Gunter Gastrock

Online optical detection of food contaminants in microdroplets

To accommodate the considerable increase of disease based on microbial food contaminants in the last decade, a modulated, fast optical fluorescence detection combined with microdevices is created. This method, which consists of five different steps, first selects contaminants, mainly bacteria, in the food matrix. This process is based on a biomagnetic separation technique developed by our collaborators at the Technical University of Dresden. By the steps of binding antibody functionalized magnetic beads and fluorescent capsules on the target cell, a magnetic bead‐target cell‐microcapsule complex (MTM) is generated. The well‐established pipe‐based bioreactors (pbb) platform enables the generation of droplets with a volume between 60 and 160 nL and the detection of the target cell with an integrated microscopic and spectroscopic detection system. The module used for generating droplets is based on the segmented flow principle and is chip‐ or probe‐based. In this context, the successful use of polydimethylsiloxane (PDMS) as a cost‐effective alternative to the well‐established glass‐chips is introduced. To quantify the detection based on a yes‐ or no‐decision, the most important step is to separate one MTM‐complex per droplet. This equalized the quantity of the fluorescent signals with the quantity of the contaminants in the cell sample. The feasibility of microscopic and spectroscopic detection with only one fluorescent capsule per droplet is shown. Also the first results of a special prototyping optical detection set‐up that is already in an advanced stage of development, will be presented. This easy‐to‐use device implemented a software‐controlled, automatic documentation for every fluorescent signal of a droplet to guarantee the quality control. Here are the advantages of an integration of microdevices in a rapid detection of food pathogens presented. Obviously, the modular set‐up of this detection platform enables a wide range of high‐throughput applications.

Hydrodynamic influence on sampling systems in bioreactors

A special challenge for bioprocess technology has been interfacing new analytical equipment to the bioreactor. The main component of this interface is a sterile barrier between the biological process and the monitoring system. The connecting link is a sampling unit with micro- or ultrafiltration membranes. In order to prevent biofouling on top of the membranes, we have developed an oscillating sampling system. A model can describe the hydrodynamics, and the distribution forces in the region between membrane and medium. We compare a static sampling device to an oscillating one during a yeast cultivation process, with regard to filtration rates and the analysis of the withdrawn samples via Scanning Electron Microscopy (SEM) and Fourier Transformed Infrared Spectroscopy (FTIR). The results indicate that the oscillating sampling device shows better sampling flow behaviour.

A modular segmented-flow platform for 3D cell cultivation.

In vitro 3D cell cultivation is promised to equate tissue in vivo more realistically than 2D cell cultivation corresponding to cell-cell and cell-matrix interactions. Therefore, a scalable 3D cultivation platform was developed. This platform, called pipe-based bioreactors (pbb), is based on the segmented-flow technology: aqueous droplets are embedded in a water-immiscible carrier fluid. The droplet volumes range from 60 nL to 20 μL and are used as bioreactors lined up in a tubing like pearls on a string. The modular automated platform basically consists of several modules like a fluid management for a high throughput droplet generation for self-assembly or scaffold-based 3D cell cultivation, a storage module for incubation and storage, and an analysis module for monitoring cell aggregation and proliferation basing on microscopy or photometry. In this report, the self-assembly of murine embryonic stem cells (mESCs) to uniformly sized embryoid bodies (EBs), the cell proliferation, the cell viability as well as the influence on the cell differentiation to cardiomyocytes are described. The integration of a dosage module for medium exchange or agent addition will enable pbb as long-term 3D cell cultivation system for studying stem cell differentiation, e.g. cardiac myogenesis or for diagnostic and therapeutic testing in personalized medicine.

Generation of Cardiomyocytes in Pipe-Based Microbioreactor Under Segmented Flow.

Background/Aims: Embryonic stem (ES) cells have got a broad range differentiation potential. The differentiation is initiated via aggregation of non-differentiated ES cells into embryoid body (EB) capable of multi-lineage development. However experimental variables present in standard differentiation techniques lead to high EB heterogeneity, affecting development into the cells of desired lineage, and do not support the process automatization and scalability. Methods: Here we present a novel pipe based microbioreactor (PBM) setup based on segmented flow, designed for spatial maintenance of temperature, nutrition supply, gas supply and sterility. Results: We verified PBM feasibility for continuous process generating cardiac cells starting from single ES cell suspension followed by EB formation for up to 10 days. The ES cells used in the study were genetically modified for cardiac-specific EGFP expression allowing optical monitoring of cardiomyocytes while EBs remained within PBM for up to 10 days. Efficiency of cardiac cells formation within PBM was similar compared to a standard hanging drop based protocol. Conclusion: Our findings ensure further development of microfluidic bioreactor technology to enable robust cardiomyocytes production for needs of drug screening, tissue engineering and other applications.

Electrical Sensing in Segmented Flow Microfluidics

Microfluidic systems require measurement techniques to assess the bioprocesses taking place within these systems. In droplet-based microfluidics, sensing systems have thus far been dominated by the use of optical sensing techniques. Direct electrical measurement of droplets has been quite rare. This chapter describes the use of electrical sensors in droplet-based microfluidics. Electrical sensors offer a possibility to measure the presence/position of droplets but also their electrical properties.

A modified 384-well-device for versatile use in 3D cancer cell (co-)cultivation and screening for investigations of tumor biology in vitro

Pancreatic cancer exhibits a worst prognosis owed to an aggressive tumor progression i.a. driven by chemoresistance or tumor‐stroma‐interactions. The identification of candidate genes, which promote this progression, can lead to new therapeutic targets and might improve patients outcome. The identification of these candidates in a plethora of genes requires suitable screening protocols. The aim of the present study was to establish a universally usable device which ensures versatile cultivation, screening and handling protocols of cancer cells with the 3D spheroid model, an approved model to study tumor biology. By surface modification and alternative handling of a commercial 384‐well plate, a modified device enabling (i) 3D cultivation either by liquid overlay or by a modified hanging drop method for (ii) screening of substances as well as for tumor‐stroma‐interactions (iii) either with manual or automated handling was established. The here presented preliminary results of cell line dependent dose‐response‐relations and a stromal‐induced spheroid‐formation of the pancreatic cancer cells demonstrate the proof‐of‐principle of the versatile functionality of this device. By adapting the protocols to automation, a higher reproducibility and the ability for high‐throughput analyses were ensured.

Parametric studies on droplet generation reproducibility for applications with biological relevant fluids

Although the great potential of droplet based microfluidic technologies for routine applications in industry and academia has been successfully demonstrated over the past years, its inherent potential is not fully exploited till now. Especially regarding to the droplet generation reproducibility and stability, two pivotally important parameters for successful applications, there is still a need for improvement. This is even more considerable when droplets are created to investigate tissue fragments or cell cultures (e.g. suspended cells or 3D cell cultures) over days or even weeks. In this study we present microfluidic chips composed of a plasma coated polymer, which allow surfactants‐free, highly reproducible and stable droplet generation from fluids like cell culture media. We demonstrate how different microfluidic designs and different flow rates (and flow rate ratios) affect the reproducibility of the droplet generation process and display the applicability for a wide variety of bio(techno)logically relevant media.