Frank Bunge

3D Printing Solutions for Microfluidic Chip-To-World Connections

The connection of microfluidic devices to the outer world by tubes and wires is an underestimated issue. We present methods based on 3D printing to realize microfluidic chip holders with reliable fluidic and electric connections. The chip holders are constructed by microstereolithography, an additive manufacturing technique with sub-millimeter resolution. The fluidic sealing between the chip and holder is achieved by placing O-rings, partly integrated into the 3D-printed structure. The electric connection of bonding pads located on microfluidic chips is realized by spring-probes fitted within the printed holder. Because there is no gluing or wire bonding necessary, it is easy to change the chip in the measurement setup. The spring probes and O-rings are aligned automatically because of their fixed position within the holder. In the case of bioanalysis applications such as cells, a limitation of 3D-printed objects is the leakage of cytotoxic residues from the printing material, cured resin. This was solved by coating the 3D-printed structures with parylene-C. The combination of silicon/glass microfluidic chips fabricated with highly-reliable clean-room technology and 3D-printed chip holders for the chip-to-world connection is a promising solution for applications where biocompatibility, optical transparency and accurate sample handling must be assured. 3D printing technology for such applications will eventually arise, enabling the fabrication of complete microfluidic devices.

Microfluidic Platform for the Long-Term On-Chip Cultivation of Mammalian Cells for Lab-On-A-Chip Applications

Lab-on-a-Chip (LoC) applications for the long-term analysis of mammalian cells are still very rare due to the lack of convenient cell cultivation devices. The difficulties are the integration of suitable supply structures, the need of expensive equipment like an incubator and sophisticated pumps as well as the choice of material. The presented device is made out of hard, but non-cytotoxic materials (silicon and glass) and contains two vertical arranged membranes out of hydrogel. The porous membranes are used to separate the culture chamber from two supply channels for gases and nutrients. The cells are fed continuously by diffusion through the membranes without the need of an incubator and low requirements on the supply of medium to the assembly. The diffusion of oxygen is modelled in order to find the optimal dimensions of the chamber. The chip is connected via 3D-printed holders to the macroscopic world. The holders are coated with Parlyene C to ensure that only biocompatible materials are in contact with the culture medium. The experiments with MDCK-cells show the successful seeding inside the chip, culturing and passaging. Consequently, the presented platform is a step towards Lab-on-a-Chip applications that require long-term cultivation of mammalian cells.

Experimental and numerical analysis of complete acoustic band gaps in three-dimensional phononic crystals

In this contribution, we focus on the analysis of complete omnidirectional acoustic band gaps in additively manufactured three-dimensional (3D) phononic crystals. We present a numerical analysis of band structure and phononic band gaps of different cubic unit cell geometries. For validation, we report experimental results for transmission of acoustic waves in different characteristic spatial directions through various phononic crystal samples. These results are supplemented by numerical transmission analysis. The elements form the building blocks of wideband, high-resolution phononic-fluidic systems for measuring physical properties such as fluid density, speed of sound, and concentration.

Travelling-wave dielectrophoresis allowing flexible microchannel design for suspended cell handling

In this contribution, we present travelling-wave based dielectrophoretic (twDEP) microfluidic devices for the handling of suspended grown cells. Travelling-wave based dielectrophoretic devices rely on a moving electric field gradient, which can be realised by applying phase-shifted AC-voltages between sets of parallel electrodes. The distance between these electrodes can be reduced to a few micrometres. In optimised conditions, channels with a height of even hundreds of micrometres are applicable. Two microfluidic devices have been realised to investigate the advantages of travellingwave dielectrophoresis for cell handling.

Optimization of on-chip bacterial culture conditions using the Box-Behnken design response surface methodology for faster drug susceptibility screening

Optimized culture conditions are essential for the investigation of biological processes. In this work, on-chip optimization of bacterial culture conditions by combining microfluidics with the Box-Behnken design response surface methodology is presented. With this methodology, the effects of several cultivation variables and their interactions were investigated enabling very fast drug susceptibility screening. The proposed measurement protocol for the determination of minimum inhibitory concentration (MIC) consist of three steps: i) single factor experiments to determine the effect of pH, nutrient concentration, and temperature on the bacterial culture; ii) analyses of the relationship between variables and the effect of the individual variables by means of the Box-Behnken design and response surface methodology (BBD-RSM) optimization; and iii) bacterial susceptibility screening of drugs and drug combinations. BBD-RSM is efficient to determine the optimal growth conditions of bacteria species with a strongly reduced amount of required experiments. On top of that, these experiments can in principle all be performed at the same time, yielding significant time-savings. The found optimized culture conditions of E. coli were applied to determine the MIC values of the drugs penicillin-streptomycin and baicalein, and combinations of those. MIC values were obtained within 8-14 h, including the 6-8 h required to determine the optimal growth parameters. The microfluidic BBD-RSM method results in a significant time reduction compared to the standard 2-4 days required to determine MIC values and is, therefore, a potential alternative in the management of bacterial infections.

Neue anwenderfreundliche Konzepte zur Zellkultivierung in Mikrochips

Microfluidic cell culture chips enable 3D-cultures or co-cultures as well as integrated sensors. Though, such devices are not yet utilized in biolabs. The reasons such as incompatibilities or inappropriate materials are addressed in this contribution by presenting two novel concepts with integrated porous membranes for gas and nutrient supply. The combination of clean-room technologies and 3D-printing enable production at reasonable costs as well as adapted interfaces for standard equipment.

A novel on-chip element to provide mammalian cell cultivation and passaging to Labs-on-Chips

We present for the first time the long-term cultivation, passaging, and harvesting of mammalian cells outside of an incubator but inside a microfluidic chip fabricated by standard processes out of hard materials like silicon and glass. The presented device includes structures to supply the cells continuously but separately with gases and medium. Together with the harvesting option, this enables the future integration of analysis tools towards real Labs-on-Chips (LoC) applications. The cell culture chamber is separated from the supply channels by permeable membranes of agarose hydrogel. Consequently, the medium diffuses into the culture chamber which avoids shear stress on the cells. Our experiments show the successful seeding, culturing over 24 hours and harvesting of MDCK-cells.

Integration of Silica Aerogels in Microfluidic Chips

This paper reports a method to integrate silica aerogels monolithically in microfluidic chips. Silica Aerogel is a highly porous bulk material. The gel was synthesized from tetraethyl orthosilicate by a sol-gel process. Polyethylene glycol and an extended aging period were used to strengthen the matrix minimizing gel shrinkage. This technique allows alcogel structures with high strength and stiffness that withstand high pressure during the subcritical drying process. Hexamethyldisilazane provides for hydrophobizing and prevents the formation of siloxane bonds during the drying process. The resulting transparent aerogels reach porosities of 85%, pore diameters around 50 nm and contact angles of 136°.

Easy-to-use microfluidic chip for long-term 3D-cell cultures

We present a microfluidic chip for an easy setup of a 3D-culture of mammalian cells. The chip contains feeding structures and gas supply for long-term cultivation of mammalian cells. The device is fabricated out of hard materials like silicon and glass that are all highly biocompatible. The chip uses the concept of surficial phaseguides that allows the partial filling of a microfluidic chip with liquids based on hydrophobic and hydrophilic surfaces. Here, a suspension of mammalian cells and melted agarose is filled into the chip and is pulled by the capillary pressure on the hydrophilic areas but not on the hydrophobic phaseguides. Consequently, only a part of the chip is filled with the agarose which gels by cooling a form the 3D-cell culture. The unfilled areas are used as supply structures for nutrition and gases. So the supply is based on diffusion and the supply of nutrition and gases is controlled independently. We cultured HaCaT-cells over 24 hours in our device and achieve a good viability.

Gas Supply through Agarose Walls in Cell Culturing Microchips

We present a novel structure to supply gases to microchambers in microfluidic chips. An exemplary application is the continuous feeding of oxygen and CO2 for on-chip cell cultivation of mammalian cells. In our device, the surrounding air diffuses into the culture medium inside the chip through a porous wall of agarose hydrogel resulting in an easy and robust design. One common method is the usage of gas permeable PDMS chips. However, liquid medium in which the cells grow is absorbed by PDMS causing unknown concentrations and memory effects. Another possibility is a complex setup where medium with already dissolved gas is pumped constantly through the chip. We designed and realized a silicon and borosilicate glass chip containing a gas permeable wall of agarose preventing leakage of medium. In order to precisely position the walls in the chip, we made use of surficial phaseguides (50nm high). The blue-bottle-experiment makes the effective dissipation of oxygen visible when the colorless leucomethylen-blue reacts to methylene-blue. Successful results were achieved when applying 0.5 g/l methylene blue, 10 g/l glucose and a pH of 12.6 set by a buffer solution. As a result a continuous color gradient through the chip was obtained, which reflects the oxygen gradient and confirms the oxygen diffusion.