Andreas Grodrian

Generation of larger numbers of separated microbial populations by cultivation in segmented-flow microdevices

The high speed production of fluid segments for the highly parallelized cultivation of monoclonal cell populations was carried out by the use of microchip segmentor modules. Aqueous fluid segments, embedded in a non-miscible carrier liquid, were produced with frequencies up to 30 s(-1) and showed a high homogeneity in size. This corresponds with the production of about 2.5 million samples per day. The segment volumes can be adapted between about 4 nl and 100 nl. The typical segment size for cultivation experiments is in the range between 40 nl and 80 nl. Nutrient medium can be applied instead of pure water. It is possible to aliquot a cell suspension in such a way that most of the aqueous fluid segments contain only one cell. In model experiments with four microbial species chip-produced aliquots of 60 nl, each containing one or a few cells, were incubated in Teflon capillary tubes. Rapid growth of the microcultures was observed. Cell densities were found to be as high as in conventional shake flask cultures.

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.

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.

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.