Ralf Ahrens

Microstructuring of multiwell plates for three-dimensional cell culture applications by ultrasonic embossing

Since three-dimensional (3D) cell culture models better reflect tissues in vivo in terms of cell shape and microenvironment compared to conventional monolayer cultures, 3D tissue culture substrates gain more importance for a wide range of biological applications like drug discovery, toxicological studies, cancer and stem cell research. In this study we developed a method for the fabrication of 3D cell culture substrates in a multiwell plate format by microstructuring the bottom of 96-well cell culture plates using an ultrasonic embossing process. The resulting microstructured area consists of cubic microcavities in which adherent multicellular aggregates can be formed. We performed the biological evaluation of the system with the liver-derived human cell-line HepG2 and compared the novel substrate with a commercially available 3D culture system comprising porous alginate sponges. Metabolic activity (alamarBlue® reduction) and induction of four biotransformation enzymes (EROD, ECOD, UGT, SULT) were determined by fluorimetry or HPLC. Our results revealed that HepG2 cells in microstructured plates showed a higher mitochondrial activity, as well as enzyme activity of ECOD and UGT after treatment with an inducer when compared to cells cultured in alginate sponges at otherwise comparable conditions. Since we have modified standard cell culture plates, the obtained system is adaptable to automated screening and might be useful for all kinds of cultures including adult, progenitor and stem cells which need a 3D culture configuration to restore or maintain the differentiated status.

Development of a novel two-channel microfluidic system for biomedical applications in cancer research

In this paper we present a novel two-channel microfluidic system which acts as an artificial blood capillary vessel to examine the migration steps of cancer cells in the microvasculature. The system consists of polycarbonate and is fabricated by combining hot embossing and thermal bonding. The optically transparent polycarbonate allows the use of live cell and fluorescence microscopy. The main feature of this device is that all processes take place under continuous laminar flow conditions at distinct tuneable shear rates.

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.

Fabrication of polymeric microfluidic devices with tunable wetting behavior for biomedical applications

We demonstrate the fabrication of microchannels with specific fluidic behavior due to micro- and/or nanostructures on the surfaces. With a combination of hot embossing and microthermoforming it is possible to produce microchannels with specific surface properties. These surface properties are highly dependent on the micro- and nanostructures embossed into the material. Different structure sizes and geometries where examined by contact angle measurements. Here the dependency of diameter and pitch of the structures on the contact angle is examined as well as the material impact. These results enable the fabrication of highly specific surfaces tunable to an application.

Investigation of endothelial growth using a sensors-integrated microfluidic system to simulate physiological barriers

Abstract In this paper we present a microfluidic system based on transparent biocompatible polymers with a porous membrane as substrate for various cell types which allows the simulation of various physiological barriers under continuous laminar flow conditions at distinct tunable shear rates. Besides live cell and fluorescence microscopy, integrated electrodes enable the investigation of the permeability and barrier function of the cell layer as well as their interaction with external manipulations using the Electric Cell-substrate Impedance Sensing (ECIS) method.

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)