Rapid Prototyping of Microfluidic Devices

With the growing importance of miniaturized energy applications and the development of micro Total Analysis Systems (μTAS), we have realized microfluidic devices, namely, magnetic micropumps, microfluidic fuel cells and membrane-based protein preconcentrators, all having high application potential in future. The choice of rapid prototyping microfabrication technologies and the selection of affordable materials are important aspects, when thinking of commercialization. Thus, we have employed powder blasting, polymer molding and assembly technologies during devices fabrication throughout the thesis. The first type of microfluidic device that we present is a poly(methyl methacrylate) (PMMA) ball-valve micropump with two different designs of the electromagnetic actuator, as optimized by the finite element method. The integration of a permanent magnet in a flexible polydimethylsiloxane (PDMS) membrane, which is clamped into PMMA structure, is proposed for providing a large stroke of the pumping membrane, making the micropump bubble-tolerant and self-priming Focusing on low power consumption for μTAS integration, another type of magnetic micropump with active valves is realized. It consists of a microfluidic chamber structure in glass that is assembled with a PDMS sheet, which comprises two valving membranes and a central actuation membrane, having each an integrated permanent magnet that is peristaltically actuated by a rotating arc-shaped permanent magnets assembly. A lumped circuit model is developed to predict and describe the frequency-dependent flow rate behavior for this type of pump. Powder blasting and PDMS molding rapid prototyping technologies are employed for realization of these two types of micropumps. Fuel cells with fluid delivery and removal options, having chemical reaction sites and electrode structures that can be realized in a microfluidic format, have high potential for applications. Therefore, microfluidic direct methanol fuel cells with embedded ion- permselective medium are studied and such type of fuel cell is realized by integrating a narrow Nafion strip in a molded elastomeric structure. A mechanical clamping assembly technology enables leakage-free operation and stable performance. The characterization reveals its output power density, using H2O2-based oxidant, is among the high-performance direct methanol fuel cells in microscale. Re-using the technology of the fuel cell chip, with its particular ion-permselective Nafion membrane and assembly method, we also have developed a protein preconcentrator with high purification performance. Our device can preconcentrate negatively charged biomolecules located at the anodic compartment side of the Nafion strip within only a few minutes with a high preconcentration factor. Moreover, a complex microfluidic finite element model is proposed to study and understand the physics of the preconcentration effect. Finally, we conclude the thesis with an outlook on future developments based on our work of the project and on the assembly technologies for microfluidic device integration.

Integrated microfluidic system for non-invasive electrophysiological measurements on Xenopus oocytes

Elodie Deborah Dahan

We developed a new non-invasive integrated microsystem for electrophysiological measurements on Xenopus laevis oocytes. The Xenopus oocyte is a well-known expression system for various kinds of ion channels that are of potential interest for drug screening. In the traditional "Two Electrode Voltage Clamp" (TEVC) method, delicate micromanipulation is required to impale an oocyte with two microelectrodes. In our system, a non-invasive electrical access to the cytoplasm is provided by permeabilizing the cell membrane with an ionophore (e.g. nystatin). Unlike for the classical patch-clamp or "macropatch" techniques, this method does not require removal of the fibrous vitelline membrane. Cell handling is significantly simplified, resulting in more robust recordings with increased throughput. Moreover, because only part of the oocyte surface is exposed to reagents, the required volume of reagent solutions could be reduced by an order of magnitude compared to the TEVC method. In a second part of this work, a rapid fluidic exchange system was implemented on-chip to allow recording of fast kinetic events of exogenous ion channels expressed in the cell membrane. Reducing fluidic exchange times of extracellular reagent solutions is a great challenge with these large millimeter-size cells. Fluidic switching is obtained by shifting the laminar flow interface in a perfusion channel under the cell by means of integrated poly-dimethylsiloxane (PDMS) microvalves. Reagent solution exchange times down to 20 ms have been achieved. An on-chip purging system allows to perform complex pharmacological protocols, making the system suitable for screening of ion channel ligand libraries. The fabrication process for this disposable microchip, based on PDMS micromolding, is cost-efficient and simple. Moreover, an innovative integration of agar gel-based reference electrodes was developed. A conductive liquid junction is injected by capillary force filling of suitable microchannels, overcoming the problems encountered with the integration of Ag/AgCl thin film or wire reference electrodes. We tested this new microdevice by performing electrophysiological measurements on oocytes expressing the human Epithelial Sodium Channel (hENaC). Transmembrane currents could be recorded with a large signal-to-noise ratio. The performance of the integrated rapid fluidic exchange system was demonstrated by investigating the self-inhibition of hENaC. Our results show that the response time of this ion channel to a specific reactant is about one order of magnitude faster than estimated with the traditional TEVC technique. We conclude that this new microdevice has high potential for improved electrophysiological investigations with oocytes in the field of pharmacology and toxicology.

EPFL2008

Magnetically actuated micropumps

Christophe Yamahata

"Lab-On-a-Chip" (LOC) systems are intended to transpose complete laboratory instrumentations on the few square centimetres of a single microfluidic chip. With such devices the objective is to minimize the time and cost associated with routine biological analysis while improving reproducibility. At the heart of these systems, a fluid delivery unit controls and transfers tiny quantities of liquids enabling the biological assays. This explains the need for robust integrated micropumps as a precondition for the development of many LOC devices. In this context, we have developed a rapid prototyping method for the fabrication of microfluidic chips in plastic and glass materials. The microfabrication principle, which is based on the powder blasting microstructuring process, was used to build devices in either polymethylmethacrylate (PMMA) or borosilicate glass. Various types of micropumps have been developed which were all based on external magnetic actuation. The use of ferrofluids (or magnetic liquids) has been the subject of the first part of the research. A piston pump using a ferrofluid plug moved by an external magnet has been studied. The integration of a rare-earth material (NdFeB) in a flexible polydimethylsiloxane (PDMS) membrane, in the form of a powder or as a classical permanent magnet, has then been proposed. An external electromagnet was used to actuate the magnet-containing diaphragm of a reciprocating micropump. Different types of valves, which constitute the critical element in reciprocating micropumps, have also been investigated. We have studied silicone membrane valves, nozzle-diffuser elements and ball valves. While nozzle-diffuser elements present the simplest valving solution from a manufacturing point of view, ball valves have been proposed as a very promising alternative due to their high efficiency. Together with the detailed characterization of the prototypes, we have proposed analytical models that predict the hydrodynamic behaviour of the micropumps. The performances of our micropumps indicate that magnetic actuation is well adapted for LOC microsystems. While we have demonstrated that our proposed microfabrication technique is an excellent rapid prototyping method for disposable plastic devices, our glass micropumps present a competitive low-cost alternative satisfying criteria of biocompatibility and high temperature (130 °C) resistance.

EPFL2005

A novel cell culture approach for tissue engineering based on surface expansion

Hicham Majd

Most tissue engineering approaches include the delivery of reparative cells to a damaged host tissue. Generally these cells are sought to be of a stem cell character since they retain a high potential for proliferation and differentiation into diverse phenotypes. To restrict cell function to the desired phenotype and to avoid anti-immune responses, autologous stem cells are frequently isolated from intact regions of the same (damaged) organ, such as keratinocytes from the basal layer of the skin and hair follicles or from the same germ layer origin such as mesenchymal stem cells (MSC) that are obtained from the bone marrow. This approach faces two major problems: 1) The number of source cells obtained from biopsies is generally too low for direct implantation, which requires cell multiplication in culture. 2) The standard culture conditions, including growth in plastic dishes, passaging, and the composition of the growth medium, never correspond to the natural microenvironment and influence cell differentiation, often resulting in non-desired phenotypes. The objective of my thesis was to provide a solution for both problems that is applicable for the average cell culture laboratory. Our technique reduces traumatic enzymatic passaging, provides constant cell densities to keep cells in the exponential growth phase and at the same time avoids contact inhibition of cell growth. This novel approach comprises the development of a new elastic culture surface, produced from a high extension silicone rubber (HESR) that we here for the first time applied for cell culture. The HESR is enlarged by a novel mechanical approach permitting to visualize and control cells under expansion. Untreated silicone, the polymer component of the HESR, does not promote cell attachment and must therefore be functionalized. In the first part of my thesis (Chapter 2) we establish a surface treatment for silicone membranes that will resist large surface expansions. We compare established coating techniques that have originally been developed to provide static silicone surfaces with cell adhesive molecules by 1) hydrophobic, 2) electrostatic and 3) covalent interactions. Cell attachment, spreading and proliferation was evaluated between these three treatments as a function of the coating in static and stretched conditions. We show that covalent immobilization of collagen type I is most suitable to stretch cells cultured on elastic silicone substrates. We applied this technique to functionalize the HESR culture surface in all later applications. The second central part of my thesis (Chapter 3) develops a new culture method consisting of a motorized device that isotropically expands the coated HESR surface to yet unmatched 1000% of its initial surface. Proof-of-principle of this method is given by culturing human MSC, which are sensitive to passaging during expansion in vitro and that are therapeutically relevant cells to repair bone, cartilage, and cardiovascular tissue. MSC were cultured three consecutive passages on the culture surface that has been expanded in steps of 5% every 6 h for 20 days, respectively. After nine weeks of expansion culture, MSC exhibited consistently high proliferation rates, fully retained their pluripotent stem cell character and were inducible to differentiate into adipogenic, chondrogenic, osteogenic, and myogenic lineages. The cell surface expansion culture surface produced ∼10-times higher cell numbers compared with nine weeks of standard culture, which required nine events of passaging to attain the same culture surface. We propose our method to produce MSC for clinical cell therapy. In the third part of my thesis (Chapter 4) we implement our novel surface expansion method to culture fibroblasts with the aim to: 1) rapidly expand primary fibroblasts, 2) achieve higher cell yields than obtained with conventional culture, and 3) obtain homogeneous cell populations with non-fibrogenic character. The expansion of primary autologous dermal fibroblasts in culture is a pivotal step to obtain sufficiently high cell numbers for application in skin esthetics and for the treatment of large burn wounds. Dynamically enlarging the culture surface by 800% over 20 d, yielded ∼1.5 higher fibroblasts numbers than the equivalent three standard passages. By further optimizing the surface expansion speed according to the cell growth rates, the same high cell numbers were produced after only 9 d of culture. The highly compliant nature of the HESR surface had the beneficial effect of completely abolishing the formation of 'fibrotic' myofibroblasts. Hence, our novel method for fibroblast culture is faster and yields higher numbers of more homogeneous cells than standard culture passaging. It delivers a fibroblast quality and quantity that is appealing for cell therapeutic purposes. The aim of the last part of my thesis (Chapter 5) was to provide highly compliant silicone elastomers, such as the HESR, with a micropattern of cell adhesive proteins. In particular we aim to produce arrays of extracellular matrix proteins with a size and morphology to promote formation of single focal adhesions on highly elastic and expandable surfaces. This will allow future studies on cell mechanosensing through focal adhesions by discriminating between effects of focal adhesion size changes and stretch. An existing method to pattern rigid surfaces with proteins, so-called microcontact printing, is not suitable to transfer proteins onto surfaces that are softer than the stamp. We here establish a new transfer technique that is based on a stencil with micron-sized openings. The new stencil is used to expose the surface of compliant silicone substrates with Young's modulus of 10-100 kPa to a protein solution of fibronectin, collagen I and protein-repellent polymers, leaving behind the desired protein pattern. Fibroblasts were successfully attached onto the resulting focal islet arrays on compliant substrates.

EPFL2008

A novel cell culture approach for tissue engineering based on surface expansion

Hicham Majd

EPFL2008

Integrated microfluidic system for non-invasive electrophysiological measurements on Xenopus oocytes

Elodie Deborah Dahan

EPFL2008

Magnetically actuated micropumps

Christophe Yamahata

EPFL2005