A modular microfluidic bioreactor to investigate plant cell-cell interactions.

Plants produce a wide variety of secondary metabolites, which often are of interest to pharmaceutical and nutraceutical industry. Plant-cell cultures allow producing these metabolites in a standardised manner, independently from various biotic and abiotic factors difficult to control during conventional cultivation. However, plant-cell fermentation proves to be very difficult, since these chemically complex compounds often result from the interaction of different biosynthetic pathways operating in different cell types. To simulate such interactions in cultured cells is a challenge. Here, we present a microfluidic bioreactor for plant-cell cultivation to mimic the cell-cell interactions occurring in real plant tissues. In a modular set-up of several microfluidic bioreactors, different cell types can connect through a flow that transports signals or metabolites from module to module. The fabrication of the chip includes hot embossing of a polycarbonate housing and subsequent integration of a porous membrane and in-plane tube fittings in a two-step ultrasonic welding process. The resulting microfluidic chip is biocompatible and transparent. Simulation of mass transfer for the nutrient sucrose predicts a sufficient nutrient supply through the membrane. We demonstrate the potential of this chip for plant cell biology in three proof-of-concept applications. First, we use the chip to show that tobacco BY-2 cells in suspension divide depending on a "quorum-sensing factor" secreted by proliferating cells. Second, we show that a combination of two Catharanthus roseus cell strains with complementary metabolic potency allows obtaining vindoline, a precursor of the anti-tumour compound vincristine. Third, we extend the approach to operationalise secretion of phytotoxins by the fungus Neofusicoccum parvum as a step towards systems to screen for interorganismal chemical signalling.

Initial Bacterial Adhesion Properties of Anodically Oxidized Ti6Al4V.

This paper reports about the initial interaction of bacteria with anodically oxidized Ti6Al4V for the use as dental implant abutment surfaces. Ti6Al4V samples are anodically oxidized in hydrofluoric acid using different voltages. The resulting nanotopographies are characterized by atomic force microscopy, scanning electron microscopy and contact angle measurements. The topographies reach from micro-porous structures with small nanoporosities on top to fully hexagonally aligned nanotubes. For initial bacterial adhesion tests, Escherichia coli and Staphylococcus aureus are used. Samples are incubated for 2 h and afterwards non-adherent cells are washed off. The results of live/dead staining and cell counts are presented. Gram-negative and Gram-positive strains show different behavior in respect to total number of initially adherent cells on different micro/nanotopographies. The observed reduction of adhered microorganisms is mainly based on underlying microporous topographies.

Miniaturized instrument systems for minimally invasive diagnosis and therapy.

Microsystems technologies allow to considerably improve the functionality of existing medical instruments and produce novel devices. Using extremely miniaturized operation systems based on micro-technically processed nickel-titanium alloys, minimally invasive therapeutic interventions can be accomplished in the most sensitive parts of the human body. This has not been possible so far. Fields of use presently comprise among others minimally invasive surgery, endoscopic neurosurgery, interventional cardiology, gynaecology, urology, and ophthalmology.

[Plastic micro-tips for drug delivery]. / Mikrospitzen aus Kunststoff für die Medikamentenapplikation.

Removal or exact transfer of minimum substance volumes from reservoirs or microfluidic systems may be accomplished by means of miniaturized tips with integrated through-going capillaries. Applications in biomedical engineering, e.g. for the application of drugs, or in life sciences, e.g. equipping of microarrays, require the use of disposable plastic products for hygienic reasons and reasons of costs. For this purpose, a method to fabricate microtips out of plastic by doublesided molding has been developed at the Forschungszentrum Karlsruhe.

[Lab-On-A- Chip--systems for biomedical research and diagnosis]. / Lab-On-A-Chip--Systeme für Die Biomedizinische Forschung und Diagnostik.

In today's biomedical research and diagnosis, a number of substances and agents have to be checked. Frequently, plastic micro titer plates are used for this purpose as large-area test platforms. For the first time, plastic micro titer plates with 96 identical microfluidic labon-a-chip structures for simultaneous capillary electrophoresis (CE) have now been produced using microtechnical fabrication methods. Such structures are suited for e.g. the separation of biomolecules. In completely sealed microfluidic channel systems, smallest sample volumes can be processed, separated, mixed with other substances, or detected. Due to the small channel dimensions, these microfluidic systems are characterized by very small sample volumes needed.

Further development of microstructured culture systems and their use in tissue engineering.

The Forschungszentrum Karlsruhe aims at improving its CellChip. Its main feature is the 1 cm2 core, subdivided into 900 cubic microcontainers (300 x 300 x 300 microns). It is manufactured by injection molding using biodegradable (polylactide) as well as non-degradable (PMMA or PC) polymers. The CellChips will be modified such that membranes will be mounted at the bottom of the CellChip, thus facilitating backend processing. Furthermore, the membranes can be adapted ideally to the assay system of interest by various surface modification techniques.

Fabrication of metal and polymer microstructures.

Microelectrical discharge machining (microEDM) is an innovative manufacturing technique for producing multifunctional metal microcomponents from difficult to machine materials such as nitinol and stainless steel. In addition, the microEDM technique allows the microstructurisation of stainless steel mould inserts for low-cost mass production of components made from various types of polymers.

Microthermoforming as a novel technique for manufacturing scaffolds in tissue engineering (CellChips).

The CellChip is a microstructured polymer scaffold, which favours a three-dimensional cultivation of cells within an array of cubic microcontainers. The manufacturing process used so far is microinjection moulding combined with laser-based perforation. In a first attempt to simplify the process, costly perforation was avoided by using commercially available, inexpensive microfiltration membranes for the bottom of the microcavities. Microthermoforming is a promising novel technique which allows the CellChip to be produced from thin film. Working pressures of approximately 4000 kPa were required for the adequate moulding of 50 microm thick films from three different polymers (polystyrene, polycarbonate, cyclo-olefin polymer). Integrating drafts and chamfers in micromoulds is not going to eliminate an uneven thickness profile, but reduces demoulding forces. Microthermoformed CellChips of polycarbonate were perforated by an ion track technique to guarantee a sufficient supply of medium and gases to the cells. The prestructured CellChips were irradiated with 1460 MeV xenon ions at a fluence of a few 10(6) ions/cm2. The tracks were etched in an aqueous solution of 5 N NaOH at 30 degrees C, which resulted in cylindrical pores approximately 2 microm in diameter. Microinjection-moulded, membrane-bonded and thermoformed CellChips were subjected to comparative examination for viability in a cell culture experiment with parenchymal liver cells (HepG2). The cells stayed viable over a period of more than 20 days. No significant differences in viability between injection-moulded, membrane-bonded, and thermoformed CellChips were observed.