Natanel Korin

A parametric study of human fibroblasts culture in a microchannel bioreactor

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.

Design of well and groove microchannel bioreactors for cell culture.

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 µm depth) and well bioreactors (35 µm 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. Biotechnol. Bioeng. 2009;102: 1222–1230.

Experimental and theoretical study of selective protein deposition using focused micro laminar flows

The present work describes an experimental method and design tools which enable the precise localization of an analyte, a few microns in width, both temporally and spatially using laminar flows and thus improves previous methods in hydrodynamic focusing. The technique is used to adsorb proteins to selected regions within a microfluidic device without any contamination of the surroundings and may serve in applications requiring selective conveying of other reagents. The regions not coated by proteins are modified with poly(ethylene glycol; PEG), known to efficiently resist protein and cell adhesion. Human endothelial and fibroblast cells are later introduced into the device selectively attaching to the protein coated regions and cultured for a few days. A simulation of the convection–diffusion characteristics of the system is presented and compared to the known T-sensor. The results reveal that, by proper design, reagents concentration may be kept nearly constant along the flow direction. This phenomenon is demonstrated here by achieving particularly precise patterning of cells but may be utilized for numerous other applications as well.

The culture of human embryonic stem cells in microchannel perfusion bioreactors

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.

Modeling and Studying Human Embryonic Stem Cell Culture Conditions in Pulsed Flow Micro-Reactors

Embryonic stem (ES) cells research is a promising field for tissue engineering due to their proliferative capacity and differentiation abilities. The culture of Human Embryonic Stem Cells (hESC) in microchannel bioreactors can be valuable for hESC cell biology studies and hESC tissue engineering applications. We have previously demonstrated the long-term culture of mammalian (HFF-Human Foreskin Fibroblasts) cells in a microchannel (130μm) bioreactor under constant perfusion in a simple approach. However, hESC were found to be highly sensitive to flow and did not grow under flow rates which were proper for HFF long-term culture. Here, we propose the use of a novel automated periodic perfusion system to co-culture hESC with HFF in a microchannel bioreactor. The method is based on short temporal pulsed flows of medium renewal followed by long static incubation periods. The short pulsed exposure to shear enables shear sensitive cells (e.g., hESC) to withstand the medium flow. The present work studies experimentally and via numerical simulations the conditions required for hESC culture in a microchannel bioreactor using the periodic perfusion method. Conventional soft-lithography techniques were used to fabricate PDMS microchannels (100 μm) sealed reversibly with glass cover slides. HESC were seeded in the microchannel with HFF, incubated for several hours and then connected to a perfusion system which contained: a syringe pump, a permeable tube oxygenator, and waste container. The ability of the periodic perfusion protocols to prevent hESC de-attachment and maintain their culture was examined. Mass transport and fluid mechanics models were used to evaluate the culture conditions within the micro-bioreactor (shear stress, oxygen level, nutritious etc.). 3D finite element mass transport analysis (Comsol 3.3) was preformed to examine the oxygen levels in the microchannel as a function of time and design parameters. Altogether, the experimental results and the theoretical model indicate that the use of a periodic perfusion bioreactor is a suitable and promising method to culture hESC in a microreactor. Culturing undifferentiated human ES cell colonies in a micro-bioreactor is an initial step toward utilizing microfluidic techniques to investigate embryonic stem cell biology.Copyright

A microfabricated bio-sensor for erythrocytes deformability and volume distributions analysis

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.