biological and medical applications. Many approaches have been proposed to date in order to increase the efficiency and accuracy of inertial microfluidic systems. However, the effects of channel cross-section and solution properties (Newtonian or non-Newtonian) have not been fully explored, primarily due to limitations in current microfabrication methods. In this study, we overcome many of these limitations using wax 3D printing technology and soft lithography through a novel workflow, which eliminates the need for the use of silicon lithography and polydimethylsiloxane (PDMS) bonding. We have shown that by adding dummy structures to reinforce the main channels, optimizing the gap between the dummy and main structures, and dissolving the support wax on a PDMS slab to minimize the additional handling steps, one can make various non-conventional microchannels. These substantially improve upon previous wax printed microfluidic devices where the working area falls into the realm of macrofluidics rather than microfluidics. Results revealed a surface roughness of 1.75 µm for the printed channels, which does not affect the performance of inertial microfluidic devices used in this study. Channels with complex cross-sections were fabricated and then analyzed to investigate the effects of viscoelasticity and superposition on the lateral migration of the particles. Finally, as a proof of concept, microcarriers were separated from human mesenchymal stem cells using an optimized channel with maximum cell-holding capacity, demonstrating the suitability of these microchannels in the bioprocessing industry.

Liver microsystems in vitro for drug response.

Engineering approaches were adopted for liver microsystems to recapitulate cell arrangements and culture microenvironments in vivo for sensitive, high-throughput and biomimetic drug screening. This review introduces liver microsystems in vitro for drug hepatotoxicity, drug-drug interactions, metabolic function and enzyme induction, based on cell micropatterning, hydrogel biofabrication and microfluidic perfusion. The engineered microsystems provide varied microenvironments for cell culture that feature cell coculture with non-parenchymal cells, in a heterogeneous extracellular matrix and under controllable perfusion. The engineering methods described include cell micropatterning with soft lithography and dielectrophoresis, hydrogel biofabrication with photolithography, micromolding and 3D bioprinting, and microfluidic perfusion with endothelial-like structures and gradient generators. We discuss the major challenges and trends of liver microsystems to study drug response in vitro.

Cost analysis of oil cake-to-biodiesel production in packed-bed micro-flow reactors with immobilized lipases.

Biodiesel production depends to a great extent on the use of cheap raw materials, since biodiesel itself is a mass product, not a high-value product. New processing methods, such as micro-flow continuous processing combined with enzymatic catalysis, open doors to the latter. As reported here, the window of opportunity in enzyme-catalyzed biodiesel production is the conversion of waste cooking oil. The main technological challenge for this is to obtain efficient immobilization of the lipase catalyst on beads. The beads can be filled into tubular reactors where designed packed-bed provide porous channels, forming micro-flow. It turns out, that in this way, the immobilization costs become the decisive economic factor. This paper reports a solution to that issue. The use of oil cake enables economic viability, which is not given by any of the commercial polymeric substrates used so far for enzyme immobilization. The costs of immobilization are mirrored in the earnings and cash flow of the new biotechnological process.

Organic Electrochemical Transistor Arrays for In Vitro Electrophysiology Monitoring of 2D and 3D Cardiac Tissues.

Here, a multichannel organic electrochemical transistor (OECT) array is reported for electrophysiological monitoring and mapping of action potential propagation of a wide range of cardiac cells, including cell lines, primary cell lines, and human-sourced stem cell derivatives in 2D and 3D structures. The results suggest that the ability to exploit this OECT-based platform to map 2D action potential propagation provides a viable strategy to better characterize cardiac cells in response to various chronotropic drugs. The effects of chronotropic agents Isoproterenol and Verapamil on cardiac tissues validate the utility of OECT for drug screening capability, and a preliminary demonstration of a 64-channel OECT array to monitor the cardiac action potentials for better spatial resolution is presented. The study demonstrates that OECT will be a viable and versatile platform for applications in medical and pharmacological industries.

[Medicine 4.0: examples of applications of electronics, information technology and microsystems in modern medicine]. / « Médecine 4.0 ¼ - Exemples d'application de l'électronique, de la technologie de l'information et des microsystèmes dans la médecine moderne.

The electronic advances of the last hundred years have made enormous contributions to medical research and the development of new therapeutic methods. In recent years in particular, it has been demonstrated that intelligent sensors, with appropriate radio interfaces, will soon allow diagnostic and therapeutic processes in medicine to be linked to one another - this will enable the development of completely new forms of therapy [1]. This new "Medicine 4.0" was the subject of a first article in the series, which presented the progress achieved through the merging of microsensor technology, microelectronics, information and communication technologies, with a particular focus on the case of personalized chemotherapy. The purpose of this new article is to present more practical applications of these new therapeutic methods.

Preparation of the Multifunctional Liposome-Containing Microneedle Arrays as an Oral Cavity Mucosal Vaccine Adjuvant-Delivery System.

Recently, the multifunctional liposome-constituted microneedle arrays (LiposoMAs) have been proven to be an interesting vaccine adjuvant-delivery system (VADS) that are stable and can be vaccinated via oral cavity mucosal route. When given to mice at oral mucosa, the LiposoMAs can effectively eliminate the ingredient loss caused by chewing, swallowing, and saliva flowing and can, thus, elicit robust systemic as well as mucosal immunoresponses against the loaded antigens. In addition, the LiposoMAs can induce a mixed Th1/Th2 immunoresponse and strong cellular/humoral immunity due to special adjuvanticity and targeting delivery functions of the nanoparticulate VADS. In this chapter, the preparation, characterization as well as mucosal vaccination of the LiposoMAs are introduced. In addition, the methods for sampling mouse organs, tissues, and cells and for evaluation of the immunization efficacy are mainly included.

A Microfluidic Bioreactor for Toxicity Testing of Stem Cell Derived 3D Cardiac Bodies.

Modeling tissues and organs using conventional 2D cell cultures is problematic as the cells rapidly lose their in vivo phenotype. In microfluidic bioreactors the cells reside in microstructures that are continuously perfused with cell culture medium to provide a dynamic environment mimicking the cells natural habitat. These micro scale bioreactors are sometimes referred to as organs-on-chips and are developed in order to improve and extend cell culture experiments. Here, we describe the two manufacturing techniques photolithography and soft lithography that are used in order to easily produce microfluidic bioreactors. The use of these bioreactors is exemplified by a toxicity assessment on 3D clustered human pluripotent stem cells (hPSC)-derived cardiomyocytes by beating frequency imaging.

Wound healing potential of antibacterial microneedles loaded with green tea extracts.

This study evaluates the utility of an antibacterial microneedle composed of green tea (GT) extract and hyaluronic acid (HA), for the efficient delivery of GT. These microneedles have the potential to be a patient-friendly method for the conventional sustained release of drugs. In this study, a fabrication method using a mold-based technique to produce GT/HA microneedles with a maximum area of ~50mm(2) with antibacterial properties was used to manufacture transdermal drug delivery systems. Fourier transform infrared (FTIR) spectrometry was carried out to observe the potential modifications in the microneedles, when incorporated with GT. The degradation rate of GT in GT/HA microneedles was controlled simply by adjusting the HA composition. The effects of different ratios of GT in the HA microneedles were determined by measuring the release properties. In HA microneedles loaded with 70% GT (GT70), a continuous higher release rate was sustained for 72h. The in vitro cytotoxicity assays demonstrated that GT/HA microneedles were not generally cytotoxic to Chinese hamster ovary cells (CHO-K1), human embryonic kidney cells (293T), and mouse muscle cells (C2C12), which were treated for 12 and 24h. Antimicrobial activity of the GT/HA microneedles was demonstrated by ~95% growth reduction of gram negative [Escherichia coli (E. coli), Pseudomonas putida (P. putida), and Salmonella typhimurium (S. typhimurium)] and gram positive bacteria [Staphylococcus aureus (S. Aureus) and Bacillus subtilis (B. subtilis)], with GT70. Furthermore, GT/HA microneedles reduced bacterial growth of infected wound sites in the skin and improved wound healing process of skin in rat model.

Mitochondrial membrane studies using impedance spectroscopy with parallel pH monitoring.

A biological microelectromechanical system (BioMEMS) device was designed to study complementary mitochondrial parameters important in mitochondrial dysfunction studies. Mitochondrial dysfunction has been linked to many diseases, including diabetes, obesity, heart failure and aging, as these organelles play a critical role in energy generation, cell signaling and apoptosis. The synthesis of ATP is driven by the electrical potential across the inner mitochondrial membrane and by the pH difference due to proton flux across it. We have developed a tool to study the ionic activity of the mitochondria in parallel with dielectric measurements (impedance spectroscopy) to gain a better understanding of the properties of the mitochondrial membrane. This BioMEMS chip includes: 1) electrodes for impedance studies of mitochondria designed as two- and four-probe structures for optimized operation over a wide frequency range and 2) ion-sensitive field effect transistors for proton studies of the electron transport chain and for possible monitoring other ions such as sodium, potassium and calcium. We have used uncouplers to depolarize the mitochondrial membrane and disrupt the ionic balance. Dielectric spectroscopy responded with a corresponding increase in impedance values pointing at changes in mitochondrial membrane potential. An electrical model was used to describe mitochondrial sample's complex impedance frequency dependencies and the contribution of the membrane to overall impedance changes. The results prove that dielectric spectroscopy can be used as a tool for membrane potential studies. It can be concluded that studies of the electrochemical parameters associated with mitochondrial bioenergetics may render significant information on various abnormalities attributable to these organelles.

Miniature electrical stimulator for hemorrhage control.

Noncompressible hemorrhage is currently the most common cause of preventable death in battlefield and in civilian trauma injuries. Tourniquets, specialized wound dressings, and hemorrhage-inhibiting biomaterials are not sufficiently effective in arrest of noncompressible hemorrhage and often cause collateral tissue damage. An effective, easy-to-use, portable device is needed to reduce blood loss in trauma patients immediately following injury and to maintain hemorrhage control up to several hours-until the injured is evacuated to a medical facility. We developed a miniature electrical stimulator to induce vascular constriction and, thereby, reduce hemorrhage. Vasoconstriction of the rat femoral arteries and veins was studied with pulse durations in the range of 1 µs to 10 ms and repetition rate of 10 Hz. Pulse amplitude of 20 V, duration of 1 ms, and repetition rate of 10 Hz were found sufficient to induce rapid constriction down to 31 ± 2% of the initial diameter, which could be maintained throughout a two-hour treatment. Within one minute following treatment termination the artery dilated back to 88 ± 3% of the initial diameter, providing rapid restoration of blood perfusion. Histology indicated no damage to the vessel wall and endothelium seven days after stimulation. The same treatment reduced the blood loss following complete femoral artery resection by 68 ± 11%, compared to untreated vessels. Very low power consumption during stimulation (<10 mW per 1.6 mm electrode) allows miniaturization of the stimulator for portable battery-powered operation in the field to control the blood loss following vascular trauma.

Electronics for better healthcare.

Microelectronics and microsystem technology have changed our daily lives considerably in the past 50 years. Countless everyday objects contain microelectronic components. In healthcare up to the present, however, it has not been possible to make major alterations in introducing electronics and information technology that would lead to innovative improvements and greater transparency. This paper describes initial steps in diagnostics and oncological therapy including telematic healthcare systems which can, for example, assist patients with cardiovascular diseases and shows, through these areas, how electronics and microsystems technology can contribute to better healthcare.

Microfabrication of electrode patterns for high-frequency ultrasound transducer arrays.

High-frequency ultrasound is needed for medical imaging with high spatial resolution. A key issue in the development of ultrasound imaging arrays to operate at high frequencies (≥30 MHz) is the need for photolithographic patterning of array electrodes. To achieve this directly on 1-3 piezocomposite, the material requires not only planar, parallel, and smooth surfaces, but also an epoxy composite filler that is resistant to chemicals, heat, and vacuum. This paper reports, first, on the surface finishing of 1-3 piezocomposite materials by lapping and polishing. Excellent surface flatness has been obtained, with an average surface roughness of materials as low as 3 nm and step heights between ceramic/polymer of ∼80 nm. Subsequently, high-frequency array elements were patterned directly on top of these surfaces using a photolithography process. A 30-MHz linear array electrode pattern with 50-µm element pitch has been patterned on the lapped and polished surface of a high-frequency 1-3 piezocomposite. Excellent electrode edge definition and electrical contact to the composite were obtained. The composite has been lapped to a final thickness of ∼55 µm. Good adhesion of electrodes on the piezocomposite has been achieved and electrical impedance measurements have demonstrated their basic functionality. The array was then packaged, and acoustic pulse-echo measurements were performed. These results demonstrate that direct patterning of electrodes by photolithography on 1-3 piezocomposite is feasible for fabrication of high-frequency ultrasound arrays. Furthermore, this method is more conducive to mass production than other reported array fabrication techniques.

Tolerant chalcogenide cathodes of membraneless micro fuel cells.

The most critical issues to overcome in micro direct methanol fuel cells (µDMFCs) are the lack of tolerance of the platinum cathode and fuel crossover through the polymer membrane. Thus, two novel tolerant cathodes of a membraneless microlaminar-flow fuel cell (µLFFC), Pt(x)S(y) and CoSe(2), were developed. The multichannel structure of the system was microfabricated in SU-8 polymer. A commercial platinum cathode served for comparison. When using 5 M CH(3)OH as the fuel, maximum power densities of 6.5, 4, and 0.23 mW cm(-2) were achieved for the µLFFC with Pt, Pt(x)S(y), and CoSe(2) cathodes, respectively. The Pt(x)S(y) cathode outperformed Pt in the same fuel cell when using CH(3)OH at concentrations above 10 M. In a situation where fuel crossover is 100 %, that is, mixing the fuel with the reactant, the maximum power density of the micro fuel cell with Pt decreased by 80 %. However, for Pt(x)S(y) this decrease corresponded to 35 % and for CoSe(2) there was no change in performance. This result is the consequence of the high tolerance of the chalcogenide-based cathodes. When using 10 M HCOOH and a palladium-based anode, the µLFFC with a CoSe(2) cathode achieved a maxiumum power density of 1.04 mW cm(-2). This micro fuel cell does not contain either Nafion membrane or platinum. We report, for the first time, the evaluation of Pt(x)S(y)- and CoSe(2)-based cathodes in membraneless micro fuel cells. The results suggest the development of a novel system that is not size restricted and its operation is mainly based on the selectivity of its electrodes.

A miniaturized system for spike-triggered intracortical microstimulation in an ambulatory rat.

This paper reports on a miniaturized system for spike-triggered intracortical microstimulation (ICMS) in an ambulatory rat. The head-mounted microdevice comprises a previously developed application-specific integrated circuit fabricated in 0.35-µm two-poly four-metal complementary metal-oxide-semiconductor technology, which is assembled and packaged on a miniature rigid-flex substrate together with a few external components for programming, supply regulation, and wireless operation. The microdevice operates autonomously from a single 1.55-V battery, measures 3.6 cm × 1.3 cm × 0.6 cm, weighs 1.7 g (including the battery), and is capable of stimulating as well as recording the neural response to ICMS in biological experiments with anesthetized laboratory rats. Moreover, it has been interfaced with silicon microelectrodes chronically implanted in the cerebral cortex of an ambulatory rat and successfully delivers electrical stimuli to the second somatosensory area when triggered by neural activity from the rostral forelimb area with a user-adjustable spike-stimulus time delay. The spike-triggered ICMS is further shown to modulate the neuronal firing rate, indicating that it is physiologically effective.

Robust penetrating microelectrodes for neural interfaces realized by titanium micromachining.

Neural prosthetic interfaces based upon penetrating microelectrode devices have broadened our understanding of the brain and have shown promise for restoring neurological functions lost to disease, stroke, or injury. However, the eventual viability of such devices for use in the treatment of neurological dysfunction may be ultimately constrained by the intrinsic brittleness of silicon, the material most commonly used for manufacture of penetrating microelectrodes. This brittleness creates predisposition for catastrophic fracture, which may adversely affect the reliability and safety of such devices, due to potential for fragmentation within the brain. Herein, we report the development of titanium-based penetrating microelectrodes that seek to address this potential future limitation. Titanium provides advantage relative to silicon due to its superior fracture toughness, which affords potential for creation of robust devices that are resistant to catastrophic failure. Realization of these devices is enabled by recently developed techniques which provide opportunity for fabrication of high-aspect-ratio micromechanical structures in bulk titanium substrates. Details are presented regarding the design, fabrication, mechanical testing, in vitro functional characterization, and preliminary in vivo testing of devices intended for acute recording in rat auditory cortex and thalamus, both independently and simultaneously.

The heterogeneous integration of single-walled carbon nanotubes onto complementary metal oxide semiconductor circuitry for sensing applications.

A simple methodology for integrating single-walled carbon nanotubes (SWNTs) onto complementary metal oxide semiconductor (CMOS) circuitry is presented. The SWNTs were incorporated onto the CMOS chip as the feedback resistor of a two-stage Miller compensated operational amplifier utilizing dielectrophoretic assembly. The measured electrical properties from the integrated SWNTs yield ohmic behavior with a two-terminal resistance of approximately 37.5 kOmega and the measured small signal ac gain (-2) from the inverting amplifier confirmed successful integration of carbon nanotubes onto the CMOS circuitry. Furthermore, the temperature response of the SWNTs integrated onto CMOS circuitry has been measured and had a thermal coefficient of resistance (TCR) of -0.4% degrees C(-1). This methodology, demonstrated for the integration of SWNTs onto CMOS technology, is versatile, high yield and paves the way for the realization of novel miniature carbon-nanotube-based sensor systems.

Simulations of electrode placement for a thalamic visual prosthesis.

In this paper, placement parameters for microstimulation electrodes in a visual prosthesis are evaluated based on retinotopic models of macaque and human lateral geniculate nucleus. Phosphene patterns were simulated for idealized microwire electrodes as well as for currently available clinical electrodes. For idealized microwire electrodes, spacing as large as 600 microm in three dimensions would allow for over 250 phosphenes per visual hemifield in macaques, and over 800 in humans.

Single-chip detector for electron spin resonance spectroscopy.

We have realized an innovative integrated detector for electron spin resonance spectroscopy. The microsystem, consisting of an LC oscillator, a mixer, and a frequency division module, is integrated onto a single silicon chip using a conventional complementary metal-oxide-semiconductor technology. The implemented detection method is based on the measurement of the variation of the frequency of the integrated LC oscillator as a function of the applied static magnetic field, caused by the presence of a resonating sample placed over the inductor of the LC-tank circuit. The achieved room temperature spin sensitivity is about 10(10) spinsGHz(12) with a sensitive volume of about (100 microm)(3).