[Show abstract][Hide abstract] ABSTRACT: The present study examines the use of automated periodic "flow-stop" perfusion systems for long-term culture of mammalian cells in a microchannel bioreactor. The method is used to culture Human Foreskin Fibroblasts (HFF) and Human Umbilical Vein Endothelial Cells (HUVEC) for long periods of time (>7 d) in a microchannel (height 100 mum). Design parameters, mass transport and shear stress issues are theoretically examined via numerical simulations. Cell growth and morphology are experimentally monitored and an enhanced growth rate was measured compared to constant perfusion micro-reactors and to traditional culture in Petri dishes. Moreover, we demonstrate the use of the method to co-culture undifferentiated colonies of human Embryonic Stem Cells (hESC) on HFF feeder cells in microchannels. The successful hESC-HFF co-culture in the microbioreactor is achieved due to two vital characteristics of the developed method-short temporal exposure to flow followed by long static incubation periods. The short pulsed exposure to shear enables shear sensitive cells (e.g., hESC) to withstand the medium renewal flow. The long static incubation period may enable secreted factors (e.g., feeder cells secreted factors) to accumulate locally. Thus the developed method may be suitable for long-term culture of sensitive multi-cellular complexes in microsystems.
[Show abstract][Hide abstract] ABSTRACT: Microfluidic bioreactors have been shown valuable for various cellular applications. The use of micro-wells/grooves bioreactors, in which micro-topographical features are used to protect sensitive cells from the detrimental effects of fluidic shear stress, is a promising approach to culture sensitive cells in these perfusion microsystems. However, such devices exhibit substantially different fluid dynamics and mass transport characteristics compared to conventional planar microchannel reactors. In order to properly design and optimize these systems, fluid and mass transport issues playing a key role in microscale bioreactors should be adequately addressed. The present work is a parametric study of micro-groove/micro-well microchannel bioreactors. Operation conditions and design parameters were theoretically examined via a numerical model. The complex flow pattern obtained at grooves of various depths was studied and the shear protection factor compared to planar microchannels was evaluated. 3D flow simulations were preformed in order to examine the shear protection factor in micro-wells, which were found to have similar attributes as the grooves. The oxygen mass transport problem, which is coupled to the fluid mechanics problem, was solved for various groove geometries and for several cell types, assuming a defined shear stress limitation. It is shown that by optimizing the groove depth, the groove bioreactor may be used to effectively maximize the number of cells cultured within it or to minimize the oxygen gradient existing in such devices. Moreover, for sensitive cells having a high oxygen demand (e.g., hepatocytes) or low endurance to shear (e.g., human embryonic stem cells), results show that the use of grooves is an enabling technology, since under the same physical conditions the cells cannot be cultured for long periods of time in a planar microchannel. In addition to the theoretical model findings, the culture of human foreskin fibroblasts in groove (30 microm depth) and well bioreactors (35 microm depth) was experimentally examined at various flow rates of medium perfusion and compared to cell culture in regular flat microchannels. It was shown that the wells and the grooves enable a one order of magnitude increase in the maximum perfusion rate compared to planar microchannels. Altogether, the study demonstrates that the proper design and use of microgroove/well bioreactors may be highly beneficial for cell culture assays.
Biotechnology and Bioengineering 10/2008; 102(4):1222-30. · 4.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In current devices, dielectrophoresis (DEP) is implemented by connecting microelectrodes to external voltage signals. This study presents an alternative approach for implementing DEP in which some electrodes are left electrostatically floating. It is demonstrated experimentally and theoretically that these electrodes produce effective DEP forces. Floating electrodes do not require a connection to an external signal. Therefore, DEP devices can be constructed as hybrids of excited microelectrodes and floating nanoelectrodes. This approach can overcome the significant difficulties involved in connecting nanoelectrodes to an external signal. The study demonstrates that floating nanoelectrodes can increase local DEP forces by at least three orders of magnitude without having to increase the voltages applied to the excited microelectrodes. Therefore, floating nanoelectrodes can significantly broaden the range of DEP devices capable of manipulating nanoparticles. Moreover, floating nanoelectrodes enable nanoparticles to be manipulated with nanometer spatial resolution. As a result, hybrid devices that employ floating nanoelectrodes may make bottom-up DEP fabrication possible.
[Show abstract][Hide abstract] ABSTRACT: The culture of human Embryonic Stem (ES) cells in microchannel bioreactors can be highly beneficial for ES cell biology studies and ES tissue engineering applications. In the present study we examine the use of Human Foreskin Fibroblasts (HFF) cells as feeder cells for human ES culture in a microchannel perfusion bioreactor. PDMS microchannels (depth:130 micron) were fabricated using conventional soft-lithography techniques. The channels were sterilized, coated with a human fibronectin solution and seeded with cells. Following a period of static incubation, culture medium was perfused through the channels at various flow rates and cell growth was monitored throughout the culture process. Mass transport and fluid mechanics models were used to evaluate the culture conditions (shear stress, oxygen levels within the micro-bioreactor as a function of the medium flow rate. The conditions for successful long-term culture (>7 days) of HFF under flow were established. Experiments with human embryonic stem cells cultured in microchannels show that the conditions essential to co-culture human ES cell on HFF cells under perfusion differ from the conditions necessary for HFF cell culture. Human ES cells were found to be highly sensitive to flow and culture conditions and did not grow under flow rates which were suitable for HFF long-term culture. Successful culture of undifferentiated human ES cell colonies in a perfusion micro-bioreactor is a basic step towards utilizing microfluidic techniques to explore stem cell biology.
[Show abstract][Hide abstract] ABSTRACT: The deformability of erythrocytes is of great importance for oxygen delivery in the microcirculation. Reduced RBC deformability is associated with several types of hemolytic anaemias, malaria, sepsis and diabetes. Aging of erythrocytes is also associated with loss of deformability as well as reduction in cell volume. An automated rheoscope has been developed, utilizing a microfabricated glass flow cell, high speed camera and advanced image-processing software. RBCs suspended in a high viscosity medium were filmed flowing through a microchannel. The system produces valuable data such as velocity profiles of RBCs, spatial distribution within the microchannel, cell volume and deformation index (DI) curves. The variation of DI across the channel height, due to change in shear stress, was measured for the first time. Such DI curves were obtained for normal and Thalassemia RBCs and their diagnostic potential was demonstrated. The spatial distribution and velocity of RBCs and rigid microspheres were measured. Both RBC and rigid spheres showed enhanced inward lateral migration, however the RBCs form a depletion region at the center of flow. The volume and surface area of the flowing cells have been estimated based on a fluid mechanics model and experimental results and fell within the normal range. Hence, the system developed, provides means for examining the behavior of individual RBCs in microchannels, and may serve as a microfabricated diagnostic device for deformability and volume measurements.
[Show abstract][Hide abstract] ABSTRACT: In this Letter we describe a novel method for tunable viscoelastic focusing of particles flowing in a microchannel. It is proposed that some elasticity, inherently present in dilute polymer solutions, may be responsible for highly nonuniform spatial distribution of flowing particles across the channel cross section, yielding their "focusing" in the midplane of the channel. A theory based on scaling arguments is presented to explain the lateral migration and is found to be in a very good agreement with the experimental observations. It was found that, in agreement with the theoretical prediction, the particles would have different spatial distribution depending on their size and rheology of the suspending medium. We demonstrate how the viscoelastic focusing can be precisely controlled by proper rheological design of the carrier solution.
[Show abstract][Hide abstract] ABSTRACT: The culture of cells in a microbioreactor can be highly beneficial for cell biology studies and tissue engineering applications. The present work provides new insights into the relationship between cell growth, cell morphology, perfusion rate, and design parameters in microchannel bioreactors. We demonstrate the long-term culture of mammalian (human foreskin fibroblasts, HFF) cells in a microbioreactor under constant perfusion in a straightforward simple manner. A perfusion system was used to culture human cells for more than two weeks in a plain microchannel (130 microm x 1 mm x 2 cm). At static conditions and at high flow rates (>0.3 ml h(-1)), the cells did not grow in the microchannel for more than a few days. For low flow rates (<0.2 ml h(-1)), the cells grew well and a confluent layer was obtained. We show that the culture of cells in microchannels under perfusion, even at low rates, affects cell growth kinetics as well as cell morphology. The oxygen level in the microchannel was evaluated using a mass transport model and the maximum cell density measured in the microchannel at steady state. The maximum shear stress, which corresponds to the maximum flow rate used for long term culture, was 20 mPa, which is significantly lower than the shear stress cells may endure under physiological conditions. The effect of channel size and cell type on long term cell culture were also examined and were found to be significant. The presented results demonstrate the importance of understanding the relationship between design parameters and cell behavior in microscale culture system, which vary from physiological and traditional culture conditions.
Lab on a Chip 06/2007; 7(5):611-7. · 5.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The motion and deformation of red blood cells (RBCs) flowing in a microchannel were studied using a theoretical model and a novel automated rheoscope. The theoretical model was developed to predict the cells deformation under shear as a function of the cells geometry and mechanical properties. Fluid dynamics and membrane mechanics are incorporated, calculating the traction and deformation in an iterative manner. The model was utilized to evaluate the effect of different biophysical parameters, such as: inner cell viscosity, membrane shear modulus and surface to volume ratio on deformation measurements. The experimental system enables the measurement of individual RBCs velocity and their deformation at defined planes within the microchannel. Good agreement was observed between the simulation results, the rheoscope measurements and published ektacytometry results. The theoretical model results imply that such deformability measuring techniques are weakly influenced by changes in the inner viscosity of the cell or the ambient fluid viscosity. However, these measurements are highly sensitive to RBC shear modulus. The shear modulus, estimated by the model and the rheoscope measurements, falls between the values obtained by micropipette aspiration and laser trapping. The study demonstrates the integration of a theoretical model with a microfabricated device in order to achieve a better understanding of RBC mechanics and their measurement using microfluidic shear assays. The system and the model have the potential of serving as quantitative clinical tools for diagnosing deformability disorders in RBCs.
Journal of Biomechanics 02/2007; 40(9):2088-95. · 2.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The deformability of erythrocytes is of great importance for oxygen delivery in the microcirculation [Lipowsky, H.H., 2005. Microvascular rheology and hemodynamics. Microcirculation 12, 5-15]. Aging of erythrocytes is associated with a reduction in deformability and also in size. The present work describes an automated cell analyzer which utilizes a glass microchannel and advanced image processing software. Erythrocytes suspended in a high viscosity medium are filmed flowing through the microchannel. Under these conditions, the cells assume different orientations and undergo varying deformations according to their location in the velocity profile. The cell analyzer enables the measurement of individual erythrocyte velocity, deformability and volume at varying depths within the microchannel. The volume of the cells is calculated based on the experimental data and a fluid mechanics model. The results obtained show that, on average, the deformability of the cells increases with increase in their size. Additionally, the behavior of RBCs in a microchannel is investigated, showing promising diagnostic possibilities.
Microvascular Research 02/2007; 73(1):7-13. · 2.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In practice, dielectrophoresis (DEP) devices are based on micropatterned electrodes. When subjected to applied voltages, the electrodes generate nonuniform electric fields that are necessary for the DEP manipulation of particles. In this study, electrically floating electrodes are used in DEP devices. It is demonstrated that effective DEP forces can be achieved by using floating electrodes. Additionally, DEP forces generated by floating electrodes are different from DEP forces generated by excited electrodes. The floating electrodes' capabilities are explained theoretically by calculating the electric field gradients and demonstrated experimentally by using test-devices. The test-devices show that floating electrodes can be used to collect erythrocytes (red blood cells). DEP devices which contain many floating electrodes ought to have fewer connections to external signal sources. Therefore, the use of floating electrodes may considerably facilitate the fabrication and operation of DEP devices. It can also reduce device dimensions. However, the key point is that DEP devices can integrate excited electrodes fabricated by microtechnology processes and floating electrodes fabricated by nanotechnology processes. Such integration is expected to promote the use of DEP devices in the manipulation of nanoparticles.
[Show abstract][Hide abstract] ABSTRACT: An automated rheoscope has been developed, utilizing a microfabricated glass flow cell, high speed camera and advanced image-processing software. RBCs suspended in a high viscosity medium were filmed flowing through a microchannel. Under these conditions, RBCs exhibit different orientations and deformations according to their location in the velocity profile. The rheoscope system produces valuable data such as velocity profile of RBCs, spatial distribution within a microchannel and deformation index (DI) curves. The variation of DI across the channel height, due to change in shear stress, was measured carrying implications for diffractometry methods. These curves of DI were taken at a constant flow rate and cover most of the relevant shear stress spectrum. This is an improvement of the existing techniques for deformability measurements and may serve as a diagnostic tool for certain blood disorders. The DI curves were compared to measurements of the flowing RBCs velocity profile. In addition, we found that RBCs flowing in a microchannel are mostly gathered in the center of the flow and maintain a characteristic spatial distribution. The spatial distribution in this region changes slightly with increasing flow rate. Hence, the system described, provides means for examining the behavior of individual RBCs, and may serve as a microfabricated diagnostic device for deformability measurement.
Biosensors and Bioelectronics 09/2006; 22(2):165-9. · 5.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The use of microfabricated elements and microfluidics, offer a great promise in the field of clinical blood tests. An automated rheoscope has been developed, utilizing a microfabricated glass flow cell, high speed camera and advanced image-processing software. Red Blood Cells (RBCs) and rigid microspheres (1-8 mum) suspended in a high viscosity media were filmed flowing through a microchannel (Re
[Show abstract][Hide abstract] ABSTRACT: A novel electrostatically actuated valveless micropump is presented whereby an actuation voltage is applied across a working fluid, which takes advantage of the higher relative electrical permittivity of water and many other fluids with respect to air. The device is fabricated in silicon and the diaphragm is made of electroplated nickel, while the assembly is carried out using flip–chip bonding. A reduced-order model is used to describe the micropump's performance in terms of electrical properties of the fluid, the residual stress in the diaphragm, geometrical features and the actuation voltage. The tested prototype featured a ~1 µl min−1 flow rate at 50 V actuation voltage. The model predictions show the possibility of achieving flow rates >1 µl min−1 with the actuation voltage <10 V for devices with 3 mm diaphragm size.
Journal of Micromechanics and Microengineering 11/2005; 15(12):2309. · 1.79 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This work presents new concepts of ISFET encapsulations for catheters in brain in-vivo monitoring. Plain-chip and flip-chip bonding techniques are introduced. In both techniques, a pseudo-reference electrode is implemented.
Electronics, Circuits and Systems, 2004. ICECS 2004. Proceedings of the 2004 11th IEEE International Conference on; 01/2005
[Show abstract][Hide abstract] ABSTRACT: This work presents the main challenges in ISFET encapsulation. It analyzes SU8 drawbacks as an encapsulant and presents a novel flip-chip bonding packaging concept.
Electronics, Circuits and Systems, 2004. ICECS 2004. Proceedings of the 2004 11th IEEE International Conference on; 01/2005
[Show abstract][Hide abstract] ABSTRACT: The paper presents a novel readout configuration for ISFET sensors based on Wheatstone-Bridge connection. This design technique allows on-chip integration, temperature compensation and measurements from ISFET/REFET pairs. The circuit is capable of operating in differential mode, and can also perform common mode and combined measurements, while improving the immunity to noise and interferences. The Wheatstone-Bridge interface benefits from enhanced operational flexibility, due to the ability of relative sensitivity control of the output signal. Direct and indirect feedback configurations are presented with operational analysis, simulations and application options. Simulation results showing 9 μV accuracy are presented. A mm test chip in 1.6 μm CMOS technology was used for laboratory experiments using MOSFETs for emulation of ISFET/REFET sensors.
[Show abstract][Hide abstract] ABSTRACT: CIMP (complementary ISFET/MOSFET pair)—a novel technique for implementation of readout interface in CMOS ISFET-based microsystems is described. This design technique allows body effect elimination, temperature compensation and design simplicity, while maintaining constant biasing of ISFET sensor. Several configurations of CIMP interface provide applicability for array-type sensors. The general concept presentation is followed by a detailed analysis, test results showing 0.1% accuracy and layout implementations in a standard 1.6 μm CMOS technology.
Sensors and Actuators B Chemical 01/2004; · 3.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Highly integrated ion-sensitive field-effect transistor (ISFET) microsystems require the monolithic implementation of ISFETs, CMOS electronics, and additional sensors on the same chip. This paper presents new ISFETs in standard CMOS, fabricated by post-processing of a standard CMOS VLSI chip. Unlike CMOS compatible ISFETs fabricated in a dedicated process, the new sensors are directly combined with state-of-the-art CMOS electronics and are subject to continuous technology upgrading. The ISFETs presented include an intermediate gate formed by one or more conducting layers placed between the gate oxide and the sensing layer. The combination of the highly isolating gate oxide of the MOS with a leaky or conducting sensing layer allows the use of low temperature materials that do not damage the CMOS chip. The operation of ISFETs with an intermediate gate and sensing layers fabricated at low temperature is modeled. ISFETs with a linear pH response and drift as low as 0.3 mV/h are reported.
[Show abstract][Hide abstract] ABSTRACT: Summary form only given. This paper presents a novel catheter for brain pH monitoring. The catheter consists of a tiny silicone rubber tube and includes several ion sensitive field effect transistors. Similar catheters without the sensors are regularly introduced during neurosurgery through a hole in the skull, and are placed subdurally or inside the cerebral ventricles. With the addition of the sensors, the catheter provides brain local information on pH at the cerebro-spinal fluid without modifying the therapeutic process in use today. The main objective is the diagnosis of secondary brain local ischemia. It is expected that the information provided by the catheter will help to save life and extend the life expectancy of patients undergoing neurosurgery. Preliminary clinical tests are presented.
[Show abstract][Hide abstract] ABSTRACT: The paper presents a complete microsystem for pH measurement based on ISFET sensors. On-chip integration of ISFET sensors, readout, signal processing and serial communication systems was performed in a standard 1.6µm CMOS fabrication process. System architecture allows compatibility for clinical catheter applications, 4-channel sensibility and digital serial output with 2-LSB accuracy. Integration of various interfaces and sensors on 3X0.9 mm 2 area makes the design suitable for implantable sensor systems. Fig. 1. Microsystem architecture (a) and implantable catheter application (b) Keywords: ISFET, CMOS, microsystem. A schematic structure of a typical application of implantable system is given in Fig. 1b, where a sensor system applied together with reference electrode into a catheter. Because of the limited internal space (typical diameter of 1mm), the demand for minimal area and number of wires is obvious.