Kristina Kreppenhofer

Formation of a Polymer Surface with a Gradient of Pore Size Using a Microfluidic Chip

Here we demonstrate the generation of polymer monolithic surfaces possessing a gradient of pore and polymer globule sizes from ~0.1 to ~0.5 μm defined by the composition of two polymerization mixtures injected into a microfluidic chip. To generate the gradient, we used a PDMS microfluidic chip with a cascade micromixer with a subsequent reaction chamber for the formation of a continuous gradient film. The micromixer has zigzag channels of 400 × 680 μm(2) cross section and six cascades. The chip was used with a reversible bonding connection, realized by curing agent coating. After polymerization in the microfluidic chip the reversible bond was opened, resulting in a 450 μm thick polymer film possessing the pore size gradient. The gradient formation in the microfluidic reaction chamber was studied using microscopic laser-induced fluorescence (μLIF) and different model fluids. Formation of linear gradients was shown using the fluids of the same density by both diffusive mixing at flow rates of 0.001 mL/min and in a convective mixing regime at flow rates of 20 mL/min. By using different density fluids, formation of a two-dimensional wedge-like gradient controlled by the density difference and orientation of the microfluidic chip was observed.

Time-resolved NMR metabolomics of plant cells based on a microfluidic chip.

The plant secondary metabolism generates numerous compounds harbouring pharmaceutical activity. In plants, these compounds are typically formed by different and specialised cell types that have to interact constituting a metabolic process chain. This interactivity impedes biotechnological production of secondary compounds, because cell differentiation is suppressed under the conditions of a batch bio-fermenter. We present a novel strategy to address this limitation using a biomimetic approach, where we simulate the situation in a real tissue by a microfluidic chamber system, where plant cells can be integrated into a process flow. We show that walled cells of the plant model tobacco BY-2 can be successfully cultivated in this system and that physiological parameters (such as cell viability, mitotic index and division synchrony) can be preserved over several days. The microfluidic design allows to resolve dynamic changes of specific metabolites over different stages of culture development. These results serve as proof-of-principle that a microfluidic organisation of cultivated plant cells can mimic the metabolic flows in a real plant tissue.

Microfluidic polycarbonate chip for long-term cell analyses

Differentiation of stem cells to more specific tissue like heart, skin or nerve cells is influenced by long-range signaling molecules (morphogens). We designed a three-stacked microfluidic chip for long-term cultivation of cells to be probed with morphogen gradients to analyze this influence. We chose polycarbonate as material, which is commonly used in cytology. The microfluidic chip is made of two microstructured polycarbonate parts by hot embossing in a commercial polycarbonate foil. Each part contains one fluidic circuit: (1) the cell chamber part to cultivate and continuously supply the cells in and (2) the mixer part to form and provide a morphogen step gradient to these cells. A nanoperforated polycarbonate membrane embedded in-between the two parts allows exposing the cells to the provided step gradient. The two parts and the nanoperforated polycarbonate membrane of the microfluidic chip are assembled by a two-step thermal bonding process. We observed living and proliferating HeLa cells in the cell chamber part after six days of long-term cultivation. The activation of the Wnt/beta-catenin signaling pathway of HeLa cells in the cell chamber part was shown by applying a gradient of the Wnt pathway activator 6-bromoindirubin-3’-oxime (BIO) to the mixer part. We monitored the expected endogenous nuclear beta-catenin accumulation by fluorescence microscopy for those HeLa cells being exposed to a BIO concentration above the threshold. The presented microfluidic chips showed in a reproducible manner an adequate mechanical and chemical stability during the experiments. Polycarbonate is a material allowing for industrial mass production. Due to the three-stack design of the microfluidic chip, cells can be cultured under shear stress-free conditions and supplied continuously with culture medium, allowing for long-term experiments, while they are exposed to varying morphogen step gradients. We are convinced, that the differentiation of stem cells can be analyzed in a likewise microfluidic chip. Biomed Tech 2012; 57 (Suppl. 1)